Novel Sol-Gel Nanoporous Materials, Nanocomposites and Their
Applications in Bioscience
A Thesis
Submitted to the Faculty
of
Drexel University
by
Zhengfei Sun
in partial fulfillment of the
requirements for the degree
of
Doctor of Philosophy
September 2005
© Copyright 2005
Zhengfei Sun. All Rights Reserved.
2 Dedications
This dissertation is dedicated to my parents, Mr. Chongzhen Sun and Mrs. Xiuqing
Nie for their encouragement, support and love.
3 Acknowledgments
In retrospect as I approach the completion of my doctorate, I feel a deep gratitude
towards many people for their assistance and support. I would like to express my genuine
gratitude to each of them, although it would be impossible for me to name all.
First of all, I would like to sincerely thank my advisor, Dr. Yen Wei, for his
tremendous time and effort spent in leading, supporting and encouraging me during the
last five years. His passion for challenges has given me inspiration; his knowledge of
science has given me guidance; his perseverance in research has given me confidence.
Without his help and effort, it would be impossible for me to even get close to this point.
I want to express my gratitude to all the committee members in my candidacy
examination and/or my dissertation defense, Dr. Anthony Addison, Dr. Jean-Claude
Bradley, Dr. Joe Foley, Dr. Susan Jansen-Varnum, Dr. Caroline Schauer, Dr. Sally
Solomon and Dr. Jian-Min Yuan for their time and valuable suggestions. Special thanks
are due to the committee chair, Dr. Anthony W. Addison for insightful discussions on
many topics, including my oral proposal and research. I am also grateful to Dr. Sally
Solomon, Dr. Caroline Schauer and Dr. Jian-Min Yuan for their detailed comments on
my oral proposal and thesis.
I would like to thank many of my collaborators. I specially thank Dr. Jian-Min Yuan
for his valuable suggestion and assistance, especially in protein folding and amyloid β aggregation projects. I also would like to thank Dr. Karl Sohlberg for giving me the opportunities to work with him on an important project. I would also thank Dr. Reinhard
Schweitzer-Stenner for his wonderful insights on many of my research works, especially
on the project of aggregation of Amyloid β peptide. I also thank Dr. Solomon Praveen for
4 his help in dental materials projects and suggestions in thesis writing.
Many thanks are due to Dr. Thomas G. Spiro of Princeton University and his student Dr. Gurusamy Balakrishnan on assistance in Raman spectroscopy. I am grateful to many people for their selfless support in many areas. I especially acknowledge Dr.
Patrick Loll for his assistance in circular dichroism spectroscopy, Dr. Guoliang Yang for
AFM studies, Ms. Edith Smith for her kindly help and coordination in obtaining the chemicals and instruments for our research work.
Many thanks are due to my friends in the Department of Chemistry who make my life here memorable. I thank Dr. Shuxi Li, Dr. Qiuwei Feng, Dr. Shan Cheng, Dr. Hua
Dong, Dr. Houping Yin, Ms. Alpa Patel, Mr. Yi Guo, Dr. Jim Tu, Ms. Stephanie Schuster for their collaboration, discussions and help during these years. I also want to thank all the professors, staff and students in Department of Chemistry, Drexel University for making the Department such a joyful working and studying environment.
Finally, I am greatly grateful to my parents, Mr. Chongzhen Sun and Mrs. Xiuqing
Nie, for their continuous encouragement and unconditional love throughout in my life.
5 Table of Contents
Table of Contents...... 6
List of Tables ...... 15
List of Figures...... 17
Abstract...... 24
Chapter 1: An Overview to Nanoporous Sol-Gel Materials...... 27
1.1 Introduction...... 27
1.2 Fundamentals of Sol-Gel Process ...... 29
1.2.1 Sol-Gel Reactions ...... 30
1.3 Nanoporous Sol-Gel Materials from Surfactant Templated Pathways...... 31
1.4 Nanoporous Sol-Gel Materials from Nonsurfactant Templated Pathway ... 35
1.5 Characterizations of Nanoporous Materials...... 37
1.5.1 Gas sorption measurement...... 37
1.5.2 X-Ray Diffraction (XRD)...... 40
1.5.3 Electron Microscopy...... 41
1.6 Organic/Inorganic Hybrid Materials by Sol-Gel Approach...... 42
1.7 Sol-Gel Encapsulation of Biomolecules ...... 45
1.8 References...... 49
Chapter 2: Rigid Matrix Artificial Chaperon (RMAC) – Mediated Refolding of
Cytochrome c...... 63
2.1 Introduction...... 63
2.1.1 Fundamentals of Protein Folding Unfolding ...... 63
2.1.2 Chaperone and Protein Folding ...... 64
6 2.1.3 Rigid Matrix Artificial Chaperone (RMAC) ...... 65
2.1.4 Why Cytochrome c? ...... 67
2.1.5 Analytical Methods to Monitor Cytochrome c’s Folding Unfolding ...... 68
2.1.5.1 Fluorescence Spectroscopy...... 68
2.1.5.2 Circular Dichroism (CD) ...... 69
2.2 Experimental...... 71
2.2.1 Materials ...... 71
2.2.2 Encapsulation of Unfolded Cc into Silica Matrix...... 71
2.2.3 Removal of Templates ...... 73
2.2.4 Characterizations of Nanoporous Silica Matrix...... 73
2.2.5 Fluorescence Spectroscopy of Encapsulated Cc...... 73
2.2.6 Circular Dichroism of Cc...... 74
2.2.7 Fourier Transform Infrared Spectroscopy (FTIR) of Silica Matrix...... 75
2.2.8 UV-Vis Spectroscopy (UV) of Encapsulated Cc...... 76
2.2.9 Attempts of Making Silica Thin Film...... 77
2.3 Results and Discussion ...... 78
2.3.1 Characterization of Silica Matrix...... 79
2.3.2 FT-IR Spectroscopy on Silica Matrix...... 80
2.3.3 Fluorescence Spectroscopy of Encapsulated Cc...... 80
2.3.4 Leakage Tests...... 81
2.3.5 CD Spectroscopy ...... 82
2.4 Conclusion ...... 83
2.5 Acknowledgement ...... 83
7 2.6 References...... 83
Chapter 3: Using Resonance Raman Spectroscopy to Study Folding Unfolding Behavior of Encapsulated Heme Proteins in Silica Matrix with Controlled Pore Sizes ...... 99
3.1 Introduction...... 99
3.1.1 Resonance Raman Spectroscopy and Protein Folding...... 99
3.1.2 Heme Protein ...... 102
3.1.2.1 Cytochrome c and its Resonance Raman Spectroscopy ...... 102
3.1.2.1.1 Marker Band Region...... 103
3.1.2.1.2 Fingerprint Region...... 104
3.1.2.2 Hemoglobin (Hb) and Its Resonance Raman Spectra...... 104
3.1.2.3 Myoglobin (Mb) and Its Resonance Raman Spectra ...... 105
3.1.2.4 Protein Encapsulation ...... 108
3.2 Experimental...... 109
3.2.1 Materials ...... 109
3.2.2 Encapsulation of Cc into Silica Matrix by Using Urea as Template ..... 109
3.2.3 Encapsulation of Cc into Silica Matrix by Using Glucose as Template 110
3.2.4 Encapsulation of Hb into Silica Matrix by Using Glucose as Template 111
3.2.5 Encapsulation of DeoxyMb into Silica Matrix by Using Glucose as
Template ...... 112
3.2.6 Resonance Raman Spectroscopy ...... 113
3.2.7 Characterizations of Nanoporous Silica Matrix...... 114
3.2.8 Fourier Transform Infrared Spectroscopy (FTIR) of Silica Matrix...... 114
3.3 Results and Discussion ...... 115
8 3.3.1 Characterization of Silica Matrix...... 115
3.3.1.1 Ccg series...... 115
3.3.1.2 Ccu series...... 116
3.3.2 Resonance Raman Spectroscopy of Ccu series ...... 117
3.3.3 Resonance Raman Spectroscopy of Ccg series ...... 119
3.4 Conclusions...... 123
3.5 Acknowledgements...... 124
3.6 References...... 124
Chapter 4: A Novel Method to Study Aggregation of Amyloid β1-42 - A Key Peptide
Associated with Alzheimer’s Disease...... 146
4.1 Introduction...... 146
4.1.1 Alzheimer’s Disease and Amyloid β ...... 146
4.1.2 The Aggregation of Aβ peptides...... 148
4.1.3 Detection of Aβ aggregation...... 149
4.1.3.1 The Thioflavine T Fluorescence Assay ...... 149
4.1.3.2 The Congo Red Birefringence Assay ...... 150
4.1.3.3 Negative staining of amyloid fibrils for TEM ...... 150
4.1.4 Bioencapsulation of Aβ peptide in silica matrix with controlled pore size
...... 151
4.2 Experimental...... 152
4.2.1 Materials ...... 152
4.2.2 Redissolving of Aβ peptide in their monomeric state ...... 153
4.2.3 Bioencapsulation of Aβ1-42 in silica matrix with controlled pore size... 154
9 4.2.4 pH changing of suspension by adding NaOH...... 155
4.2.5 Fluorescence Spectroscopy Study...... 156
4.2.5.1 Steady State Fluorescence Spectroscopy...... 156
4.2.5.2 Time Scale Fluorescence Spectroscopy...... 157
4.2.6 Congo Red Birefringence Assay...... 157
4.2.6.1 Preparation of the Staining Solution...... 157
4.2.6.2 Incubation of Silica Biogel Samples in Buffer Solution...... 157
4.2.6.3 Mounting Silica Biogel Samples on Glass Slides...... 158
4.2.7 Negative staining of amyloid fibrils for TEM ...... 158
4.3 Result and Discussion...... 159
4.3.1 Characterization of Silica Matrix...... 159
4.3.2 Steady State Fluorescence Spectroscopy...... 159
4.3.2.1 Abata42 Series Samples...... 160
4.3.3 Time Scale Fluorescence Spectroscopy...... 162
4.3.4 Congo Red Birefringence Assay...... 164
4.4 Conclusion ...... 165
4.5 Acknowledgements...... 166
4.6 References...... 167
Chapter 5: Fabrication of Poly (2-hydroxyethyl methacrylate)-Silica Nanoparticle Hybrid
Nanofibers via Electrospinning...... 182
5.1 Introduction...... 182
5.1.1 Organic-Inorganic Nanocomposite: Opportunities to Advanced Materials
...... 182
10 5.1.2 Electrospinning ...... 184
5.2 Experimental...... 186
5.2.1 Materials ...... 186
5.2.2 Synthesis of the Hybrid Material ...... 187
5.2.3 Set-up of Electrospinning Apparatus...... 188
5.2.4 Electrospinning of PHEMA-Silica Hybrids...... 189
5.2.5. Instrumentation and Characterization...... 189
5.2.5.1 FTIR Spectroscopy ...... 189
5.2.5.2 Thermal Gravimetric Analysis (TGA)...... 190
5.2.5.3 SEM and TEM ...... 190
5.3 Results and Discussion ...... 190
5.4 Conclusion ...... 195
5.5 Acknowledgements...... 196
5.6 References...... 196
Chapter 6: Synthesis and Characterization of Dental Composite Containing Nanoporous
Silica as Fillers...... 211
6.1 Introduction...... 211
6.1.1 Resin Matrix...... 211
6.1.2 Filler System ...... 212
6.1.3 Coupling Agent...... 213
6.1.4 Limitations of Coupling Agent ...... 213
6.1.5 Porous Filler without Coupling Agent...... 214
6.1.6 Non-surfactant Templated Sol-Gel Process...... 215
11 6.2 Experimental...... 216
6.2.1 Materials ...... 216
6.2.2 Preparation of Nanoporous Silica Filler ...... 216
6.2.3 Characterization of Nanoporous Silica Filler ...... 217
6.2.4 Preparation of Dental Resin...... 218
6.2.5 Silanization of Non-Porous Silica Particles...... 218
6.2.6 Preparation of Dental Composite...... 219
6.2.7 Evaluation of Mechanical Properties...... 220
6.2.8 Aging Test...... 220
6.3 Results and Discussion ...... 220
6.3.1 BET Analysis...... 221
6.3.2 Compression Testing ...... 221
6.3.2.1 Comparison of Non-Porous, Nanoporous and Neat Resin Materials
...... 221
6.3.2.2 Comparison of Heat Treatment Temperature Effects on
Nanoporous Filler ...... 222
6.3.2.3 Aging Test...... 223
6.4 Conclusion and Future Works ...... 224
6.5 Acknowledgements...... 226
6.6 References...... 226
Chapter 7: Dense Packing of Vinyl Modified Silica Nanoparticles and Its Potential
Application as Low Shrinkage Dental Materials...... 235
7.1 Introduction...... 235
12 7.2 Experimental...... 238
7.2.1 Materials ...... 238
7.2.2 Separation of Nanoparticle from OG100-31...... 239
7.2.2.1 Reduced Pressure Distillation...... 239
7.2.2.2 Ultracentrifuging...... 239
7.2.3 Atomic Force Microscopic (AFM) Measurements...... 241
7.2.4 Thermal Gravimetric Analysis (TGA)...... 241
7.2.5 FTIR Spectroscopy ...... 241
7.2.5.1 Solid Sample ...... 241
7.2.5.2 Liquid Sample...... 241
7.2.6 Isolation of Silica Nanoparticles...... 242
7.2.7 Preparation of Dental Resin...... 242
7.2.8 Preparation of Dental Composite...... 242
7.2.9 Evaluation of Mechanical Properties...... 244
7.3 Results and Discussion ...... 245
7.4 Conclusion and Future Work...... 248
7.5 Acknowledgements...... 249
7.6 References...... 250
Chapter 8: Summary and Conclusions...... 259
8.1 Nanoporous Materials and Their Application in Bioscience...... 259
8.2 Organic-Inorganic Hybrid Nanocomposites...... 262
Appendix A: Supplemental Data of Chapter 2 ...... 266
Appendix B: Supplemental Data of Chapter 4...... 268
13 Appendix C: Supplemental Data of Chapter 5...... 273
Appendix D: Fabrication of a New Type Molecularly Imprinted Polymer Membrane
Sensor for Atrazine ...... 279
14 List of Tables
Table 2- 1. Pore parameters of water-extracted Ccu series prepared at various urea
concentrations ...... 89
Table 3- 1 Pore parameters of water-extracted Ccu series prepared at various urea
concentrations ...... 129
Table 3- 2 Pore parameters of water-extracted Ccg series prepared at various urea
concentrations...... 130
Table 4- 1 Summary of porous parameters of Abeta series samples after removel of
templates...... 172
Table 4- 2 Relative fluorescence intensity of immobilized Amyloid β 1-42 and free
at difference pH...... 173
Table 6- 1 BET analysis of mesoporous fillers at different fructose concentration.
...... 230
Table 6- 2 The pore parameters of the nanoporous silica fillers after template
removal of fructose by water extraction and heat treatments at different
temperatures...... 231
Table 6- 3 Comparison of compressive properties of composites with different types
of fillers. The number of specimens tested is given in parenthesis...... 232
15 Table 6- 4 The effect of aging (in water at 37o C) on the compressive properties of
composites prepared using mesoporous and SiO2 fillers. The number of
specimens tested is given in parenthesis...... 233
Table A-1 Data of relative difference, (IU-IT)/IT, in fluorescence intensity between
unfolded and refolded Cc for the samples with increasing pore size up to free
Cc in solution...... 266
Table B- 1 Data of time scale fluorescence study of Abeta42 series samples when
the pH value jumping from 2.35 to 7.02...... 272
16 List of Figures Figure 1- 1 Sol-gel process and their products. (www.chemat.com/ html/solgel.html)
...... 58
Figure 1- 2 Diagrams of sol-gel reactions: (a) Hydrolysis reaction; (b) Condensation
reaction...... 59
Figure 1- 3 Possible mechanisms for formation of MCM-41: (1) liquid crystal phase
initiated and (2) silicate anion initiated. 20,21...... 60
Figure 1- 4 IUPAC classification of physisorption isotherms.16 ...... 61
Figure 1- 5 IUPAC classification of hysteresis loops.16 ...... 62
Figure 2- 1 Schematic diagram of a folding energy landscape. Denatured molecules
at the top of the funnel might fold to the native state by a myriad of different
routes, some of which involve transient intermediates (local energy minima)
whereas others involve significant kinetic traps (misfolded states). For proteins
that fold without populating intermediates, the surface of the funnel would be
smooth.23 ...... 90
Figure 2- 2 Illustrations of artificial chaperones assisting protein folding...... 91
Figure 2- 3 N2 adsorption-desorption isotherm at –196°C...... 92
Figure 2- 4 BJH pore size distributions for the sol-gel material synthesized in the
presence of 0-50% wt% of urea...... 93
Figure 2- 5 Relationship between BJH average pore diameter, pore volume and
amount of urea template used in the synthesis. For Ccu0 (wt%=0), pore
diameter was taken as 1.7 nm...... 94
Figure 2- 6 Representative IR spectra of (a) Ccu50 before template extraction; (b)
Ccu0 before template extraction; (c) Cc50 after template extraction...... 95
17 Figure 2- 7 Plot of relative difference, (IU-IT)/IT, in fluorescence intensity between
unfolded and refolded Cc for the samples with increasing pore size up to free
Cc in solution...... 96
Figure 2- 8 Circular dichroism in far UV range at 25 oC of (A) unfolded cytochrome
c (dashed line) in 9M urea and native cytochrome c (solid line) and (B)
cytochrome c entrapped in Ccu0 (dashed line) and cytochrome c entrapped in
Ccu50 (solid line) after washing out urea. All the curves are normalized by
concentrations of Cc in solution or suspension but not convert to mean residue
ellipticity because of strong light scattering in suspension...... 97
Figure 2- 9 A cartoon presentation of cytochrome c refolding upon removal of urea
template/denaturant by water extraction. (a) When pore size is large, Cc refolds
to its native state. (b) When pore size is small, Cc cannot refold to its native
state. The small dots represent urea molecules...... 98
Figure 3- 1 Diagram of a typical heme group...... 131
Figure 3- 2 Deconvoluted resonance Raman spectra of the various heme coordinated
forms of Cc.25...... 132
Figure 3- 3 N2 adsorption-desorption isotherm of Ccg at –196°C...... 133
Figure 3- 4 BJH pore size distributions for Ccg samples synthesized in the presence
of 0-60 wt% of glucose...... 134
Figure 3- 5 N2 adsorption-desorption isotherm of Ccu at –196°C...... 135
Figure 3- 6 BJH pore size distributions for the Ccu samples synthesized in the
presence of 0-50% wt% of urea...... 136
18 Figure 3- 7 Low frequency region of Resonance Raman Spectra of Ccu series and
native cytochrome c samples at pH 7.4...... 137
Figure 3- 8 Relationship between relative intensity (I567/I418) and BJH average pore
diameter. For Ccu0 (wt%=0), pore diameter was taken as 1.7 nm...... 138
Figure 3- 9 Relationship between relative intensity (I397/I418) and BJH average pore
diameter. For Ccu0 (wt%=0), pore diameter was taken as 1.7 nm...... 139
Figure 3- 10 Resonance Raman spectra of Ccu50 and Ccu0 at pH 7.0 and pH 3.5.
...... 140
Figure 3- 11 Resonance Raman spectra of Ccu50 and Ccu0 at different
temperatures...... 141
Figure 3- 12 Resonance Raman spectra of Ccg series samples when first immersed
in water. (a: in high frequency region; b: in low frequency region)...... 142
Figure 3- 13 Resonance Raman spectra of Ccg series samples when immersed in 9M
urea solution. (a: in high frequency region; b: in low frequency region) ...... 143
Figure 3- 14 Resonance Raman spectra of Ccg series samples when washed out urea
and reimmersed in water for 24 hours. (a: in high frequency region; b: in low
frequency region)...... 144
Figure 3- 15 Ratio of intensity of peak 1503 cm-1 over peak 1494 cm-1 for Ccg series
samples in different conditions...... 145
Figure 4- 1 Model of alternate aggregation pathways.33...... 174
Figure 4- 2 Molecular structure of Thioflavin T...... 175
19 Figure 4- 3 Relationship between average pore diameter and amount of DMA
template used in the synthesis...... 176
Figure 4- 4 (a) Relative fluorescence intensity of immobilized Amyloid β 1-42 at
different pH...... 177
Figure 4- 5 Time scale fluorescence study of Abeta42 series samples when the pH
value jumping from 2.35 to 7.02...... 178
Figure 4- 6 The aggregation of Aβ1-42 peptides into amyloid fibrils typically begins
with a “lag phase” in which no aggregation is observed. During this time, the
entropically unfavorable process of initial association occurs. Once the
aggregation process begins and a critical nucleus is formed, the aggregation
proceeds rapidly into amyloid fibrils (solid line). The lag phase, however, can
be overcome (dotted line) by the addition of a pre-formed nucleus (i.e., an
aliquot of solution containing pre-formed fibrils). This schematic represents the
“nucleation–polymerization” kinetics for amyloid fibril formation.39 ...... 179
Figure 4- 7 Microscope images of control silica samples without Aβ1-42 inside: (a)
under ordinary light (b) under polarized light...... 180
Figure 4- 8 Microscope images of Abeta42-50 samples with encapsulated Aβ1-42
inside after incubation in pH 7.1 buffer for 2 hours: (a) under ordinary light (b)
under polarized light...... 181
Figure 5- 1 Schematic diagram to show polymer nanofibers by electrospinning... 202
Figure 5- 2 The picture of the set-up of electrospinning apparatus in our group. .. 203
20 Figure 5- 3 FTIR spectra of pure PHEMA electrospun fiber and hybrid electrospun
fiber...... 204
Figure 5- 4 TGA spectra of pure PHEMA electrospun fiber and hybrid electrospun
fiber...... 205
Figure 5- 5 SEM picture of hybrid electrospun fiber when the molecular weight of
hybrid material was not high enough...... 206
Figure 5- 6 SEM (a) and TEM (b) pictures of hybrid electrospun fiber...... 207
Figure 5- 7 EDX analysis of hybrid electrospun fiber. (The white cross in the SEM
picture indicates the detection spot)...... 208
Figure 5- 8 SEM pictures of electrospun hybrid fiber under different composition of
solvents...... 209
Figure 5- 9 SEM pictures of electrospun hybrid fiber in solutions with different
concentrations...... 210
Figure 6- 1 The effect of filler heat treatment on the compressive modulus of post
cured composites...... 234
Figure 7- 1 FTIR spectra of (a) HEMA monomer; (b) dry silica nanoparticles
modified with vinyl groups on surface; ...... 252
Figure 7- 2 FTIR spectra of silica nanoparticle sediments after ultracentrifuge (a)
with EtOH as solvent; (b) with acetone as solvent...... 253
Figure 7- 3 FTIR spectrum of the new dental composite with vinyl modified silica
nanoparticles as inorganic fillers...... 254
21 Figure 7- 4 TGA spectra of silica nanoparticle sediments after ultracentrifuge (a)
with EtOH as solvent; (b) with acetone as solvent...... 255
Figure 7- 5 TGA spectrum of the new dental composite with vinyl modified silica
nanoparticles as inorganic fillers...... 256
Figure 7- 6 Picture of the new dental composite with vinyl modified silica
nanoparticles as inorganic fillers. For this particular sample, the loading
percentage is 51 wt%...... 257
Figure 7- 7 AFM picture of nanoparticle sediments after ultracentrifuge...... 258
Figure A- 1 Representative plot of free Cc in its refolded and unfolded state...... 267
Figure B- 1 Representative fluorescence spectra of free Aβ 1-42 in buffer with
different pH value...... 268
Figure B- 2 Representative fluorescence spectra of Abeta42-0 in buffer with
different pH value...... 269
Figure B- 3 Representative fluorescence spectra of Abeta42-30 in buffer with
different pH value...... 270
Figure B- 4 Representative fluorescence spectra of Abeta42-50 in buffer with
different pH value...... 271
Figure C- 1 SEM pictures of electrospun hybrid fiber when the ratio of DMF to
EtOH in solvent mixture was 20:80...... 273
22 Figure C- 2 SEM pictures of electrospun hybrid fiber when the ratio of DMF to
EtOH in solvent mixture was 30:70...... 274
Figure C- 3 SEM pictures of electrospun hybrid fiber when the ratio of DMF to
EtOH in solvent mixture was 40:60...... 275
Figure C- 4 SEM pictures of electrospun hybrid fiber when the ratio of DMF to
EtOH in solvent mixture was 60:40...... 276
Figure C- 5 SEM pictures of electrospun hybrid fiber when the ratio of DMF to
EtOH in solvent mixture was 70:30...... 277
Figure C- 6 SEM pictures of electrospun hybrid fiber when the ratio of DMF to
EtOH in solvent mixture was 80:20...... 278
23 Abstract Sol-Gel Nanoporous Materials, Hybrid Nanocomposites and Their Applications in Bioscience Zhengfei Sun Advisor: Yen Wei
Transparent, nanoporous silica materials have been prepared successfully via the
acid-catalyzed hydrolysis and condensation of tetramethyl orthosilicate using the
nonsurfactant templated sol-gel process. The synthetic conditions have been
systematically studied and optimized. The effects of template and synthetic process,
especially template removal steps on pore structure, have been investigated. The
composition and pore structures were thoroughly characterized with various
spectroscopic and microscopic methods such as IR, TGA, SEM, TEM and BET. The
obtained nanoporous materials usually exhibit high surface area, large pore volume and
narrowly distributed pore diameter. The porosity can be fine tuned to a certain extent simply by adjusting the template concentration. The convenient synthesis, as well as the distinctive structure and physical-chemical properties, render these sol-gel materials suitable for a wide range of potential applications, such as chemical and biological sensors, catalysts, drug delivery and functional coatings.
Because of excellent biocompatibility of this novel sol-gel technology, the method was used to study behaviors of encapsulated biospecies in silica matrix within a confined space. We call such nanoporous materials “rigid matrix artificial chaperone” because they mediate protein folding process in many aspects like a chaperone. In the studies included in this thesis, cytochrome c, hemoglobin, myoglobin as well as amyloid
β peptide were encapsulated in sol-gel nanoporous silica matrix with controlled pore size.
24 Then their folding-unfolding and aggregation behaviors were investigated by a variety of analytical methods, such as fluorescence spectroscopy, circular dichroism and resonance
Raman spectroscopy. It was found that the size of pore had great effects on the folding and aggregation process of those encapsulated biospecies.
In the second part of this thesis, the synthesis, processing and characterization of hybrid nanocomposites and their applications in dental materials are described. Two approaches have been developed to achieve homogenous hybrid nanocomposites: the first approach involves the use of vinyl modified silica nanoparticle as inorganic component and the other one employs nanoporous silica particle as inorganic filler. The materials synthesized by both of these two approaches demonstrated promising potential in dental applications.
25
26 Chapter 1: An Overview to Nanoporous Sol-Gel Materials
1.1 Introduction
In the past few years, nanomaterials have attracted tremendous international
interest, investment and effort both in scientific research and in industrial development
because of their potential applications in various fields. 1-4 Nanoporous materials are one of subset of nanomaterials. With their unique porous structure in nanometer dimensions, they can be used in various applications such as ion exchange, separation, catalysis, sensor, biological molecular recognition and purification. 5-15
According to the International Union of Pure and Applied Chemistry (IUPAC) 16 porous materials can be classified into three categories: 1) micropores are smaller than 2 nm in diameter; 2) mesopores are between 2 to 50 nm; 3) macropores are larger than 50 nm. But this definition is somewhat conflict with the more broadened definition of nanoporous materials. The term of “nanoporous” currently refers to the class of porous materials having pore diameters between 1 and 100 nm. It is noted that nanoporous materials actually encompass some micro and macro porous materials and all mesoporous materials. 1
Nanoporous materials can bring us many interesting unique properties. The high surface area to volume ratio, large surface area and porosity, versatile surface composition and properties enable the nanoporous materials to be used widely in applications, such as catalysis, chromatography, separation, sensing and so on.
Furthermore, nanoporous materials, especially inorganic nanoporous materials, which are made of mostly metal oxides, are usually non-toxic, inert, chemically and thermally
27 stable, so they have wide applications where biocompatibility or thermal stability
requirements are essential.
Sol-gel technology has been used extensively in the synthesis of nanoporous
materials as illustrated in Figure 1-1.17-19 The overall sol-gel process, as the name implies, usually involves two stages: precursors initially form high molecular weight but still soluble oligmeric intermediates, a sol, and the intermediates further link together to form a three-dimensional crosslinked network, a gel. The precursors for a sol-gel reaction could be either inorganic salts or organic compounds, such as metal alkoxides.
The synthesis of M41-S family brought a great breakthrough in the research of nanoporous materials.20-23 The first example in this family is MCM41, which was
developed in the early 1990s by researchers at Mobil Corporation.20 In this approach,
surfactant molecules were used as template to direct mesophase formation during sol-gel
process, thus high surface areas (> 1000m2 g-1), tunable, uniform and long-range ordered
porous structure (2-10 nm) can be achieved after removal of the templates. Afterwards,
this method has been extended to the synthesis of a wide range of porous silica materials,
as well as many other metal oxides species, like alumina, titania, zirconia etc.24-27
A novel non-surfactant pathway to synthesize nanoporous materials via sol-gel
process has been developed in our group.28-32 In this method, instead of using surfactant molecules as a template, we chose non-surfactant molecules, such as glucose, fructose and urea, etc, to direct the mesophase formation during the sol-gel process. This method turned out to be an effective way to synthesize nanoporous materials with high surface area ( e.g. 1000 m2/g) and pore volume ( e.g. 0.5-1.0 cm3/g), as well as pore size in the
range of 2-12 nm with narrow size distributions. Most importantly, this process has been
28 proved to be convenient, mild and biofriendly method. In our group, this approach has
been exploited to immobilize enzymes and other biological substances for biosensing and
biocatalysis applications.33-36 A main part of my research work has been focused on
further developing this novel non-surfactant template sol-gel technology and their
applications in bioscience, especially in study of protein folding unfolding and peptide
aggregation.37-40 Besides that, the synthesis of novel nanostructured organic-inorganic
hybrid materials based on the sol-gel process have also been done.41,42 For the convenience of further discussion, some background knowledge, like sol-gel process, surfactant and non-surfactant pathways to nanoprous materials and fundamental of enzyme immobilization will be introduced in the following sections. Due to the importance of establishment of structure-properties relationship in porous materials, major characterization methods will also be discussed with emphasis on gas adsorption measurements.
1.2 Fundamentals of Sol-Gel Process
In general, sol-gel process involves a transition of a system from a liquid "sol"
(mostly colloidal) into a solid "gel" phase. The starting materials used in the preparation of the "sol" are usually inorganic metal salts or metal organic compounds such as metal alkoxides. The first metal alkoxide was prepared from SiCl4 and alcohol by Ebelmen in
1844, who found that the compound gelled on exposure to ambient environment. In a typical sol-gel process, the precursor is subjected to a series of hydrolysis and polymerization reactions to form a colloidal suspension, or a "sol". When the "sol" is cast into a mold, a wet "gel" will form. With further drying and heat-treatment, the "gel" is
29 converted into dense ceramic or glass particles.43,44 The whole process is illustrated in
Figure 1-2.
1.2.1 Sol-Gel Reactions
The precursors for a sol-gel reaction could be either inorganic salts or organic
compounds, known as metal alkoxides. Compared with inorganic salts, reactions of
alkoxide precursors have easily controlled reaction kinetics and generate byproducts of
alcohols and water, which can be readily removed during the drying process.
Tetraethoxysilane (TEOS), the most widely used alkoxide precursor, is used as an
example below to illustrate the sol-gel process. In the first step, TEOS is hydrolyzed by
mixing with water:
OH OC2H5
HO OH + 4 C H OH C2H5O Si OC2H5 + 4 H2O Si 2 5
OH OC2H5
In this reaction, either an acid or a base can serve as catalyst.
The silanols, i.e., hydrolyzed TEOS undergoes further condensation reaction forming siloxane bonds, that is the second step of the sol-gel process:
Si OH + HO Si Si O Si + H2O
or
Si OR + HO Si Si O Si + ROH
30 Linkage of additional Si(OH)4 tetrahedra occurs as a polycondensation reaction
and eventually results in a three dimensional SiO2 network. The water and alcohol
byproduct molecules generated from the reactions remain in the pores of the silica’s three
dimensional networks. They can also, as most often done, be removed during the reaction.
The hydrolysis and polycondensation reactions happen at numerous sites within
the TEOS and water mixture upon mixing. When sufficient interconnected Si-O-Si bonds
are formed in region they respond cooperatively as colloidal particles, e. g. a sol. The size
of the sol particles and the crosslinking within the particles depend upon the pH and
[H2O]/Si(OR)4 ratio in the solution.
Since sol is a relatively low-viscosity liquid, it can be cast into any shape
according to its applications. With time, the colloidal particles and condensed silica
species link together to become a three-dimensional network. The physical characteristics
of the gel network depend greatly on the size of particles and extent of crosslinking prior
to gelation. At the gelation step, the viscosity increases sharply and the system losses its fluidity to form a solid-like object resulting in the general shape of the mold.
The ultrastructure and texture of a gel are established at the time of gelation.
Subsequent processing such as aging, drying can also contribute to the forming of a gel ultrastructure.
1.3 Nanoporous Sol-Gel Materials from Surfactant Templated Pathways
MCM-41 was the first example of nanoporous sol-gel material synthesized through surfactant templated pathway in the early 1990’s.20,21 Since then, tremendous attention and effort have been made in this research field. This surfactant-templated
31 method proved to be an extremely useful strategy to synthesize nanoporous materials with the following characteristics:
⎯ High surface areas (> 1000 m2g-1);
⎯ Tunable, uniform pore sizes (2-10 nm);
⎯ Long-range ordered pore structures;
⎯ Structural stability and so on.
Due to those interesting properties, nanoporous materials synthesized by surfactant templated pathways have been studied for various applications, such as heterogeneous catalysis, separation, optics, electronics and sensing.
The synthesis process always involves two conceptually simple steps: first forming the surfactant/inorganic mesophase and second removing the surfactant template molecules from the mesostructure after metal oxide framework formation. But the actual kinetics are much more complicated. A variety of parameters can affect the mesophase formation, such as solvent, temperature, pH value, aging time, initial precursor/water/catalyst ratio, etc.
Surfactants are bifunctional molecules that contain both hydrophilic and hydrophobic groups.45,46 As a result of their amphiphilic nature, surfactants can associate into supramolecular arrays. For example, cetyltrimethylammonium bromide
+ - (CH3(CH2)15N(CH3)3 Br ) in water will self-assemble into spherical micelle forms incorporating about 90 molecules. In the micelle, hydrophilic ends point toward outside and form an outer surface, while hydrophobic ends point toward center. The concentration of surfactant molecules in solution is the key factor to determine the extent of micellization, the shape of the micelles, and the aggregation of micelles into liquid
32 crystals. When the concentration of surfactant is very low, these molecules distribute
individually in the solution or absorb at the interfaces. With the concentration going up
beyond the critical micelle concentration (CMC1), the separated surfactant molecules
form small, spherical aggregates (micelles). At higher concentrations (CMC2), those
micelles can coalesce to form elongated cylindrical micelles. If the concentrations
increase, liquid-crystalline (LC) phases will form. Initially, rod like micelles aggregate to
form hexagonal close-packed LC arrays. As the concentration increases, cubic
bicontinuous LC phases form, followed by LC lamellar phases.23
Though several mechanisms have been proposed to explain the formation of
mesophase during surfactant-templated sol-gel process with slightly differences, the
“liquid-crystal templating” (LCT) mechanism suggested by Beck, et al. includes many of
the proposed mechanisms.20,21 In LCT theory, two possible pathways were proposed. In the first, the liquid-crystal phase is intact before the silicate species are added; in the second, the addition of the silicate results in the ordering of the subsequent silicate- encased surfactant micelles. The difference between the two pathways rises from changes in surfactant properties, depending on the surfactant concentrations in water and the presence of other ions. This theory is illustrated in Figure 1-3.
Dependent on their end group chemistry and charge, the surfactant can be classified into three categories:
⎯ Anionic surfactant: the hydrophilic end group carries a negative charge, e.g.
47 - 47 - sulfate (CnH2n+1OSO3 ( n = 12, 14, 16, 18), sulfonates (C16H33SO3 and
47,48 C12H25C6H4SO3-Na), phosphates (C12H25OPO3H2, C14H29OPO3K), and carboxylic
acids (C17H35COOH and C14H29COOH);
33 ⎯ Cationic surfactant: the hydrophilic end group carries a positive charge, e. g.
alkylammonium salts, such as (CnH2n+1(CH3)3NX, n = 6 (nonmesophase), 8, 9, 10, 12, 14,
16, 18, 20, 22; X = OH/Cl, OH, Cl, Br, HSO4, and CnH2n+1(C2H5)3N, n = 12, 14, 16, 18),
47 gemini surfactants [CmH2m+1(CH3)2N-CsH2s-N(CH3)2CmH2m+1]Br2, m= 16, s = 2-12)
47 + 47 cetylethylpiperidinium salts (C16H33N(C2H5)(C5H10) ); and bichain salts
(dialkyldimethylammonium);
⎯ Nonionic surfactant: the hydrophilic group is not charged; examples include
49 50 primary amines (CnH2n+1NH2) and poly(ethylene oxides), octaethylene glycol
51 monodecyl ether (C12EO8), and octaethylene glycol monohexadecyl ether (C16EO8).
Six templating pathways have been identified: S+ I-, S- I+, S+ X- I+, S- X+ I-, S-I, and
S0 I0,49,52,53 where S is the surfactant, I is the inorganic phase, and X is the mediating ion.
For example, in S+ I- type, a cationic surfactant is chosen and the pH is set such that the inorganic precursors will carry negative charge.
Beside silicate, the surfactant-templated pathway can be applied in synthesis the other periodic porous materials. Various inorganic oxide frameworks, some of which may have important technological applications, have been realized, including silica doped with Al,20,21,54-56 Ti,49,57 V,58 B,59 Fe,60 Mn,61 Ga,62 and transition metal and main-group materials based on tungsten oxide,63 antimony oxide,63 titanium oxide,47 zirconium
oxophosphate,64 and zirconium oxide, vanadium oxide,63 vanadium phosphate,65 and
tantalum oxide.66 In addition, a large number of lamellar mesophases have been
synthesized including those based on Si, Zn, Pb, Fe, Mg, Mn, Co, Ni, Al, and Ga oxides
and Sn, W, and Mo sulfides.47,67
34 1.4 Nanoporous Sol-Gel Materials from Nonsurfactant Templated Pathway
Even though the surfactant templated pathway has proved to be successful for
synthesis of nanoporous materials, some drawbacks have prevented their application in some areas. As an example, some cationic surfactants are toxic and expensive; the synthesis is often achieved under harsh conditions during reactions or the templated removal processes, such as high temperature and pressure, strongly acidic or basic media.
For some applications, especially in bioscience and biotechnology, those drawbacks are fatal.
To solve these problems, a novel nonsurfactant templating pathway has been developed in our group.28,29 Instead of using surfactant molecules as template to direct the nanoporous structure formation, non-surfactant small molecules are applied in this new method to function as template molecules. Those small nonsurfactant molecules include glucose, fructose, maltose, urea, dibenzoyl-L-tartaric acid (DBTA), etc..28-32,42,68-80 In general, the nonsurfactant pathway starts with the sol-gel reactions of inorganic precursors, e.g., tetraethyl orthosilicate (TEOS) for silica, in the presence of a non- surfactant compound, e.g. glucose. Upon gelation and drying, nonporous, transparent and monolithic solids can be obtained. The template removal can be easily achieved by simple solvent extraction, which means template molecules can be washed out from silica matrices and leave nanoporous structure.
As described above, the nonsurfactant templating pathway has several important advantages over surfactant-templating pathway. First, the whole process can be done at room temperature. Because the interactions between non-surfactant molecules and inorganic phase are much weaker than surfactant/inorganic phase, the removal of
35 templates can be easily accomplished by solvent extraction at room temperature, avoiding
high temperature calcination step in the surfactant-templated pathway. Second, many
non-surfactant template molecules, like glucose, fructose and maltose are non-toxic and
biofriendly. That makes applications in bioscience and biotechnology feasible. Third,
during the whole non-surfactant templated process, no acid, alkaline or even organic
solvent is necessary at the point when biological substances are introduced. All these characteristics of non-surfactant templating pathway provide a convenient and effective way to prepare nanoporous materials for various applications, especially in biological applications.
The nanoporous materials synthesized via the non-surfactant templating pathway share some characteristics with those from surfactant templating pathway. They have high surface area (e.g. 1000 m2/g) and pore volume (e.g. 0.5-1.0 cm3/g) as well as pore size in the range of 2-12 nm with narrow size distributions, which are all similar to surfactant templated materials. But they do not have long ordered range of nanoporous structure, discernable packing or orientation of the nanopores/channels. In fact, the porous structures inside non-surfactant templated samples are made of interconnected channels of regular diameters. Indeed, such a worm-hole like structure can be observed in the TEM images of all nanoporous materials prepared via the non-surfactant templating pathway. Though the long range ordered structure is missing from the materials, it is interesting to point out that may be advantageous for many applications, such as catalysis or sensing, because the nanoporosity is accessible from all directions.
The mechanism of forming mesophase through the non-surfactant templating pathway is still not very clear. After investigating nearly 100 template compounds, we
36 found only those compounds with highly polar functional groups can serve as the templates. Because the size of pores are much bigger than the single non-surfactant templated molecule, those small molecules must work in some aggregation or assembly form when function as the template. We believe that strong polar interactions and hydrogen bonding between the non-surfactant molecules or their aggregations and inorganic phase may play an important role in directing the mesophase formation.
1.5 Characterizations of Nanoporous Materials
Reliable characterization methods are very important for developing nanoporous materials. Various structural parameters, such as surface area, pore size, pore volume and pore size distribution are needed to evaluated nanoporous materials. Gas adsorption, X- ray diffraction, electron microscopy are among the most important experimental methods used.
1.5.1 Gas sorption measurement
It has long been known that a porous solid can take up a relatively large volume of condensable gas. Because surface area and porosity of the porous materials play complementary roles in adsorption phenomena, measurements of adsorption of gases can be made to yield information as to surface area and the pore structure of a solid.
The term of “adsorption” was first introduced by Kayser in 1881 to connote the condensation of gases on free surfaces; while the term “absorption” refers to the phenomenon where gas molecules penetrate into the mass of the absorbing solid. Since it
37 is sometime difficult, impossible or irrelevant to distinguish between these two terms, the
wider term “sorption” which embraces both types of phenomena is uesd.81
When a highly dispersed solid is exposed in a closed space to a gas or vapor at
some definite pressure, the solid begins to adsorb the gas, resulting in a gradual reduction in the gas pressure. After some time, the gas pressure would become constant. The amount of adsorbed gas can be calculated from the decrease of pressure by application of the gas laws.
The amount of adsorbed gas per gram of solid depends on the equilibrium pressure p, the temperature T, and also on the nature of the gas and of the solid. For a given gas adsorbed on a given solid, maintained at a fixed temperature:
X = f (P)T, gas, solid
If the gas is below its critical temperature, i.e. if it is a vapor, the alternative form:
X = f (P/P0)T, gas, solid
These two equations are expressions of the adsorption isotherm, which can be
defined as the relationship at constant temperature, between the adsorbed and the
equilibrium pressure of the gas and can be used to determine the general pore structure of
a porous solid. The adsorption isotherm is usually constructed point-by-point by the
admission to the adsorbent of successive charges of gas with the aid of a volumetric
dosing technique and application of the gas laws.
Though there are recorded tens of thousands of adsorption isotherms, the majority
of those isotherms can be grouped into six classes as illustrated in Figure 1-4:16
38 Type I isotherms are given by microporous solids having relatively small external
surfaces, the limiting uptake being governed by accessible micropore volume rather than
by internal surface area.
Type II isotherms are commonly forms of isotherms obtained with a non-porous
or macroporous solid. It demonstrates unrestricted monolayer-multilayer adsorption. The
point B labeled in the graph normally means the conversion form monolayer adsorption
to multilayer adsorption.
Type III isotherms are not common. In this case, the adsorbate-adsorbate interaction plays an important role.
Type IV isotherms have a characteristic hysteresis loop, which is associated with capillary condensation taking place in mesoporous solids. The initial part of the Type IV isotherm is attributed to monolayer-multilayer adsorption since it follows the same path as the corresponding part of a Type II isotherm. Type IV isotherms are given by many mesoporous materials.
Type V isotherms are also not common ; they are related to the Type III isotherms in that the adsorbate-adsorbate interactions are weak, but is obtained with certain porous adsorbents.
Hysteresis appearing in the multilayer range of physisorption isotherms is usually associated with capillary condensation in mesopores structures.16,82,83 There are four
types of hysteresis loops as shown in Figure 1-5: H1 loop is always associated with pores with regular shape and narrow size distribution; H2 loop is especially difficult to interpret: it was originally attributed to a difference in mechanism between condensation and evaporation processes occurring in pores with narrow necks and wide bodies before, but
39 now it is believed that the role of network effects must play an important role too; H3
loop, which does not exhibit any limiting adsorption at high P/P0, is observed with aggregates of plate-like particles and H4 loop is often assigned narrow slit-like pores.
The Brunauer-Emmett-Teller (BET) gas adsorption method has become the most widely used standard procedure for the determination of the surface area of finely-divided and porous materials. The BET equation can be described in the linear form:
P 1 (C −1) ⋅ P a = a + a n ⋅ ()P0 − P nm ⋅C nm ⋅C ⋅ P0
a a where n is the amount adsorbed at the relative pressure P/P0 and nm is the monolayer capacity.
1.5.2 X-Ray Diffraction (XRD)
X-ray diffraction is one of the cornerstones of twentieth century science.84 It has been widely used to characterize sol-gel nanoporous materials. X-rays are relatively short wavelength, high energy electromagnetic radiations. After they are generated from a source and focused into a fine beam, the X-ray can be shined on a solid sample. Though various interactions happen between X-ray and the inspected matter, such as heat conversion, photoelectric effect, fluorescence, auger electron production and Compton scattering, the most important mechanism of X-ray absorption in matter, which leads to the phenomenon of diffraction is coherent scattering. It is analogous to a perfectly elastic collision between a photon and an electron. The photon changes direction after colliding with the electron but transfers one of its energy to the electron. The result is that the scattered photon leaves in a new direction but with the same phase and energy as that of
40 the incident photon. Structural information can therefore be deduced from the knowledge
of scattering intensity and angle.85 For example, approximated as the repeating distance in the porous materials, the sum of a pore diameter and a pore wall thickness can be estimated base on the d spacing calculated from the Bragg equation.
nλ = 2d sinθ
where λ is the wavelength, d is the separation between planes and θ is the diffraction
angle. In the case of nanoporous materials with regular pore diameter and wall thickness,
the d spacing is the sum of the pore diameter and wall thickness.
1.5.3 Electron Microscopy
Electron microscopic observations can provide a straightforward evaluation on
pore size, shape and distribution in the nanoporous materials.86-89 Generally, electron
microscopes use a beam of highly energetic electrons to examine objects on a very fine
scale. This examination can yield the following information:
Topography: The surface features of an object or "how it looks", its texture;
Morphology: The shape and size of the particles making up the object;
Composition: The elements and compounds that the object is composed of and the
relative amounts of them;
Crystallographic Information: How the atoms are arranged in the object;
There are two types of electron microscopy: The transmission electron
microscope (TEM) was the first type of electron microscope to be developed and is
patterned exactly on the light transmission microscope except that a focused beam of
electrons is used instead of light to "see through" the specimen; the first scanning electron
41 microscope (SEM) debuted in 1942. Its late development was due to the electronics
involved in "scanning" the beam of electrons across the sample. For mesoporous
materials, TEM is the most useful method to provide direct observation of pore structures
and parameters.
1.6 Organic/Inorganic Hybrid Materials by Sol-Gel Approach
Hybrid materials formed by the combination of inorganic materials and organic
polymers are attractive for the purpose of creating high-performance or high-functional
polymeric materials.90-95 Both pure organic polymer and pure inorganic materials have their own advantages and disadvantages. Generally, organic polymer materials possess of the following merits, light weight, good flexibility and excellent moldability. However they usually lack of hardness and strength; on the other hand inorganic materials, such as silica glass are often in the different ways: they have good mechanical and thermal stability but sometime too brittle. So, if the homogeneous combination of inorganic and organic moieties in a single-phase material can be achieved, this material may provide unique possibilities, which combined advantages of both organic and inorganic materials and let us to tailor the mechanical, electrical, and optical properties with respect to numerous applications.
Sol-gel technology, which is mainly based on inorganic polymerization reactions, is an important way to synthesize organic/inorganic hybrid materials,79,91,94,96,97 because of its unique low temperature processing characteristic, providing opportunities to let organic and inorganic phases mix well and incorporate with each other at temperatures under which the organic phase can survive. Since the last few decades, the preparation,
42 characterization and application of those organic/inorganic hybrid materials based on sol-
gel process are a fast growing research field in materials science. By using the sol-gel
technology, the hybrid nanocomposite hybrid materials provide some new and interesting
properties which conditional macroscale composites do not have. For example, unlike
conventional composites with inorganic or organic phase domains at millimeter or
micrometer scale, the most of hybrid materials by sol-gel process are nanoscopic, with
phase domain size in nanometer scale. Therefore, they are often optically transparent.
Furthermore, via sol-gel process, various kinds of bonds between organic and inorganic
phases can be introduced in the system and consequently enhance the interactions
between these two phases.
Dependent on structural differences, organic/inorganic hybrid materials can be
grossly divided into two classes:92,93
Class I: The organic phase is physically embedded inside the inorganic matrix.
The synthesis of this class of materials is usually carried by formation of inorganic
network backbone in presence of preformed organic phase, like prepolymer. Thus, only
weak bonds can be formed between these two phases.
Class II: The organic and inorganic phases are covalent bonded. In this approach, the inorganic precursors must carry functional groups, which can react with organic phase during or after sol-gel process.
Based on sol-gel technology, there are several different synthetic techniques by incorporating various starting inorganic and organic components with varied molecular structures:
43 (1) Organic groups are introduced into hybrid network by using low
molecular weight organoalkoxysilanes as one or more of precursors for
sol-gel reaction.98-100
(2) Organic/inorganic hybrid materials can be also be formed via the co-
condensation of functionalized polymers with metal alkoxide, such as
trialkoxysilyl groups. So, covalent bonding can be established between
inorganic and organic phases.101,102
(3) In situ formation of inorganic phase domain within organic polymer
matrix can be another way to synthesize hybrid materials.103-105 Those
inorganic species with homogenous particle size in nanometer scales
can be obtained by using sol-gel reaction of the inorganic precursors.
(4) Just opposite to method (3), organic phase domain can be formed by
either infiltrating reformed oxide gels with polymerizable monomer or
mixing the polymers with metal alkoxide in a common solvent.106
(5) Simultaneous formation of inorganic and organic phases together
provides another way to synthesize hybrid materials. For example,
triethoxysilance R’Si(OR)3 where R’ is a polymerizable group like
epoxy group, can be used as a precursor in sol-gel reaction, so by
either photo- or thermal- initiation, organic network can be formed
within inorganic network.100,107
To date, only a few sol-gel hybrid materials have been commercialized, but the future of these materials is promising. A larger number of potential applications have appeared, such as scratch and abrasive-resistant coatings,108,109 electrical and nonlinear
44 optical (NLO) materials,110,111 contact lens,112 reinforcement of elastomers and plastic,113-
115 catalyst and sensor materials,116,117 porous supports, adsorbents etc.
1.7 Sol-Gel Encapsulation of Biomolecules
Though sol-gel science has been developed for many decades, bioencapsulation
via sol-gel technology was not realized until 1980s.118,119 Since then, research on this field thrives and a large number of examples of bioencapsulation by sol-gel technology have emerged out.120-132 In comparison with conventional bioimmobilization methods, in which biospecies are often covalently bonded to organic polymer matrix, the sol-gel bioencapsulation methods have the following advantages:
(1) Inorganic matrix synthesized by sol-gel process usually has much
better thermal and chemical stability than organic polymer system,
which can allow the sol-gel bioencapsulated system working in an
elevated temperature and harsh environment.
(2) The synthesis of sol-gel materials can be done at room temperature.
That makes directly encapsulation of temperature sensitive
biomolecules feasible.
(3) The surface area and porosity of sol-gel materials can easily be
controlled so the suitable pore size can be designed, so leakage of
biomolecules is reduced while penetration of small required reagent
molecules is not prohibited.
(4) The good optical transparency of most of sol-gel materials enable their
usages with optical requirements, such as optical sensors.
45 Since the first example of sol-gel bioencapsulation introduced by Avnir and co-
workers,133 several types of sol-gel matrices have been developed to be used as substrates:
Inorganic sol-gels: The pure inorganic xerogels, such as aluminum, titanium, zirconium and tin oxides as well as their mixed oxides with silica, are always hard, transparent glasses with microporous structure. They are chemically robust, but limited by their brittleness and too small pore size, which prevents small molecule diffusion through the matrix.134
Organically modified silica sol-gels (Ormosils): In this category, organic groups, from simple alkyl, alkenyl, and aryl to those additionally bearing amino, amido, carboxy, hydroxy, thiol, and mixed functionalities as well as nicotinamides, flavins and quiniones, can be grafted on precursor silanes. Thus after sol-gel reactions, those organic functional groups are attached on the silica matrix by stable Si-C bonds. Because of those groups, tailorable properties, such as hydrophilic, hydrophobic, ionic as well as H-bonding capacities can be achieved in the silica matrix. However, the optical transparency and stability are lower than inorganic sol-gels.134
Hybrid sol-gels: Amino- or hydroxyl- functional polymers, such as polymethy silane, polyurethane, polyacrylate, and polyphosphazene are mixed with alkoxysilane during the sol-gel reaction. After polymerization, hybrid organic-inorganic structure can
be formed in the silica matrix to provide good mechanical properties and variable
hydrophilic-hydrophobic balances. But they are often not optically transparent and in
some case only are available as hydrogels. 135,136
Reinforced/filled composite sol-gels: To improve the mechanical properties and
processing behavior of the sol-gel materials, some nano-or micro-particles, such as
46 graphite powder, fume silica, clays, cellulose and so on, can be incorporated inside the
sol-gel silica.120,121,137 In addition, some active metal filler like gold, palladium, platinum can be used when conducting and redox-active materials are desired.
In our group, a novel one-step direct bioencapsulation via nonsurfactant- templated sol-gel process has been developed. This bioencapsulation is achieved by direct introduction of bioactive substances to nonsurfactant-templated sol-gel reactions
prior to the system gelation with pH adjusted to near neutral and partial removal of
organic byproducts.
In this method, instead of using surfactant as template molecules, nonsurfactant
small molecules, such as glucose, fructose, maltose, etc, are chosen to function as
template. This small change in the sol-gel process makes a big difference in
bioencapsulation in the following ways:
(1) Unlike surfactant molecules, especially some ionic surfactant
molecules, which are toxic and expensive, the small nonsurfactant
molecules used as templates are biocompatible, low cost and harmless
to most biological substances.
(2) Because of the positive or negative charges carried by surfactant
molecules, it is very difficult to remove surfactant molecules after the
silica matrix is formed. Normally a high temperature up to 1000 0C is
needed to burn surfactant template molecules away from silica matrix.
In the application of bioencapsulation, this method does not work since
no biospecies can survive at that high temperature. Whereas,
nonsurfactant molecules have high solubility in water or other solvents,
47 thus by simply solvent extraction at room temperature, nonsurfactant
molecules can be removed and leave desired porous structure.
(3) The porous structure of silica matrix, such as pore size, surface area
and pore size distribution, can be controlled by adjusting the amount of
nonsurfactant template added into sol-gel reaction. As the result,
desired pore size can be designed to entrap biological substances inside
the matrix without leakage, while still permit the small molecules, such
as reacting reagents and byproducts, penetrating through the matrix.
(4) As discussed in (3), biological substances such as enzymes and
proteins can be physically entrapped inside the pores instead of
covalently bonded to matrix. Bonding enzyme to the substrate usually
needs modifications of enzyme and reaction between enzyme and
substrate, which are always tedious works and may involve protein
denaturing. In this new method, without chemical reaction between
enzyme and silica matrix, the lost of enzyme activities during
encapsulation step can be neglected.
Bioencapsulation has been applied in a variety of fields. Applications of sol–gel derived biomaterials include selective coatings for optical and electrochemical biosensors, stationary phases for affinity chromatography, immunoadsorbent and solid-phase extraction media, solid-phase biocatalysts and controlled release agents. Besides, the bioencapsulation, especially our new method by which protein can be entrapped in its native state, may also be used in basic biochemistry studies, such as protein folding unfolding and peptide aggregations.
48 1.8 References
1. Lu, G. Q. & Zhao, X. S. Nanoporous materials- an overview. Series on Chemical Engineering 4, 1-13 (2004). 2. Rao, C. N. R., Mueller, A. & Cheetham, A. K. Nanomaterials - An introduction. Chemistry of Nanomaterials 1, 1-11 (2004). 3. Kear, B. H. & Skandan, G. Overview: status and current developments in nanomaterials. International Journal of Powder Metallurgy (Princeton, New Jersey) 35, 35-37 (1999). 4. Tolles, W. M. & Rath, B. B. General overview on nanomaterials. Nanomaterials: Synthesis, Properties and Applications, 545-553 (1996). 5. Lin, Y.-W., Huang, M.-F. & Chang, H.-T. Nanomaterials and chip-based nanostructures for capillary electrophoretic separations of DNA. Electrophoresis 26, 320-330 (2005). 6. Zecho, T. 31 pp ((Future Camp GmbH, Germany). Application, 2004). 7. Martin, C. R. & Mitchell, D. T. Nanomaterials in analytical chemistry. Analytical Chemistry 70, 322A-327A (1998). 8. Boennemann, H. & Nagabhushana, K. S. Chemical synthesis of nanoparticles. Encyclopedia of Nanoscience and Nanotechnology 1, 777-813 (2004). 9. Ledoux, M. J., Vieira, R., Pham-Huu, C. & Keller, N. New catalytic phenomena on nanostructured (fibers and tubes) catalysts. Journal of Catalysis 216, 333-342 (2003). 10. Lu, G. Q. Nanomaterials in Catalysis. (Proceedings of a Workshop from the Chemeca 2000 Conference held 9-12 July 2000 in Perth, Australia.) [In: Catal. Today, 2001; 68(1-3)] (2001). 11. Wang, Z. L. Nanomaterials for nanoscience and nanotechnology. Characterization of Nanophase Materials, 1-11 (2000). 12. Shi, J., Zhu, Y., Zhang, X., Baeyens, W. R. G. & Garcia-Campana, A. M. Recent developments in nanomaterial optical sensors. TrAC, Trends in Analytical Chemistry 23, 351-360 (2004). 13. Mahadevan, V. & Sethuraman, S. Nanomaterials and nanosensors for medical applications. ICASE/LaRC Interdisciplinary Series in Science and Engineering 9, 207-228 (2003). 14. Walcarius, A. Electrochemistry of silicate-based nanomaterials. Encyclopedia of Nanoscience and Nanotechnology 2, 857-893 (2004). 15. Ong, K. G. & Grimes, C. A. Magnetostrictive nanomaterials for sensors. Encyclopedia of Nanoscience and Nanotechnology 5, 1-27 (2004). 16. Sing, K. S. W., Everett, D. H., Haul, R. A. W., Moscou, L., Pierotti, R. A., Rouquerol, J. & Siemieniewska, T. Reporting physisorption data for gas/solid systems with special reference to the determination of surface area and porosity (Recommendations 1984). Pure and Applied Chemistry 57, 603-19 (1985). 17. Brinker, C. J., Wallace, S., Raman, N. K., Sehgal, R., Samuel, J. & Contakes, S. M. Sol-gel processing of amorphous nanoporous silicas: Thin films and bulk.
49 Access in Nanoporous Materials, [Proceedings of a Symposium on Access in Nanoporous Materials], East Lansing, Mich., June 7-9, 1995, 123-139 (1995). 18. Dave, B. C., Dunn, B. & Zink, J. I. 21 pp (Dep. Materials,California Univ.,Los Angeles,CA,USA., 1995). 19. Lakshmi, B. B., Patrissi, C. J. & Martin, C. R. Sol-gel template synthesis of semiconductor oxide micro- and nanostructures. Chemistry of Materials 9, 2544- 2550 (1997). 20. Kresge, C. T., Leonowicz, M. E., Roth, W. J., Vartuli, J. C. & Beck, J. S. Ordered mesoporous molecular sieves synthesized by a liquid-crystal template mechanism. Nature (London, United Kingdom) 359, 710-12 (1992). 21. Beck, J. S., Vartuli, J. C., Roth, W. J., Leonowicz, M. E., Kresge, C. T., Schmitt, K. D., Chu, C. T. W., Olson, D. H., Sheppard, E. W. A new family of mesoporous molecular sieves prepared with liquid crystal templates. Journal of the American Chemical Society 114, 10834-43 (1992). 22. Ciesla, U. & Schuth, F. Ordered mesoporous materials. Microporous and Mesoporous Materials 27, 131-149 (1999). 23. Raman, N. K., Anderson, M. T. & Brinker, C. J. Template-based approaches to the preparation of amorphous, nanoporous silicas. Chemistry of Materials 8, 1682-1701 (1996). 24. Zhang, W. Rare earth stabilization of mesoporous alumina molecular sieves assembled through an N DegI Deg pathway. Chemical Communications (Cambridge), 1185-1186 (1998). 25. Bagshaw, S. A. & Pinnavaia, T. J. Mesoporous alumina molecular sieves. Angewandte Chemie, International Edition in English 35, 1102-1105 (1996). 26. Alberius, P. C. A., Frindell, K. L., Hayward, R. C., Kramer, E. J., Stucky, G. D. & Chmelka, B. F. General predictive syntheses of cubic, hexagonal, and lamellar silica and titania mesostructured thin films. Chemistry of Materials 14, 3284-3294 (2002). 27. Yang, P., Zhao, D., Margolese, D. I., Chmelka, B. F. & Stucky, G. D. Generalized syntheses of large-pore mesoporous metal oxides with semicrystalline frameworks. Nature (London) 396, 152-155 (1998). 28. Wei, Y., Jin, D. L., Ding, T. Z., Shih, W. H., Liu, X. H., Cheng, S. Z. D. & Fu, Q. A non-surfactant templating route to mesoporous silica materials. Advanced Materials (Weinheim, Germany) 10, 313-316 (1998). 29. Wei, Y., Xu, J., Dong, H., Dong, J. H., Qiu, K. & Jansen-Varnum, S. A. Preparation and physisorption characterization of D-glucose-templated mesoporous silica sol-gel materials. Chemistry of Materials 11, 2023-2029 (1999). 30. Pang, J.-B., Qiu, K.-Y., Xu, J., Wei, Y. & Chen, J. Synthesis of mesoporous silica materials via nonsurfactant urea-templated sol-gel reactions. Journal of Inorganic and Organometallic Polymers 10, 39-50 (2000). 31. Pang, J.-B., Qiu, K.-Y. & Wei, Y. A new nonsurfactant pathway to mesoporous silica materials based on tartaric acid in conjunction with metallic chloride. Chemistry of Materials 13, 2361-2365 (2001). 32. Zheng, J.-Y., Pang, J.-B., Qiu, K.-Y. & Wei, Y. Synthesis of mesoporous silica materials via nonsurfactant templated sol-gel route by using mixture of organic
50 compounds as template. Journal of Sol-Gel Science and Technology 24, 81-88 (2002). 33. Wei, Y., Xu, J., Feng, Q., Lin, M., Dong, H., Zhang, W.-J. & Wang, C. A novel method for enzyme immobilization: direct encapsulation of acid phosphatase in nanoporous silica host materials. Journal of Nanoscience and Nanotechnology 1, 83-93 (2001). 34. Wei, Y., Xu, J., Feng, Q., Dong, H. & Lin, M. Encapsulation of enzymes in mesoporous host materials via the nonsurfactant-templated sol-gel process. Materials Letters 44, 6-11 (2000). 35. Ping, G., Yuan, J.-M., Sun, Z. & Wei, Y. Studies of effects of macromolecular crowding and confinement on protein folding and protein stability. Journal of molecular recognition : JMR 17, 433-40 (2004). 36. Wei, Y., Xu, J., Feng, Q., Lin, M., Dong, H., Zhang, W. J. & Wang, C. A novel method for enzyme immobilization: direct encapsulation of acid phosphatase in nanoporous silica host materials. Journal of nanoscience and nanotechnology 1, 83-93 (2001). 37. Ping, G., Yuan, J.-M., Sun, Z. & Wei, Y. Studies of effects of macromolecular crowding and confinement on protein folding and protein stability. Journal of Molecular Recognition 17, 433-440 (2004). 38. Sun, Z., Balakrishnan, G., Nielsen, S. B., Yuan, J.-M., Spiro, T. G., Wei, Y. & Venkatesh, S. A study of cytochrome c folding and unfolding in rigid silica matrices with controlled pore size by resonance Raman spectroscopy. Abstracts of Papers, 228th ACS National Meeting, Philadelphia, PA, United States, August 22-26, 2004, BIOL-069 (2004). 39. Ping, G., Yuan, J. M., Vallieres, M., Dong, H., Sun, Z., Wei, Y., Li, F. Y. & Lin, S. H. Effects of confinement on protein folding and protein stability. Journal of Chemical Physics 118, 8042-8048 (2003). 40. Wei, Y., Sun, Z., Zheng, J., Dong, H., Yuan, J. & Ping, G. Rigid matrix artificial chaperone (RMAC)-mediated refolding of heme protein. PMSE Preprints 87, 252-253 (2002). 41. Sun, Z., Li, S., Patel, A. C., Wang, C. & Wei, Y. Fabrication of poly(2- hydroxyethyl methacrylate)-silica nanoparticle hybrid nanofibers via electrospinning. Abstracts of Papers, 228th ACS National Meeting, Philadelphia, PA, United States, August 22-26, 2004, INOR-582 (2004). 42. Patel, A. C., Sun, Z. & Wei, Y. Sol-gel synthesis and characterization of silver nanoparticles in D-glucose-templated mesoporous silica materials. Abstracts, 36th Middle Atlantic Regional Meeting of the American Chemical Society, Princeton, NJ, United States, June 8-11, 270 (2003). 43. Brinker, C. Sol-gel science Academic Press, New York, N. Y. (1990). 44. Hench, L. L. Sol-Gel Silica: Properties, Processing and Technology Transfer Noyes, Westwood, N. J.(1998). 45. Rosen, M. J. Surfactants and Interfacial Phenomena (1995). 46. Myers, D. Surfactant Science and Technology (1988). 47. Huo, Q., Margolese, D. I., Ciesla, U., Demuth, D. G., Feng, P., Gier, T. E., Sieger, P., Firouzi, A., Chmelka, B. F. Organization of organic molecules with inorganic
51 molecular species into nanocomposite biphase arrays. Chemistry of Materials 6, 1176-91 (1994). 48. Antonelli, D. M. & Ying, J. Y. Synthesis of hexagonally packed mesoporous TiO2 by a modified sol-gel method. Angewandte Chemie, International Edition in English 34, 2014-17 (1995). 49. Tanev, P. T. & Pinnavaia, T. J. A neutral templating route to mesoporous molecular sieves. Science (Washington, D. C.) 267, 865-7 (1995). 50. Bagshaw, S. A., Prouzet, E. & Pinnavaia, T. J. Templating of mesoporous molecular sieves by nonionic polyethylene oxide surfactants. Science (Washington, D. C.) 269, 1242-4 (1995). 51. Attard, G. S., Glyde, J. C. & Goltner, C. G. Liquid-crystalline phases as templates for the synthesis of mesoporous silica. Nature (London) 378, 366-8 (1995). 52. Behrens, P. Voids in variable chemical surroundings: mesoporous metal oxides. Angewandte Chemie, International Edition in English 35, 515-18 (1996). 53. Huo, Q., Margolese, D. I., Ciesla, U., Feng, P., Gier, T. E., Sieger, P., Leon, R., Petroff, P. M., Schueth, F. & Stucky, G. D. Generalized synthesis of periodic surfactant/inorganic composite materials. Nature (London, United Kingdom) 368, 317-21 (1994). 54. Luan, Z., Cheng, C.-F., Zhou, W. & Klinowski, J. Mesopore Molecular Sieve MCM-41 Containing Framework Aluminum. Journal of Physical Chemistry 99, 1018-24 (1995). 55. Borade, R. B. & Clearfield, A. Synthesis of aluminum rich MCM-41. Catalysis Letters 31, 267-72 (1995). 56. Luan, Z., He, H., Zhou, w., Cheng, C.-F. & Klinowski, J. Effect of structural aluminum on the mesoporous structure of MCM-41. Journal of the Chemical Society, Faraday Transactions 91, 2955-9 (1995). 57. Corma, A., Navarro, M. T. & Perez Pariente, J. Synthesis of an ultralarge pore titanium silicate isomorphous to MCM-41 and its application as a catalyst for selective oxidation of hydrocarbons. Journal of the Chemical Society, Chemical Communications, 147-8 (1994). 58. Reddy, K. M., Moudrakovski, I. & Sayari, A. Synthesis of mesoporous vanadium silicate molecular sieves. Journal of the Chemical Society, Chemical Communications, 1059-60 (1994). 59. Sayari, A., Danumah, C. & Moudrakovski, I. L. Boron-Modified MCM-41 Mesoporous Molecular Sieves. Chemistry of Materials 7, 813-15 (1995). 60. Yuan, Z. Y., Liu, S. Q., Chen, T. H., Wang, J. Z. & Li, H. X. Synthesis of iron- containing MCM-41. Journal of the Chemical Society, Chemical Communications, 973-4 (1995). 61. Zhao, D. & Goldfarb, D. Synthesis of mesoporous manganosilicates: Mn-MCM- 41, Mn-MCM-48 and Mn-MCM-L. Journal of the Chemical Society, Chemical Communications, 875-6 (1995). 62. Cheng, C.-F., He, H., Zhou, W., Klinowski, J., Goncalves, J. A. S. & Gladden, L. F. Synthesis and characterization of the gallosilicate mesoporous molecular sieve MCM-41. Journal of Physical Chemistry 100, 390-6 (1996).
52 63. Luca, V., MacLachlan, D. J., Hook, J. M. & Withers, R. Synthesis and characterization of mesostructured vanadium oxide. Chemistry of Materials 7, 2220-3 (1995). 64. Ciesla, U., Schacht, S., Stucky, G. D., Unger, K. K. & Schueth, F. Formation of a porous zirconium oxo phosphate with a high surface area by a surfactant-assisted synthesis. Angewandte Chemie, International Edition in English 35, 541-3 (1996). 65. Abe, T., Taguchi, A. & Iwamoto, M. Non-Silica-Based Mesostructured Materials. 1. Synthesis of Vanadium Oxide-Based Materials. Chemistry of Materials 7, 1429-30 (1995). 66. Antonelli, D. M. & Ying, J. Y. Synthesis and Characterization of Hexagonally Packed Mesoporous Tantalum Oxide Molecular Sieves. Chemistry of Materials 8, 874-81 (1996). 67. Anderson, M. T. & Newcomer, P. Molecular approach to mesoporous metal sulfides. Materials Research Society Symposium Proceedings 371, 117-21 (1995). 68. Wei, Y. & Qiu, K.-Y. A novel nonsurfactant route to nanoporous materials and its biological applications. Series on Chemical Engineering 4, 873-892 (2004). 69. Wei, Y., Dong, H., Xu, J., Wang, C., Feng, Q., Qiu, K.-Y., Li, Z.-C. & Jansen, S. A. Nonsurfactant route to nanoporous phenyl-modified hybrid silica materials. Series on Chemical Engineering 4, 188-205 (2004). 70. Pang, J.-B., Dong, C.-M., Qiu, K.-Y. & Wei, Y. Tartaric acid templated synthesis of mesoporous Ti-incorporated silica and its catalytic activity for the ring-opening polymerization of e-caprolactone. Chinese Journal of Polymer Science 20, 361- 368 (2002). 71. Pang, J.-B., Qiu, K.-Y. & Wei, Y. Recent progress in research on mesoporous materials I: Synthesis. Wuji Cailiao Xuebao 17, 407-414 (2002). 72. Zheng, J.-Y., Pang, J.-B., Qiu, K.-Y. & Wei, Y. Synthesis of mesoporous titanium dioxide materials by using a mixture of organic compounds as a non-surfactant template. Journal of Materials Chemistry 11, 3367-3372 (2001). 73. Zheng, J.-Y., Pang, J.-B., Qiu, K.-Y. & Wei, Y. Synthesis and characterization of mesoporous titania and silica-titania materials by urea templated sol-gel reactions. Microporous and Mesoporous Materials 49, 189-195 (2001). 74. Wu, Q., Pang, J., Qiu, K. & Wei, Y. Synthesis of methacrylyloxypropyl-modified inorganic/organic hydrib mesoporous materials via a nonsurfactant-templated sol- gel process. Gaofenzi Xuebao, 538-540 (2001). 75. Zheng, J.-Y., Pang, J.-B., Qiu, K.-Y. & Wei, Y. Synthesis of mesoporous silica materials with hydroxyacetic acid derivatives as templates via a sol-gel process. Journal of Inorganic and Organometallic Polymers 10, 103-113 (2000). 76. Zheng, J.-Y., Qiu, K.-Y., Feng, Q., Xu, J. & Wei, Y. Sol-gel synthesis of mesoporous titania using nonsurfactant organic compounds as templates. Molecular Crystals and Liquid Crystals Science and Technology, Section A: Molecular Crystals and Liquid Crystals 354, 183-194 (2000). 77. Feng, Q., Xu, J., Dong, H., Li, S. & Wei, Y. Synthesis of polystyrene-silica hybrid mesoporous materials via the nonsurfactant-template sol-gel process. Journal of Materials Chemistry 10, 2490-2494 (2000).
53 78. Pang, J.-B., Qiu, K.-Y. & Wei, Y. Synthesis of mesoporous silica materials with ascorbic acid as template via sol-gel process. Chinese Journal of Chemistry 18, 693-697 (2000). 79. Dong, H., Xu, J., Qiu, K.-Y., Jansen, S. A. & Wei, Y. Synthesis of nonsurfactant- based hybrid mesoporous sol-gel materials. Polymer Preprints (American Chemical Society, Division of Polymer Chemistry) 41, 194-195 (2000). 80. Wei, Y., Jin, D., Ding, T. & Xu, J. 79 pp ((Drexel University, USA). Patent Number 99-US1116, 9936357 1999). 81. Gregg, S. J. & Sing, K. S. W. Adsorption, Surface Area and Porosity. 2nd Ed (1982). 82. Lee, C. K., Chiang, A. S. T. & Tsay, C. S. The characterization of porous solids from gas-adsorption measurements. Key Engineering Materials 115, 21-43 (1996). 83. Barrett, E. P., Joyner, L. G. & Halenda, P. P. The determination of pore volume and area distributions in porous substances. I. Computations from nitrogen isotherms. Journal of the American Chemical Society 73, 373-80 (1951). 84. Lifshin, E. X-ray Characterization of Materials Wiley-VCH, Germany (1999). 85. Stoeckli, H. F. A generalization of the Dubinin-Radushkevich equation for the filling of heterogeneous micropore systems. Journal of Colloid and Interface Science 59, 184-5 (1977). 86. Terasaki, O., Ohsuna, T., Liu, Z., Sakamoto, Y. & Garcia-Bennett, A. E. Structural study of meso-porous materials by electron microscopy. Studies in Surface Science and Catalysis 148, 261-288 (2004). 87. Sasaki, Y. & Kato, H. Nanostructure analysis for applied research on zeolites. Zeoraito 21, 11-17 (2004). 88. Terasaki, O. & Ohsuna, T. Structural study of microporous and mesoporous materials by transmission electron microscopy. Handbook of Zeolite Science and Technology, 291-315 (2003). 89. Terasaki, O., Sakamoto, Y., Ohsuna, T., Ohnishi, N., Yu, J. & Hiraga, K. Electron microscopy of micro- and mesoporous materials. Electron Microscopy 1998, Proceedings of the International Congress on Electron Microscopy, 14th, Cancun, Mex., Aug. 31-Sept. 4, 1998 3, 301-302 (1998). 90. Chujo, Y. Organic-inorganic hybrid materials. Current Opinion in Solid State & Materials Science 1, 806-811 (1996). 91. Chujo, Y. & Saegusa, T. Organic polymer hybrids with silica gel formed by means of the sol-gel method. Advances in Polymer Science 100, 11-29 (1992). 92. Sanchez, C. & Ribot, F. Design of hybrid organic-inorganic materials synthesized via sol-gel chemistry. New Journal of Chemistry 18, 1007-47 (1994). 93. Judeinstein, P. & Sanchez, C. Hybrid organic-inorganic materials: a land of multidisciplinarity. Journal of Materials Chemistry 6, 511-25 (1996). 94. Schubert, U., Huesing, N. & Lorenz, A. Hybrid Inorganic-Organic Materials by Sol-Gel Processing of Organofunctional Metal Alkoxides. Chemistry of Materials 7, 2010-27 (1995). 95. Novak, B. M. Hybrid nanocomposite materials - between inorganic glasses and organic polymers. Advanced Materials (Weinheim, Germany) 5, 422-33 (1993). 96. Wen, J. & Wilkes, G. L. Organic/inorganic hybrid network materials by the sol- gel approach. Chemistry of Materials 8, 1667-1681 (1996).
54 97. Schottner, G. Hybrid sol-gel-derived polymers: Applications of multifunctional materials. Chemistry of Materials 13, 3422-3435 (2001). 98. Schmidt, H. New type of non-crystalline solids between inorganic and organic materials. Journal of Non-Crystalline Solids 73, 681-91 (1985). 99. Philipp, G. & Schmidt, H. New materials for contact lenses prepared from Si- and Ti-alkoxides by the sol-gel process. Journal of Non-Crystalline Solids 63, 283- 292 (1984). 100. Schmidt, H., Scholze, H. & Kaiser, A. Principles of hydrolysis and condensation reaction of alkoxysilanes. Journal of Non-Crystalline Solids 63, 1-11 (1984). 101. Wilkes, G. L., Brennan, A. B., Huang, H. H., Rodrigues, D. & Wang, B. The synthesis, structure and property behavior of inorganic-organic hybrid network materials prepared by the sol-gel process. Materials Research Society Symposium Proceedings 171, 15-29 (1990). 102. Wilkes, G. L., Orler, B. & Huang, H. H. "Ceramers": hybrid materials incorporating polymeric/oligomeric species into inorganic glasses utilizing a sol- gel approach. Polymer Preprints (American Chemical Society, Division of Polymer Chemistry) 26, 300-2 (1985). 103. Mark, J. E., Wang, S., Xu, P. & Wen, J. Reinforcement from in-situ precipitated silica in polysiloxane elastomers under various types of deformation. Materials Research Society Symposium Proceedings 274, 77-84 (1992). 104. Mark, J. E. Generate reinforcing particles in place. Chemtech 19, 230-3 (1989). 105. Mark, J. E., Jiang, C. Y. & Tang, M. Y. Simultaneous curing and filling of elastomers. Macromolecules 17, 2613-16 (1984). 106. Pope, E. J. A., Asami, M. & Mackenzie, J. D. Transparent silica gel-PMMA composites. Journal of Materials Research 4, 1018-26 (1989). 107. Schmidt, H. & Wolter, H. Organically modified ceramics and their applications. Journal of Non-Crystalline Solids 121, 428-435 (1990). 108. Wen, J., Vasudevan, V. J. & Wilkes, G. L. Abrasion resistant inorganic/organic coating materials prepared by the sol-gel method. Journal of Sol-Gel Science and Technology 5, 115-26 (1995). 109. Tamami, B., Betrabet, C. & Wilkes, G. L. Synthesis and application of abrasion resistant coating materials based on functionalized bis and tris maleimides. Polymer Bulletin (Berlin, Germany) 30, 293-9 (1993). 110. Wung, C. J., Pang, Y., Prasad, P. N. & Karasz, F. E. Poly(p-phenylenevinylene)- silica composite: a novel sol-gel processed nonlinear optical material for optical waveguides. Polymer 32, 605-8 (1991). 111. Avnir, D., Levy, D. & Reisfeld, R. The nature of the silica cage as reflected by spectral changes and enhanced photostability of trapped Rhodamine 6G. Journal of Physical Chemistry 88, 5956-9 (1984). 112. Schmidt, H., Scholze, H. & Tunker, G. Hot melt adhesives for glass containers by the sol-gel process. Journal of Non-Crystalline Solids 80, 557-563 (1986). 113. Kojima, Y., Usuki, A., Kawasumi, M., Okada, A., Kurauchi, T. & Kamigaito, O. Synthesis of nylon 6-clay hybrid by montmorillonite intercalated with e- caprolactam. Journal of Polymer Science, Part A: Polymer Chemistry 31, 983-6 (1993).
55 114. Usuki, A., Kojima, Y., Kawasumi, M., Okada, A., Fukushima, Y., Kurauchi, T. & Kamigaito, O. Synthesis of nylon 6-clay hybrid. Journal of Materials Research 8, 1179-84 (1993). 115. Kojima, Y., Usuki, A., Kawasumi, M., Okada, A., Fukushima, Y., Kurauchi, T. & Kamigaito, O. Mechanical properties of nylon 6-clay hybrid. Journal of Materials Research 8, 1185-9 (1993). 116. Zusman, R., Rottman, C., Ottolenghi, M. & Avnir, D. Doped sol-gel glasses as chemical sensors. Journal of Non-Crystalline Solids 122, 107-109 (1990). 117. Dave, B. C., Dunn, B., Valentine, J. S. & Zink, J. I. Sol-gel encapsulation methods for biosensors. Analytical Chemistry 66, 1120A-1127A (1994). 118. Venton, D. L., Cheesman, K. L., Chatterton, R. T., Jr. & Anderson, T. L. Entrapment of a highly specific antiprogesterone antiserum using polysiloxane copolymers. Biochimica et Biophysica Acta 797, 343-7 (1984). 119. Glad, M., Norrloew, O., Sellergren, B., Siegbahn, N. & Mosbach, K. Use of silane monomers for molecular imprinting and enzyme entrapment in polysiloxane- coated porous silica. Journal of Chromatography 347, 11-23 (1985). 120. Avnir, D., Braun, S., Lev, O. & Ottolenghi, M. Enzymes and other proteins entrapped in sol-gel materials. Chemistry of Materials 6, 1605-14 (1994). 121. Dunn, B., Miller, J. M., Dave, B. C., Valentine, J. S. & Zink, J. I. Strategies for encapsulating biomolecules in sol-gel matrixes. Acta Materialia 46, 737-741 (1998). 122. Akbarian, F., Lin, A., Dunn, B. S., Valentine, J. S. & Zink, J. I. Spectroscopic determination of cholinesterase activity and inhibition in sol-gel media. Journal of Sol-Gel Science and Technology 8, 1067-1070 (1997). 123. Miller, J. M., Dunn, B., Valentine, J. S. & Zink, J. I. Synthesis conditions for encapsulating cytochrome c and catalase in SiO2 sol-gel materials. Journal of Non-Crystalline Solids 202, 279-289 (1996). 124. Zink, J. I., Dunn, B., Valentine, J. S., Yamanaka, S. & Akbarian, F. Optical sol- gel materials based on binding and catalysis by biomolecules. Polymeric Materials Science and Engineering 73, 264-5 (1995). 125. Zink, J. I., Dunn, B., Valentine, J. S., Yamanaka, S. & Akbarian, F. Optical sol- gel materials based on binding and catalysis by biomolecules. Book of Abstracts, 210th ACS National Meeting, Chicago, IL, August 20-24, PMSE-140 (1995). 126. Yamanaka, S. A., Dunn, B., Valentine, J. S. & Zink, J. I. Nicotinamide adenine dinucleotide phosphate fluorescence and absorption monitoring of enzymic activity in silicate sol-gels for chemical sensing applications. Journal of the American Chemical Society 117, 9095-6 (1995). 127. Valentine, J. S., Dunn, B., Zink, J. I., Fukuto, J. M., El Sayed, M. A., Ellerby, L. M., Chung, K. E., Dave, B. C., Nishida, C. R. Sol-gel encapsulation of metalloproteins for biosensor. Polymeric Materials Science and Engineering 71, 666-7 (1994). 128. Yamanaka, S. A., Nguyen, N. P., Ellerby, L. M., Dunn, B., Valentine, J. S. & Zink, J. I. Encapsulation and reactivity of the enzyme oxalate oxidase in a sol-gel derived glass. Journal of Sol-Gel Science and Technology 2, 827-9 (1994).
56 129. Zink, J. I., Yamanaka, S. A., Ellerby, L. M., Valentine, J. S., Nishida, F. & Dunn, B. Biomolecular materials based on sol-gel encapsulated proteins. Journal of Sol- Gel Science and Technology 2, 791-5 (1994). 130. Zink, J. I., Valentine, J. S. & Dunn, B. Biomolecular materials based on sol-gel encapsulated proteins. New Journal of Chemistry 18, 1109-15 (1994). 131. Zink, J. I., Dunn, B., Yamanaka, S., Lan, E., Valentine, J. S. & Chung, K. E. optical sol-gel materials based on binding and catalysis by biomolecules. Materials Research Society Symposium Proceedings 346, 1017-26 (1994). 132. Yamanaka, S. A., Nishida, F., Ellerby, L. M., Nishida, C. R., Dunn, B., Valentine, J. S. & Zink, J. I. Enzymatic activity of glucose oxidase encapsulated in transparent glass by the sol-gel method. Chemistry of Materials 4, 495-7 (1992). 133. Braun, S., Rappoport, S., Zusman, R., Avnir, D. & Ottolenghi, M. Biochemically active sol-gel glasses: the trapping of enzymes. Materials Letters 10, 1-5 (1990). 134. Avnir, D. Organic chemistry within ceramic matrixes: doped sol-gel materials. Accounts of Chemical Research 28, 328-34 (1995). 135. Gill, I. & Ballesteros, A. Bioencapsulation within synthetic polymers (Part 1): sol- gel encapsulated biologicals. Trends in Biotechnology 18, 282-296 (2000). 136. Gill, I. & Ballesteros, A. Encapsulation of biologicals within silicate, siloxane, and hybrid sol-gel polymers: An efficient and generic approach. Journal of the American Chemical Society 120, 8587-8598 (1998). 137. Avnir, D., Braun, S. Biochemical Aspects of Sol-Gel Science and Technology Kluwer Hingham, Mass. (1996).
57
Figure 1- 1 Sol-gel process and their products. (www.chemat.com/ html/solgel.html)
58
(a)
OH OC2H5
HO OH + 4 C H OH C2H5O Si OC2H5 + 4 H2O Si 2 5
OH OC2H5
(b)
Si OH + HO Si Si O Si + H2O
Figure 1- 2 Diagrams of sol-gel reactions: (a) Hydrolysis reaction; (b) Condensation reaction.
59
Figure 1- 3 Possible mechanisms for formation of MCM-41: (1) liquid crystal phase initiated and (2) silicate anion initiated. 20,21
60
Figure 1- 4 IUPAC classification of physisorption isotherms.16
61
Figure 1- 5 IUPAC classification of hysteresis loops.16
62 Chapter 2: Rigid Matrix Artificial Chaperon (RMAC) – Mediated Refolding of
Cytochrome c
2.1 Introduction
Besides the applications of the novel nonsurfactant templated sol-gel technology
in biosensing and biocatalysis,1-9 this new method to encapsulate biological substances
can also be used in biochemistry or biophysics studies, such as protein folding/unfolding
and protein aggregation.10-13 Protein folding is one of the most intensively studied subjects in modern biochemistry. To understand how proteins fold is both of great academic interest and industrial importance.14 Among many interesting approaches to protein refolding,15-20 one utilizes a pair of low molecular weight folding assistants, a detergent and a cyclodextrin.15,16 The detergent forms a complex with the non-native protein, thereby preventing aggregation. Then folding is induced with cyclodextrin, which strips the detergents away from the protein to initiate refolding.15,16 In this study, we use mesoporous sol-gel silica matrix as rigid matrix artificial chaperone (RMAC) to mediate refolding of a denatured (unfolded) heme protein, i.e., cytochrome c (Cc), back to its native state based on controlling cavity size around Cc molecules. Intrinsic fluorescence and circular dichroism were used to investigate conformational changes of the protein.
2.1.1 Fundamentals of Protein Folding Unfolding
Forty years ago, Anfinsen first discovered that denatured ribonuclease could be refolded into its native structure in vitro.21 Since then, protein folding has been realized as
one of key problems in bioscience attracting great interests in recent years. The essence
63 of the problem is to understand how an unstructured polypeptide chain folds into a unique
three dimensional structure with biological activity. Currently, people realize that there
may not be a single, specific pathway, as was suggested in some early models. Instead, a
multidimensional energy landscape or folding funnel better describes the folding
process.22 Thus, peptides may follow a plethora pathways leading to their native state. In
this “energy funnel”, there may be some local energy minima, which are possible
transient intermediates or misfolded states. But for some folding processes of particular
proteins, there may not be folding intermediates, thus the surface of funnel will be
smooth.23,24 This model is illustrated in Figure 2-1.
2.1.2 Chaperone and Protein Folding
In the late 1980s, it was found that many proteins require assistance in their folding pathways, especially when in cells. The assistance is usually provided by another helper protein, called chaperone. 25
The difficulties of protein folding in vivo come from the following aspects. First,
the processes of biosynthesis and folding always happen simultaneously, and the new
polypeptide chains can fold before synthesis is completed. As soon as a peptide chain
with enough amino acids is generated from the ribosome, it will try to fold to some
particular structure to gain the lowest available energy minimum for that length of
chain.26 Second, the concentration of biological substances inside cell is extremely high
[~300 (g protein) L-1]. Because the thermodynamic activity of intermediate state is
increased 10 to 100 fold in that highly crowed situation, the chance for aggregations of
partially folded polypeptides during folding process increase significantly.27,28
64 Chaperones can assist the protein folding process by involving in the biosynthetic
process and protecting proteins from aggregation. Also, they can capture misfolded states
in the cytosol post-translationally and allow them to renew the folding process.29 The chaperones include about 20 protein families of different molecular weights, structures, cellular locations and precise biological roles.29 Among them, the two best understood chaperons are the hsp70 and hsp60 families. The functions of these two proteins are different: one of hsp70’s biological activities is binding to the short hydrophobic sequences as they are generated from the ribosome. Thus, the hsp70 can prevent the aggregation of newly synthesized chains during protein biosynthesis; hsp60 helps protein folding after the protein biosynthesis is completed. These so-called chaperonins are built of two doughnut-shaped rings stacked back to back, and the central cavity forming the important folding cage. As a whole, the hsp70 and hsp60 chaperons co-operate in vivo, to help newly generated protein folding to its native state. 29,30
2.1.3 Rigid Matrix Artificial Chaperone (RMAC)
In this project, a modified nonsurfactant templated sol-gel process was used to make silica matrix with controlled pore size, which was termed by us as “Rigid Matrix
Artificial Chaperone”. The nanoporous silica matrix synthesized via this method is usually with high surface area (up to 1000 m2/g) and pore volume (e. g. 0.5- 1.0 cm3/g) as well as pore size in the range of 2-12 nm with narrow size distribution. The nonsurfactant molecules such as glucose, maltose, dibenzoyl-L-tartaric acid (DBTA), fructose, cyclodextrins, urea, glycerol, soluble starch, citric acid, hydroxylethyl methacrylate, ascorbic acid, oligopeptides, etc., can be used as templates in directing the mesostructure formation during the sol-gel reactions.9,31-38 In general, inorganic precursors such as
65 tetraethyl orthosilicate (TEOS) or tetramethyl orthosilicate (TMOS) are first hydrolyzed with hydrochloric acid as catalyst followed by mixing with nonsurfactant compounds solution. Upon gelation and drying, the transparent template-silica composites can be obtained. At this time, they are still nonporous, with removal of template by solvent extraction, the porosity of the materials increases, leading to the final mesoporous materials after complete removal of the templates.
The porosity of the materials can be adjustable to a certain extent. Generally, the values of these porous parameters increase with the template weight percentage. When the weight percentage is between 30% and 50%, mesoporosity can be achieved.
To understand the mechanism of porosity forming by nonsurfactant templates, nearly 100 compounds have been investigated. It was found that only the compounds with highly polar functional groups can work as the templates to form narrowly distributed nanopores. One of possible explanations is that strong polar interactions and hydrogen bonding between the nonsurfactant molecules or their aggregations and the inorganic species may play an important role in directing the mesophase formation. After removal of nonsurfactant templates, nanoporous silica matrix with interconnected pores and channels can be obtained. Unlike typical surfactant templated nanoporous materials, the materials formed via nonsurfactant pathway have no ordered pore structure consisted of oriented pores and channels. But it is interesting to notice that this “drawback” may be advantageous for many applications because this mesoporosity is accessible in all directions.
One of the most important advantages of this technique is its biocompatibility.
The whole synthesis and encapsulation process can be readily accomplished at room
66 temperature and near neutral pH. Nonsurfactants such as sugar compounds are highly
biocompatible. Furthermore unlike surfactant molecules which are tightly adhered to
silica matrix and difficult to remove, those nonsurfactant molecules can be easily
removed from silica matrix at room temperature by water extraction instead of
calcination at high temperature. These enabled us to develop a novel nanobiotechnology:
one-step direct encapsulation of bioactive substances, such as proteins, polypeptides,
cells, etc., inside nanoporous matrices. Adjusting pH to the desired value and partial
removal of organic byproducts (e. g. methanol or ethanol) can help the encapsulated
bioactive substances retain their bioactivities during the sol-gel process.
In this project, proteins were unfolded by urea at high concentration, e.g. 9M, followed by encapsulated in silica matrix. In this sol-gel process, urea functions as both denaturant and template. By controlling the amount of urea used, desired pore size of the silica matrix can be achieved. Washing out urea by water extraction from silica matrix and incubating in phosphate buffer (pH= 7.0) gave the proteins encapsulated in silica pores an opportunity to fold back to their native states. Because the sizes of pores where the proteins stay were different, the effect of space confinement on protein folding process can be examined. Thus, this silica matrix may be considered as artificial chaperone in assisting and mediating unfolded protein to fold back to native state. The whole process can be illustrated in Figure 2-2.
2.1.4 Why Cytochrome c?
Cytochrome c (Cc), a small single-domain protein, was used as the target protein
to study protein folding process in this project. The three dimensional structure of Cc has
been well defined.39 The protoporphyrin IX prosthetic heme group, located in the center
67 of the protein, is covalently bound to the polypeptide backbone by Cys14 and Cys17. The
heme iron is coordinated by His18 and Met80 in the native form.40 Under unfolding conditions His26 or His33 may coordinate to the heme in place of Met80. The single tryptophan with fluorescence activity is at position 59.41,42
Cc has been widely used as the model system for folding studies. There are several reasons as follows. First, the unfolding and refolding of Cc is reversible without degradation of the protein: Second, no complicated bimolecular rebinding of heme to polypeptide chain happens in its refolding process because the heme prosthetic group stays intact even in its unfolded state due to the covalent linkages between the heme and protein moiety: Third, various chemical denature methods, like the pH changing and the concentration of denaturant, can generate different folding intermediates. Thus it can help us to understand the contributions of various intermediates in folding energy landscape.40
Another reason for choosing cytochrome c in our experiment is that Cc is a rather robust protein and its diameter (~3 nm in its folded state and > 4.5 nm in its unfolded state)43 matches well with the pore size of the silica matrix.
2.1.5 Analytical Methods to Monitor Cytochrome c’s Folding Unfolding
Two analytical methods were adopted in this project to monitoring Cc folding unfolding process, steady state fluorescence and circular dichroism.
2.1.5.1 Fluorescence Spectroscopy
Fluorescence spectroscopy is one of the most sensitive and versatile of the optical techniques for studying protein structure.44-46 There are three types of fluorescent chromophores in proteins- intrinsic, co-enzymic and extrinsic. The intrinsic chromophores are the aromatic side chains of phenylalanine, tyrosine, and tryptophan
68 residues. In this project, because there is a tryptophan residue at position 59 in Cc,
intrinsic fluorescence spectroscopy was used to illustrate the changing of three
dimensional structure of Cc.
When Cc in its folded state, the distance between heme and tryptophan 59 (Trp59) are so short that the intrinsic fluorescence of tryptophan is quenched by heme because of dipole-dipole coupling energy transfer. Then the fluorescence of the tryptophan residue of native Cc is quenched by the heme group and represents only 2.0% of free Trp59 fluorescence in aqueous solution. In highly concentrated denaturant solution such as 9 M
urea solution, the Trp59 fluorescence reaches 60% of intensity level for free tryptophan.
So the difference of fluorescence intensity of Cc was used as an indication of the protein
folded state.41,42
2.1.5.2 Circular Dichroism (CD)
CD refers to the differential absorption of the left and right circularly polarized
components of plane-polarized radiation.47 This phenomenon happens when a
chromophore is chiral (optically active). The chirality of a molecule may rise from (a)
intrinsically its own chiral structure or (b) being covalently bonded to a chiral center or (c)
being placed in an asymmetric environment. In practice, the light generated from lamp is
split into two circularly polarized components by a modulator (usually a piezoelectric
crystal such as quartz) and pass through the sample. If the chromophore is chiral, the
absorption of two polarized components are different, thus the resultant (combined
component) radiation would now be elliptically polarized. In fact, the CD instrument
(spectropolarimeter) does not recombine the components but detects the two components
separately; it will then display the dichroism at a given wavelength of light expressed as
69 either the difference in absorbance of the two components (∆A =AL- AR) or as the
ellipticity in degree [θ = tan-1(b/a), where b and a are the minor and major axes of the
resultant ellipse]. The relation of θ with ∆A is θ= 32.98 ∆A.
CD is a very useful instrumental method for studying protein structure. Dependent on the spectral wavelength, a CD spectrum can be divided into three parts:
Far-UV region (240 nm to 190 nm or 180 nm): Spectrum in this region can be used to calculate quantitatively the overall secondary structure content of the protein. In this region, the absorbing groups are principally the peptide bonds. There is a weak but broad n → π* transition centered around 210 nm and an intense π → π* transition at about
190 nm.
It is known that different forms of regular secondary structure found in peptides
and proteins exhibit distinct far-UV CD spectra. But some other effects, such as aromatic
amino acid side chains, disulfide bonds and the length as well as regularity of structural
elements in the peptides and proteins can also contribute to the spectrum in this region.48-
51 Thus some sophisticated curve-fitting procedures are used to deduce the contributions of the different structural forms quantitatively. In this project, we qualitatively estimated the α-helical content in Cc by examining the characteristic negative double peaks at 208
and 222 nm. 52,53
Near-UV region (aromatic amino acid dichroism): Aromatic amino acid chains and disulfide bonds make the majority contributions in the region spectrum. Each of the aromatic amino acids tends to have a characteristic wavelength profile: tryptophan, a peak close to 290 nm with fine structure between 290 nm and 305 nm; tyrosine, a peak between 275 and 282 nm; phenylalanine, sharp fine structure between 255 and 270 nm.54
70 But the rigidity of the protein, interactions between aromatic amino acids and the number of aromatic amino acid give have effects on the spectrum.
Near-UV and visible region: CD in the near-UV, visible and near-IR regions can give a great deal of information on the environments of cofactors and other protein-bound ligands. In many situations, the free cofactor or ligand is optically inactive, but when binding to the protein becomes immobilized in an asymmetric environment, thus giving rise to CD signals in a characteristic absorption region.47
2.2 Experimental
The encapsulation of cytochrome c in silica matrix with controlled pore size was achieved by nonsurfactant templated sol-gel process which was developed originally in our group. In this project, urea was used as both template molecule and denaturant. The characterization of silica matrix was carried by BET nitrogen sorption measurement and the monitoring of Cc refolding process was done through fluorescence spectroscopy and circular dichroism (CD).
2.2.1 Materials
Tetramethyl orthosilicate (TMOS, 98%, Aldrich), urea (99%, Sigma), cytochrome c (from horse heart, 95%, Sigma), sodium phosphate (monobasic anhydrous, minimum
99.0%, Sigma), Sodium phosphate dibasic heptahydrate (minimum 99.0%, Sigma), hydrochloride acid (HCl, Fisher), Nye optical coupling gel kit (Nye Optical Products,
MA), were all used as received without further purification.
2.2.2 Encapsulation of Unfolded Cc into Silica Matrix
71 The preparation of the template (urea)-containing silica with unfolded protein was achieved by HCl-catalyzed sol-gel reactions of tetramethyl orthosilicate (TMOS) in the presence of 9 M urea and cytochrome c. In a typical procedure, 3.1 g TOMS, 0.7 g H2O and 0.03 ml HCl solution (40 mM) were mixed at room temperature for 1-2 h under stirring. Upon mixing, the organic phase (TMOS) and inorganic phase (H2O) were
separated, so the mixer was translucent. After stirring vigorously for less than 5 minutes,
a lot of heat was generated by the hydrolysis reaction and the whole solution turned to
transparent suddenly. Stirring continued until the solution cool down to room temperature.
Then the mixture was cooled to ~ 0 oC in an ice-water bath followed by addition of
appropriate amount of 9 M urea solution containing 10 mg of horse heart cytochrome c.
After mixing the whole sol turned to the color of cytochrome c, deep red. Amounts of 9
M urea were designed to yield 0-50% by weight of templates in the final dry gel products
(sample code: Ccu plus a number denoting weight percentage of urea). The reaction was
then sealed with a parafilm. Upon gelation of the system within a few hours at room
temperature, 12-15 holes were pinned in the parafilm with a hypodermic syringe needle
to allow for the slow evaporation of solvents and reaction by-products. Dependent on the
weight percentage of templates, the gelation time varied from several minutes to half an
hour. Normally, the higher weight the percentage was, the shorter time the gelation took.
During the gelation process, a large amount of byproduct solution was generated. After
24 h, the system was placed in a vacuum oven and dried to reach a constant weight at
room temperature in about 6 days. Thus the silica biogel samples containing urea and Cc
were obtained as deep red, transparent, dry, brittle, glassy monoliths. Then the samples
were grinded into fine powders and kept in sealed vials in a –15 oC freezer. Porcelain
72 pestle and mortar were used for grinding and the whole process was done by hand. For
each sample, the grinding time was maintained at around five minutes to achieve powders
with uniform size. According to SEM picture taken afterward, the average size of
particles were from several micrometer to 20 micrometers.
2.2.3 Removal of Templates
Since the solubility of urea (300 g/l) in water is very high, simple water extraction
can wash template urea out from silica matrix completely. In a typical washing procedure,
about 1-2 grams of silica sample was put into a test tube with about 10-15 ml water, and
then the test tube was fixed in an Aliquot mixer by scotch tape. To avoid losing silica
powder during washing steps, the test tubes were centrifuged at several thousand rpm in
an IEC Spinette centrifuge (Damon/ IEC Division). After 5 minutes, most of silica
powder settled down firmly in the bottom of the test tubes. Then the upper clear
supernant can be discarded carefully without losing powder. In the first two days,
washing water was changed every two hours. After that, the interval time could be
extended to 4 hours. The washing process usually took about 4 to 5 days. Then the FTIR
spectrum was taken on silica powder to examine for trace of urea.
2.2.4 Characterizations of Nanoporous Silica Matrix
The characterization of the nanoporous structure parameters of the silica matrix
was carried out with N2 adsorption-desorption isotherms on a Micromeritics 2010 system
(Micromeritics, Inc., Norcross, GA) after the removal of template by water extraction.
Prior to the measurements, the samples were degassed at 100 oC under vacuum for 6-7 h.
For each measurement, 0.1 - 0.2 gram of sample was used.
2.2.5 Fluorescence Spectroscopy of Encapsulated Cc
73 The refolding of cytochrome c was monitored with fluorescence spectroscopy on a PTI M-III spectrophotofluorometer. A 5-nm slit width setting for both excitation and emission and a cuvette with 1-cm pathlength was used throughout the experiment. The excitation wavelength was chosen at 295 nm and the maximum of emission peak was observed at near 350 nm. In order to get reliable results, magnetic stirring was kept on a constant rate and approximately the same amounts of entrapped Cc were used for fluorescence measurements.
As the first step the series of samples without washing out templates were put into
9 M urea and incubated for 24 h, followed by measuring fluorescence spectra of the samples suspended in 9 M urea for unfolded Cc. As the second step, suspensions were collected and centrifuged. Then tris(hydroxymethyl) aminomethane (Tris) buffer (pH=7.0,
50 mM) was added to wash the powder in order to remove the urea template in silica matrix as described in last section. After extraction, urea was removed completely as evidenced by comparing IR spectra of samples before and after extraction. After incubation in the buffer for about 24 hours, another set of fluorescence spectra was recorded.
2.2.6 Circular Dichroism of Cc
Circular dichroism (CD) spectroscopy was also used to investigate Cc folding in the silica matrix. As discussed in the introduction part, both far-UV and near-UV region can be used to investigate cytochrome c’s structure in solution, because there are both α- helix and β-sheet structure as well as aromatic amino acid chain (Trp59) in the polypeptide chain. Unfortunately due to low concentration of protein loading in silica matrix and strong light scattering in suspension, only the signal in far UV range at 222
74 nm which reflects secondary structure of protein can be detected because only the
negative peak at this wavelength was strong enough to be detected even in silica
suspension without burying inside the light scattering noise.
We also found the CD signal of silica powder in 9M urea solution below 250 nm is beyond the detection limit. So the CD data of Ccu series sample in 9M urea were not available, only the data of samples in Tris buffer suspension could be obtained. One of the possible explanations is that both silica and highly concentrated urea solution can absorb a lot of far-UV region radiation, when there is only either silica powder or high concentrated urea, the absorption is not strong enough to saturate the CD signal so the peak from Cc can still be detected. But when silica powders were immersed in high concentrated urea solution, the absorption would go beyond the detection limit and signals from Cc was buried in noise.
Thus in this project, we only got the CD spectra of Ccu series in Tris buffer after washing out urea above 200 nm and focused the analysis of the α-helix peak at 222 nm.
By comparing the intensity of this peak, the percentage of α-helix remained in the protein
can be estimated. The CD spectra of Ccu series were taken on a J-810 circular dichroism
instrumentation (Jasco Inc. Easton, MD).
2.2.7 Fourier Transform Infrared Spectroscopy (FTIR) of Silica Matrix
FTIR was used to investigate the urea template in silica matrix. Ccu series
samples were taken powder FTIR before and after extraction of templates. To prepare the
powder Ccu sample, the powder samples were put into high vacuum oven in room
temperature after water extraction for 5 days. By vacuuming for 2-3 days, the wetted
powder became totally dry, which was verified by weighting tests.
75 2.2.8 UV-Vis Spectroscopy (UV) of Encapsulated Cc
UV spectroscopy was also been tried to gain structural information of encapsulated Cc. Unfortunately, no data with reasonable signal-to-noise ratio can be obtained. In this project, two different methods were used to take Ccu UV spectrum. The first choice is using Ccu suspension. Because of the large noise from extremely strong light scattering of silica powder in the suspension, the absorption peaks were buried in baseline.
In the second method, we tried to embed the Ccu silica powder in optical gels with similar light refraction index to silica (Index of Refraction, 1.46 (literature vlue)).
We hoped the Ccu powder and the optical gel could become homogenous transparent, then the light scattering from the powder could be avoid. Several types of optical gels have been tried. One of gel we used was from Nye optical coupling kit, OC-431A-LVP
(Index of Refraction, 1.46) non-curing gel. Both wet and dry powders have been used to first mix with the optical gel. Then the mixed gel was made into a thin film in between two pieces of glass slides. We hoped that the silica powder could be embedded in the gel without any light refraction in the interface of silica and gel. Unfortunately, the phase separation could still be seen on the interface. Thus, the light scattering could not be eliminated completely. Though the UV spectrum seemed a little better than those in the suspension, it was still not good enough to reveal the detailed information about the structure of protein.
One of the possible reasons for the failure of the experiment was the viscosity of the optical coupling gel was too high. When the silica powders were mixed with the gel, some air bubbles attached on the surface of silica powder. Since the viscosity of the gel is
76 so high that it was not flowable, the air bubbles could not be removed from the gel. Thus,
the light scattering still happened.
In fact, another type of gel, Biomeda® mount gel for microscope, should be tried.
The original purpose of this gel is to fix the position of a cell in a glass slide to easily find it under microscope. Because of a large number of brands of mount gel with different refraction indexes available, suitable gel could be found at last. The advantage of this mount gel to optical coupling gel was the lower viscosity, which may give the air bubbles a chance to float out. Though we have not tried this gel, we believe it could be a possible way to get the UV spectrum of encapsulated Cc in the future.
2.2.9 Attempts of Making Silica Thin Film
The strong light scattering of silica powder suspension became the most serious problem in this project. The noise brought by light scattering prevented the optical
investigations in quantitative analysis. Thus only general tendencies could be obtained
from encapsulation Cc in suspensions. Further calculations and modeling were not
performed in the suspension system.
To solve the problem, another system of silica thin film materials has been tried.
In this attempt, we tried to form a flat silica thin film attached on a glass slide. Then this
piece of thin film can be placed in the middle of the light pathway for recoding its
spectrum. For UV and CD, the thin film can be simply inserted between the light source
and detector with angle of film and light beam at 90 degree. In the situation of
fluoremetry, the angle of the thin film and light beam should be kept at 45 degree. In this
angle, only emission light instead of reflecting light can reach the detector.
77 Unfortunately, this thin silica film was not easy to achieve. First, due to the
evaporation of byproducts generated from sol-gel process, the volume shrinkage could be
more than 80%. And the shrinking might lead to cracking of silica film. Second, when the
dry silica film was immersed into aqueous solution, it automatically cracked into small
pieces. So to prevent cracking of silica film was the biggest challenge in this project.
Two approaches were used to solve the cracking problem. First, before the sol
was cast on a glass slide, most byproducts such as alcohol and water molecules were
removed by evaporation under vacuum until the sol became viscous. In this way, the
volume shrinkage could be reduced. Second, once the gelation was finished, the silica
film was placed into a sealed container with a lot of water inside to let the aging process happen in environment with saturated water vapor. The silica film prepared in this way does not easy crack in water.
However problems still existed in the solutions. For example, when most of the byproducts, which can serve as solvent for both templates and the protein, evaporated, the sol became very viscous. So the templates may precipitate and the protein may be denatured by the organic components in the sol. Furthermore, the nanoporous structure might be altered when the aging process was taken in the humid environment. As the result, only some initial works has been done in this area, further investigation is worth trying to make thin film of silica matrix.
2.3 Results and Discussion
There have been many reports in the literature on immobilization55-59, fluorescence spectroscopy 60-63 and circular dichroism study64,65 of enzymes and other
78 proteins in microporous sol-gel materials and on loading of proteins to pre-synthesized
M41S type of molecular sieves.66-68 This work is the first study of an unfolded protein
which was directly encapsulated inside silica matrix with controlled pore size. The
purpose is to study the refolding behavior of unfolded protein in confined space and
examine how the size of cavity affects the refolding process. To achieve this, a novel
nonsurfactant templated sol-gel process, which was developed in our group for enzyme
immobilization, has been used in this project. The most important advantage of this
method is protein can be physically directly encapsulated in silica matrix without any
significant structure alteration. This technology and silica materials provide us a great
new technique to study protein folding unfolding.
2.3.1 Characterization of Silica Matrix
The characterization of the porous silica matrix was carried out on a
Micromeritics 2010 system (Norcross, GA) after the removal of urea template by water
extraction. Figure 2-3 shows a representative set of N2 adsorption-desorption isotherms at
–196 ºC for the biogels after the removal of urea. As the urea content is increased, the isotherm evolved from Type I, typical of microporous materials (e.g., Ccu0), to Type IV with Type H2 hysteresis loops, typical of mesoporous materials (e.g., Ccu50). The BJH pore size distributions of the biogels were obtained by plotting the differential volume of
the desorption branch of N2 sorption isotherms as a function of a pore size as shown in
Figure 2-4. Similar to the results in the literature,36,69 the pore diameter and volume tend
to increase with urea content as shown in Figure 2-5. When urea accounts for 40-50 wt%,
the materials possess narrowly distributed pore sizes centered at 33.2-34.2 Å with width
at half peak height of about 5 Å. Detailed porous parameters of Ccu series samples were
79 shown in Table 2-1. Our previous researches suggested that the aggregates of non-
surfactant molecules instead of individual molecules might be responsible for the
mesophase formation. As the sequence, with the increasing of templates amount from 0
wt% to 50 wt%, the aggregation extent of template molecules rise up to form larger pores.
2.3.2 FT-IR Spectroscopy on Silica Matrix
FT-IR spectroscopy was used to examine whether the template could be removed
completely from silica matrix after washing for 5 days. The results are shown in Figure 2-
6. The peaks at 1620 cm-1 and in the region 3400-3100 cm-1 which can be assigned
respectively to C=O bond and N-H bond in urea molecules were picked as the indications
of the presence of template, urea. Compared with these three spectra, after water
extraction, the spectra of Ccu50 was exactly same to pure silica sample; these two peaks
can be only seen in the spectra of Ccu50 before washing.
2.3.3 Fluorescence Spectroscopy of Encapsulated Cc
As shown in Figure 2-7, the relative difference, (IU-IT)/IT, in fluorescence intensity of entrapped cytochrome c between unfolded (IU, measured in 9 M urea) and folded (IT, extracted and measured in tris buffer) states is correlated to the pore volume
and pore size of silica matrix, which depends on the template content in the samples.
With the increase of pore size and volume, the change in fluorescence intensity tends to
become greater. This indicates that the average extent of Cc refolding in big cages is
higher than that in small cages. Before immobilization in silica nearly all Cc molecules
were in unfolded state in 9 M urea solution. During the gelation and drying of the sol-gel
process, the urea concentration might become higher because water and other solvent
were further removed by evaporation. Thus, cytochrome c was in its unfolded state
80 when the silica powders containing Cc were suspended in 9 M urea for fluorescence
measurements. Consequently, the fluorescence intensity was high at that time. After
removing urea by extraction with and incubation in tris buffer for 24 hours, fluorescence
intensity of Cc entrapped in silica matrix with big pores decreased dramatically,
indicating a great extent of Cc refolding. In contrast, for Cc entrapped in silica matrix
with small pores, the fluorescence intensity nearly remained at the same level or
decreased less dramatically, suggesting that most Cc molecules cannot refold back to
native state. The relative change in the fluorescence intensity, therefore, reflects the
percentage of cytochrome c refolding. The loss of powder during extraction process
could also contribute to the observed decrease in fluorescence intensity. However, such a
loss should be almost identical for all samples, because the procedures were kept the
same during experiments. Hence, it should not have significant effect on the trend
observed in Figure 2-7. Due to light scattering, the fluorescence spectra of the powder
suspension were not as smooth as those solution samples resulting in relatively large
error margins.
2.3.4 Leakage Tests
The possibility of leakage of cytochrome c from the matrix during the extraction
has been investigated. Because the silica matrix bear a net negative charge while Cc has a
net charge of 7+ at pH 7.064,65, a set of buffers with different pH values from 7.0 to 3.5 were used to wash samples and the supernatants were separated by centrifugation. It was found even for the buffer with pH=3.5, which should eliminate most adsorption of Cc on silica surface, no Soret band of Cc (at 410 nm) was observed in the UV-vis spectra of the supernatant, indicating the absence of Cc leakage.
81 2.3.5 CD Spectroscopy
Circular dichroism (CD) spectroscopy was also used to investigate Cc folding in
RMAC. Due to low protein loading in silica matrix and strong light scattering of the
suspension, only the signal in far UV range at 222 nm which indicates content of
secondary structure of protein can be detected. The CD spectroscopy is shown on Figure
2-8. After incubation in pH 7.0 buffer for 24 hours, Cc entrapped in large pores show
greater negative peak at 222 nm than in small pores. Compare to fully unfolded Cc in 9M
urea and native Cc in buffer, even Cc in small pores have partially refolding of secondary
structure but the extent of refolding is clearly less then Cc in large pores. These results
are in good agreement with those obtained from fluorescence spectroscopy.
The structure of nanoporous silica matrix consisted of three-dimensionally
interconnected pores and channels.70 Because all the samples were prepared by the same procedure and the only difference was the amount of urea template employed, the surface properties of samples should be the same. Therefore, the effect of surface on proteins should be essentially identical. It is noted that we can change surface properties, such as hydrophobicity, readily by preparing organic-inorganic hybrid mesoporous materials.
Pore size and volume should be the main factors contributing to the results. When Cc was
entrapped in the matrix in unfolded state, extended chains of unfolded proteins may be
placed in the channels or pores. So if the channels and pores are large enough to let the
protein roll back and forward, the protein would be able to refold. On the other hand, if
the channels and pores are too small and there are not enough space for the protein to fold
back, the protein would remain unfolded, which is represented in Figure 2-9.
82 2.4 Conclusion
We have described the first study of refolding of cytochrome c that was directly
entrapped in sol-gel derived mesoporous materials in its unfolded state. The extent of
refolding of Cc was found to increase with the pore size and volume of the silica host.
The ability to mediate the protein refolding process provides a new example that
mesoporous materials function like a rigid matrix artificial chaperone. Currently, we are
studying the folding/unfolding kinetics and pathways of various proteins and RNAs in
mesoporous host materials and exploring the possibility of trapping folding intermediates.
2.5 Acknowledgement
I wish to thank the Office of Vice-President for Research for a full fellowship
(2000-01) in supporting the Protein Research Initiatives at Drexel University. I thank Dr.
Patrick Loll of Medical School, Drexel University for his assistance in circular dichroism spectroscopy. This work is in collaboration with Prof. Jian-Min Yuan of Drexel
University and with Prof. George McLendon of Princeton University now at Duke
University.
2.6 References
1. Wei, Y., Dong, H., Xu, J. G. & Feng, Q. W. Simultaneous immobilization of horseradish peroxidase and glucose oxidase in mesoporous sol-gel host materials. Chemphyschem 3, 802-+ (2002). 2. Wei, Y., Xu, J. G., Feng, Q. W., Lin, M. D., Dong, H., Zhang, W. J. & Wang, C. A novel method for enzyme immobilization: Direct encapsulation of acid phosphatase in nanoporous silica host materials. Journal of Nanoscience and Nanotechnology 1, 83-93 (2001). 3. Xu, J. G., Dong, H., Feng, Q. W. & Wei, Y. Use of poly(ethylene oxide) nonionic surfactants as template for enzyme-containing mesoporous sol-gel materials. Abstracts of Papers of the American Chemical Society 220, U298-U298 (2000).
83 4. Feng, Q. W., Xu, J. G., Lin, M. D., Dong, H. & Wei, Y. One-step direct immobilization of acid phosphatase in mesoporous silica sol-gel materials. Abstracts of Papers of the American Chemical Society 220, U364-U364 (2000). 5. Dong, H., Xu, J. G., Feng, Q. W. & Wei, Y. Simultaneous immobilization of oxidase/peroxidase in the mesoporous sol-gel silicate matrix. Abstracts of Papers of the American Chemical Society 220, U364-U364 (2000). 6. Xu, J. G., Dong, H., Feng, Q. W. & Wei, Y. Direct immobilization of horseradish peroxidase in mesoporous hybrid sol-gel materials. Abstracts of Papers of the American Chemical Society 219, U421-U422 (2000). 7. Xu, J., Feng, Q. W., Dong, H. & Wei, Y. Stability of immobilized horseradish peroxidase in mesoporous silica sol-gel materials. Abstracts of Papers of the American Chemical Society 219, U422-U422 (2000). 8. Xu, J. G., Dong, H., Feng, Q. W. & Wei, Y. Immobilization and activin assay of horseradish peroxidase in mesoporous silica sol-gel materials. Abstracts of Papers of the American Chemical Society 219, U458-U459 (2000). 9. Wei, Y., Xu, J. G., Feng, Q. W., Dong, H. & Lin, M. D. Encapsulation of enzymes in mesoporous host materials via the nonsurfactant-templated sol-gel process. Materials Letters 44, 6-11 (2000). 10. Sun, Z. F., Balakrishnan, G., Nielsen, S. B., Yuan, J. M., Spiro, T. G., Wei, Y. & Venkatesh, S. Study of cytochrome c folding and unfolding in rigid silica matrices with controlled pore size by resonance Raman spectroscopy. Abstracts of Papers of the American Chemical Society 228, U182-U182 (2004). 11. Sun, Z. F., Li, S. X., Patel, A. C., Wang, C. & Wei, Y. Fabrication of poly(2- hydroxyethyl methacrylate)-silica nanoparticle hybrid nanofibers via electrospinning. Abstracts of Papers of the American Chemical Society 228, U868-U868 (2004). 12. Ping, G., Yuan, J. M., Sun, Z. F. & Wei, Y. Studies of effects of macromolecular crowding and confinement on protein folding and protein stability. Journal of Molecular Recognition 17, 433-440 (2004). 13. Wei, Y., Sun, Z. F., Zheng, J. Y., Dong, H., Yuang, J. M. & Ping, G. H. Rigid matrix artificial chaperone (RMAC)-mediated refolding of heme proteins. Abstracts of Papers of the American Chemical Society 224, U515-U515 (2002). 14. van Mierlo, C. P. M. & Steensma, E. Protein folding and stability investigated by fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy: the flavodoxin story. Journal of Biotechnology 79, 281-298 (2000). 15. Rozema, D. & Gellman, S. H. Artificial chaperone-assisted refolding of carbonic anhydrase B. Journal of Biological Chemistry 271, 3478-3487 (1996). 16. Rozema, D. & Gellman, S. H. Artificial Chaperones - Protein Refolding Via Sequential Use of Detergent and Cyclodextrin. Journal of the American Chemical Society 117, 2373-2374 (1995). 17. Lyubovitsky, J. G., Gray, H. B. & Winkler, J. R. Mapping the cytochrome c folding landscape. Journal of the American Chemical Society 124, 5481-5485 (2002). 18. Lee, J. C., Gray, H. B. & Winkler, J. R. Cytochrome c ' folding triggered by electron transfer: Fast and slow formation of four-helix bundles. Proceedings of
84 the National Academy of Sciences of the United States of America 98, 7760-7764 (2001). 19. Morar, A. S., Olteanu, A., Young, G. B. & Pielak, G. J. Solvent-induced collapse of alpha-synuclein and acid-denatured cytochrome c. Protein Science 10, 2195- 2199 (2001). 20. Hostetter, D. R., Weatherly, G. T., Beasley, J. R., Bortone, K., Cohen, D. S., Finger, S. A., Hardwidge, P., Kakouras, D. S., Saunders, A. J., Trojak, S. K., Waldner, J. C. & Pielak, G. J. Partially formed native tertiary interactions in the A-state of cytochrome c. Journal of Molecular Biology 289, 639-644 (1999). 21. Anfinsen, C. B., Haber, E., Sela, M. & White, F. H., Jr. The kinetics of formation of native ribonuclease during oxidation of the reduced polypeptide chain. Proceedings of the National Academy of Sciences of the United States of America 47, 1309-14 (1961). 22. Lazaridis, T. & Karplus, M. ''New view'' of protein folding reconciled with the old through multiple unfolding simulations. Science 278, 1928-1931 (1997). 23. Dill, K. A. & Chan, H. S. From Levinthal to pathways to funnels. Nature Structural Biology 4, 10-19 (1997). 24. Schultz, C. P. Illuminating folding intermediates. Nature Structural Biology 7, 7- 10 (2000). 25. Ellis, R. J. Discovery of molecular chaperones. Cell Stress & Chaperones 1, 155- 160 (1996). 26. Agashe, V. R. & Hartl, F. U. Roles of molecular chaperones in cytoplasmic protein folding. Seminars in Cell & Developmental Biology 11, 15-25 (2000). 27. Minton, A. P. Implications of macromolecular crowding for protein assembly. Current Opinion in Structural Biology 10, 34-39 (2000). 28. van den Berg, B., Ellis, R. J. & Dobson, C. M. Effects of macromolecular crowding on protein folding and aggregation. Embo Journal 18, 6927-6933 (1999). 29. Ellis, R. J. Molecular chaperones ten years on - Introduction. Seminars in Cell & Developmental Biology 11, 1-5 (2000). 30. Feldman, D. E. & Frydman, J. Protein folding in vivo: the importance of molecular chaperones. Current Opinion in Structural Biology 10, 26-33 (2000). 31. Cheng, S. & Wei, Y. Mesoporous silica nanospheres synthesized via the nonsurfactant templated sol-gel pathway. Abstracts of Papers of the American Chemical Society 224, U520-U520 (2002). 32. Zheng, J. Y., Pang, J. B., Qiu, K. Y. & Wei, Y. Synthesis of mesoporous silica materials via nonsurfactant templated sol-gel route by using mixture of organic compounds as template. Journal of Sol-Gel Science and Technology 24, 81-88 (2002). 33. Zheng, J. Y., Pang, J. B., Qiu, K. Y. & Wei, Y. Synthesis and characterization of mesoporous titania and silica-titania materials by urea templated sol-gel reactions. Microporous and Mesoporous Materials 49, 189-195 (2001). 34. Pang, J. B., Qiu, K. Y. & Wei, Y. A new nonsurfactant pathway to mesoporous silica materials based on tartaric acid in conjunction with metallic chloride. Chemistry of Materials 13, 2361-2365 (2001).
85 35. Zheng, J. Y., Pang, J. B., Qiu, K. Y. & Wei, Y. Synthesis of mesoporous silica materials with hydroxyacetic acidd derivatives as templates via a sol-gel process. Journal of Inorganic and Organometallic Polymers 10, 103-113 (2000). 36. Pang, J. B., Qiu, K. Y., Xu, J. G., Wei, Y. & Chen, J. Synthesis of mesoporous silica materials via nonsurfactant urea-templated sol-gel reactions. Journal of Inorganic and Organometallic Polymers 10, 39-49 (2000). 37. Pang, J. B., Qiu, K. Y. & Wei, Y. Synthesis of mesoporous silica materials with ascorbic acid as template via sol-gel process. Chinese Journal of Chemistry 18, 693-697 (2000). 38. Wei, Y., Xu, J. G., Dong, H., Dong, J. H., Qiu, K. Y. & Jansen-Varnum, S. A. Preparation and physisorption characterization of D-glucose-templated mesoporous silica sol-gel materials. Chemistry of Materials 11, 2023-2029 (1999). 39. Bushnell, G. W., Louie, G. V. & Brayer, G. D. High-Resolution 3-Dimensional Structure of Horse Heart Cytochrome-C. Journal of Molecular Biology 214, 585- 595 (1990). 40. Scott, R. A., Mauk, A. G. Cytochrome c: A Multidisciplinary Approach University Science Books, Sausalito, Calif. (1996). 41. Tsong, T. Y. Ferricytochrome c chain folding measured by the energy transfer of tryptophan 59 to the heme group. Biochemistry 15, 5467-73 (1976). 42. Tsong, T. Y. Trp-59 (tryptophan-59) fluorescence of ferricytochrome c as a sensitive measure of the over-all protein conformation. Journal of Biological Chemistry 249, 1988-90 (1974). 43. Hagen, S. J., Hofrichter, J. & Eaton, W. A. Rate of Intrachain Diffusion of Unfolded Cytochrome c. J. Phys. Chem. B 101, 2352-2365 (1997). 44. Finazzi-Agro, A. & Avigliano, L. Fluorescence as a spectroscopic probe for the study of proteins. Life Chemistry Reports 2, 97-139 (1984). 45. Cowgill, R. W. Fluorescence and the structure of protein. XX. Fluorescence of 3- aminotyrosine and other aminophenols. Photochemistry and Photobiology 13, 183-94 (1971). 46. Russell, E. C. & Cowgill, R. W. Fluorescence protein structure. XIII. Further effects of side-chain groups. Biochimica et Biophysica Acta 154, 231-3 (1968). 47. Kelly, S. M. & Price, N. C. The application of circular dichroism to studies of protein folding and unfolding. Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology 1338, 161-185 (1997). 48. Woody, R. W. in Biochemical Spectroscopy 34-71 (ACADEMIC PRESS INC, San Diego, 1995). 49. Freskgard, P. O., Martensson, L. G., Jonasson, P., Jonsson, B. H. & Carlsson, U. Assignment of the contribution of the tryptophan residues to the circular- dichroism spectrum of human carbonic-anhydrase .2. Biochemistry 33, 14281- 14288 (1994). 50. Woody, R. W. Contributions of tryptophan side-chains to the far-ultraviolet circular-dichroism of proteins. European Biophysics Journal with Biophysics Letters 23, 253-262 (1994). 51. Chaffotte, A. F., Guillou, Y. & Goldberg, M. E. Kinetic resolution of peptide- bond and side-chain far-Uv circular-dichroism during the folding of hen egg- white lysozyme. Biochemistry 31, 9694-9702 (1992).
86 52. Greenfield, N. J. Methods to estimate the conformation of proteins and polypeptides from circular dichroism data. Analytical Biochemistry 235, 1-10 (1996). 53. Johnson, W. C. Protein secondary structure and circular-dichroism - a practical guide. Proteins-Structure Function and Genetics 7, 205-214 (1990). 54. Yang, J. T., Wu, C. S. & Martinez, H. M. Calculation of protein conformation from circular dichroism. Methods in enzymology 130, 208-69 (1986). 55. Ellerby, L. M., Nishida, C. R., Nishida, F., Yamanaka, S. A., Dunn, B., Valentine, J. S. & Zink, J. I. Encapsulation of proteins in transparent porous silicate-glasses prepared by the sol-gel method. Science 255, 1113-1115 (1992). 56. Avnir, D., Braun, S., Lev, O. & Ottolenghi, M. Enzymes and other proteins entrapped in sol-gel materials. Chemistry of Materials 6, 1605-1614 (1994). 57. Samuni, U., Dantsker, D., Juszczak, L. J., Bettati, S., Ronda, L., Mozzarelli, A. & Friedman, J. M. Spectroscopic and functional characterization of T state hemoglobin conformations encapsulated in silica gels. Biochemistry 43, 13674- 13682 (2004). 58. Das, T. K., Samuni, U., Lin, Y., Goldberg, D. E., Rousseau, D. L. & Friedman, J. M. Distal heme pocket conformers of carbonmonoxy derivatives of Ascaris hemoglobi - Evidence of conformational trapping in porous sol-gel matrices. Journal of Biological Chemistry 279, 10433-10441 (2004). 59. Savini, I., Santucci, R., Di Venere, A., Rosato, N., Strukul, G., Pinna, F. & Avigliano, L. Catalytic and spectroscopic properties of cytochrome-c, horseradish peroxidase, and ascorbate oxidase embedded in a sol-gel silica matrix as a function of gelation time. Applied Biochemistry and Biotechnology 82, 227-241 (1999). 60. Bottini, M., Di Venere, A., Tautz, L., Desideri, A., Lugli, P., Avigliano, L. & Rosato, N. Structural stability of azurin encapsulated in sol-gel glasses: A fluorometric study. Journal of Sol-Gel Science and Technology 30, 205-214 (2004). 61. Brennan, J. D. Using intrinsic fluorescence to investigate proteins entrapped in sol-gel derived materials. Applied Spectroscopy 53, 106A-121A (1999). 62. Tsaprailis, G., Chan, D. W. S. & English, A. M. Conformational states in denaturants of cytochrome c and horseradish peroxidases examined by fluorescence and circular dichroism. Biochemistry 37, 2004-2016 (1998). 63. Yamanaka, S. A., Dunn, B., Valentine, J. S. & Zink, J. I. Nicotinamide adenine- dinucleotide phosphate fluorescence and absorption monitoring of enzymatic- activity in silicate sol-gels for chemical sensing applications. Journal of the American Chemical Society 117, 9095-9096 (1995). 64. Kumar, C. V. & Chaudhari, A. Proteins immobilized at the galleries of layered alpha-zirconium phosphate: Structure and activity studies. Journal of the American Chemical Society 122, 830-837 (2000). 65. Shen, C. Y. & Kostic, N. M. Kinetics of photoinduced electron-transfer reactions within sol-gel silica glass doped with zinc cytochrome c. Study of electrostatic effects in confined liquids. Journal of the American Chemical Society 119, 1304- 1312 (1997).
87 66. Yiu, H. H. P., Botting, C. H., Botting, N. P. & Wright, P. A. Size selective protein adsorption on thiol-functionalised SBA-15 mesoporous molecular sieve. Physical Chemistry Chemical Physics 3, 2983-2985 (2001). 67. Gimon-Kinsel, M. E., Jimenez, V. L., Washmon, L. & Balkus, K. J. in Mesoporous Molecular Sieves 1998 373-380 (ELSEVIER SCIENCE PUBL B V, Amsterdam, 1998). 68. Diaz, J. F. & Balkus, K. J. Enzyme immobilization in MCM-41 molecular sieve. Journal of Molecular Catalysis B-Enzymatic 2, 115-126 (1996). 69. Pang, J. B., Qiu, K. Y., Wei, Y., Lei, X. J. & Liu, Z. F. A facile preparation of transparent and monolithic mesoporous silica materials. Chemical Communications, 477-478 (2000). 70. Wei, Y., Feng, Q. W., Xu, J. G., Dong, H., Qiu, K. Y., Jansen, S. A., Yin, R. & Ong, K. K. Polymethacrylate-silica hybrid nanoporous materials: A bridge between inorganic and polymeric molecular sieves. Advanced Materials 12, 1448- 1450 (2000).
88
Table 2- 1. Pore parameters of water-extracted Ccu series prepared at various urea concentrations
Average pore Micropore data diameter Sample BET Single BET/nma BJH/nmb Surface Pore code surface point area volumec area/m2g-1 pore /m2g-1 /cm3g-1 volume /cm3g-1 Ccu0 530 0.315 2.38 2.56 120 0.067 Ccu15 294 0.249 2.53 2.72 83.8 0.047 Ccu30 524 0.341 2.60 2.61 8.74 0.002 Ccu40 494 0.443 3.58 3.07 ------Ccu50 667 0.610 3.66 3.12 ------
a b The average pore diameters calculated from 4 V/SBET by the BET method. Determined from the maxima of the BJH desorption pore size distribution curves with the Halsey equation. c Values determined from the t-plot analysis.
89
Figure 2- 1 Schematic diagram of a folding energy landscape. Denatured molecules at the top of the funnel might fold to the native state by a myriad of different routes, some of which involve transient intermediates (local energy minima) whereas others involve significant kinetic traps (misfolded states). For proteins that fold without populating intermediates, the surface of the funnel would be smooth.23
90
The detergent-cyclodextrin method in the literature:
Dilute+detergent Cyclodextrin (capture step) (“stripping step”) Protein-detergent Native protein complex (inactive) (+some aggregate)
dilute Urea-denatured protein Aggregate(inactive)
Our new technique of “rigid matrix artificial chaperone” assisted protein folding:
Entrap in Dilute silica matrix Native protein Protein-matrix Urea-denatured protein (+some Complex aggregates
Figure 2- 2 Illustrations of artificial chaperones assisting protein folding.
91
ccu50 ccu40 400 ccu30 350 ccu15 ccu0 STP)
-1 300 g 3
(cm 250
200
150
100 lume Absorbed o V 50
0.0 0.2 0.4 0.6 0.8 1.0
Relative Pressure (P/P0)
Figure 2- 3 N2 adsorption-desorption isotherm at –196°C.
92
ccu50 ccu40 0.12 ccu30 ccu15 0.10 ccu0
0.08 ) -1 A -1
g 0.06 3
0.04 D (cm d
dV/ 0.02
0.00
20 30 40 50 60
Pore Diameter(A)
Figure 2- 4 BJH pore size distributions for the sol-gel material synthesized in the presence of 0-50% wt% of urea.
93
3.5 0.7 3.3
m) 0.6 ) n 3.1 ( -1 g er
2.9 0.5 3 m c
2.7 ( amet i
0.4 e
d 2.5 e r 0.3 lum o 2.3 o v p e e r g 2.1 0.2 o a P
er 1.9 v 0.1 A 1.7 1.5 0 0 204060 wt% template in samples
Figure 2- 5 Relationship between BJH average pore diameter, pore volume and amount of urea template used in the synthesis. For Ccu0 (wt%=0), pore diameter was taken as 1.7 nm.
94
(a)
(b)
(c)
4000 3400 2800 2200 1600 1000 400 Wavenumber (cm-1)
Figure 2- 6 Representative IR spectra of (a) Ccu50 before template extraction; (b) Ccu0 before template extraction; (c) Cc50 after template extraction.
95
7 y t 6 tensi n 5
4 escence i
uor 3
2 ent of fl ffer 1 ve di ti
e 0 l
a ccu0 ccu15 ccu30 ccu40 ccu50 free cc R -1 Sample code
Figure 2- 7 Plot of relative difference, (IU-IT)/IT, in fluorescence intensity between unfolded and refolded Cc for the samples with increasing pore size up to free Cc in solution.
96
A
) 0 ry a arbit (
-50 Intensity
200 220 240 260 280 0 B ) arbitary ( -10 nsity e t n I
200 220 240 260 280 wavelength(nm)
Figure 2- 8 Circular dichroism in far UV range at 25 oC of (A) unfolded cytochrome c (dashed line) in 9M urea and native cytochrome c (solid line) and (B) cytochrome c entrapped in Ccu0 (dashed line) and cytochrome c entrapped in Ccu50 (solid line) after washing out urea. All the curves are normalized by concentrations of Cc in solution or suspension but not convert to mean residue ellipticity because of strong light scattering in suspension.
97
Figure 2- 9 A cartoon presentation of cytochrome c refolding upon removal of urea template/denaturant by water extraction. (a) When pore size is large, Cc refolds to its native state. (b) When pore size is small, Cc cannot refold to its native state. The small dots represent urea molecules.
98 Chapter 3: Using Resonance Raman Spectroscopy to Study Folding Unfolding
Behavior of Encapsulated Heme Proteins in Silica Matrix with Controlled Pore
Sizes
3.1 Introduction
The sol-gel encapsulation process has been widely employed in immobilizing proteins or enzymes in solid matrices to be used as biosensors and biocatalysts.1 With the advantage of maintaining native structure of entrapped proteins, optical clarity and accessibility by small molecules, this method has also been used to study protein reactions and protein-folding process.2,3 Previous studies have proved that silica matrix can slow, impede even prevent protein motion due to the nature of pore structure of silica matrix, demonstrating the potential of using sol-gel as a technique to study protein folding.4-7 However, very little information is available regarding the effect of
confinement on the behavior of entrapped proteins since wet gels were used in all the
above studies,8-10 for which pore parameters are not easily to be obtained. The main
objective of the present study is to use heme protein, such as cytochrome c (Cc),
hemoglobin (Hb) or myoglobin (Mb) as a model system to study the folding behavior of
proteins in pores with different sizes.
3.1.1 Resonance Raman Spectroscopy and Protein Folding
Raman spectroscopy, which is based on Raman effects named after Sir. C. V.
Raman, measures the magnitudes of frequency shifts and peak intensities because of the
99 inelastic scattering of light from matter.11-13 Information on molecular structures and dynamics can be obtained by measuring the frequency shifts as well as changes in electric field orientation of the scatted light relative to that of the incident exciting light.
The vibrational energies of molecules were measured by both infrared (IR) and
Raman spectroscopy measure but the peak intensities observed in these methods obey different selection rules. For a vibrational motion to be IR active, the dipole moment of the molecule must change. For example, the symmetric stretch in carbon dioxide is not IR active because there is not change in the dipole moment. The asymmetric stretch is IR active due to a change in dipole moment.
However for a vibration to be Raman active, the polarizability of the molecule must change with the vibrational motion. So the symmetric stretch in carbon dioxide is
Raman active because the polarizability of the molecule changes. Thus, Raman spectroscopy complements IR spectroscopy.
Usually Raman spectra can be measured by irradiating a sample with a high- intensity light beam with a well-defined frequency and a single linear polarization; the scatted light is collected over some solid angle to determine the frequency, intensity, and polarization of scattered lights.
With the advent of the photomultiplier tube and the He-Ne laser since 1980’s,
Raman spectroscopy has become a major tool for fundamental studies in physics, physical chemistry, analytical chemistry, as well as biological sciences.
Because Raman scattering of water is very weak, Raman spectroscopy has been realized as a powerful method for studying aqueous samples, especially biological samples. T. G. Spiro and T. C. Streka pioneered works in this area by studying the
100 chromophoric hemes of hemoglobin and myoglobin in 1972.14 Since then, much progress
has been made by using this technique.15-22
Among various Raman spectroscopy methods, Resonance Raman spectroscopy
(RR) is one of the most important methods applicable to bioscience. The method is based on resonance Raman effects, which can be described as follows. Some particular excitation frequencies are natural frequencies of oscillation of specific electron oscillators of the molecular electron cloud. These frequencies correspond to the molecular electronic absorption band frequencies. Excitation at these frequencies is said to be in “resonance” with the electronic transition; therefore, the Raman scattering is called to be “resonance
Raman” scattering. This resonance excitation results in an increased oscillating charge displacement and a corresponding increase in the induced dipole moment; this, in turn, results in an increased scattering efficiency for Raman scattering. The enhancement factor of resonance Raman scattering compared with that of normal Raman scattering can be as high as 108.13 As the result, the selectivity can be greatly enhanced in resonance
Raman spectroscopy.
Different information can be obtained by using excitations at different ranges of
wavelength when studying protein structure by RR spectroscopy. Absorption in the near-
UV and visible spectral regions derives solely from the heme group. When excited at
600nm, RR scattering occurs within a heme band that contains charge transfer transitions
involving the metal and its ligand.23 Excitation within the heme 400 nm Soret band transition enhances only heme in-plane ring vibrations. Though in this range, RR spectroscopy does not provide information about the three-dimensional architecture of the protein, it can give deep insight into the structure of the chromophore and its
101 immediate environment down to a molecular level, which may be beyond the resolution
the x-ray crystallography or NMR spectroscopy.24
The absorption between 270 and 300 nm is contributed mainly by tryptophan and tyrosine. But much greater UV-Raman enhancement occurs for excitation with the strong tryptophan and tyrosine absorption band between 220 and 230 nm, and the spectra are easily measured with very high signal to noise ratio.12
In this project, we used the Soret band (410 nm) as the excitation wavelength.
3.1.2 Heme Protein
Heme proteins owe their name due to their functional group, which consists of a porphyrin molecule with a central iron atom (the heme). The iron atom is attached to the porphyrin via four bonds to the central nitrogen atoms and uses its fifth binding position to establish a link to the protein via the N-atom of a histidine molecule situated below the porphyrin plane (Figure 3-1). The sixth coordination shell of the iron is available for ligand binding.
The most commonly known heme proteins are hemoglobin (four hemes) which binds and transports oxygen in blood, myoglobin (one heme) with the same function in the muscles of vertebrates and cytochrome c (one heme), an essential component of the mitochondrial respiratory chain.
3.1.2.1 Cytochrome c and its Resonance Raman Spectroscopy
The folding process of cytochrome c (Cc), a small single-domain protein, was studied in this project. The three dimensional structure of Cc has been well determined.24
102 The protoporphyrin IX prosthetic heme group, located in the center of the protein, is
covalently bound to the polypeptide backbone by Cys14 and Cys17. The heme iron is
coordinated by His18 and Met80 in the native form. Under unfolding conditions His26 or
His33 may coordinate to the heme in place of Met80. The single tryptophan with
fluorescence activity is at position 59.25
Because RR spectroscopy was the major analytical method in this project instead
of fluorescence and CD spectroscopy, the assignment of peaks is a prerequisite for
extracting structural information form the RR spectra of cytochrome c.26-28 Since the
porphyrin chromophore, which is the main part of the heme belongs to D4h symmetry, normal mode analysis calculations have been carried out and supported by a large amount of experimental data.29-37 But all naturally occurring porpyrins including heme in
Cc have an effective symmetry lower than D4h because of various effects imposed by surrounding polypeptide chain, such as asymmetric substitution of the vinyl groups by thioether bridges24, deformations of planar structure through ruffling or doming of the
porphyrin etc.35-37
3.1.2.1.1 Marker Band Region
The bands in the high-frequency range (1300-1700 cm-1) originate from modes, which include predominantly C-C and C-N stretching vibrations of the porphyrin.29
These modes are particularly useful since they are sensitive to both the axial coordination and spin state of the iron at the center of the heme.25 Based on previous literature,38-40 four folding intermediates of ferric Cc with differing heme coordination states were identified which were shown on Figure 3-2: (1) the native form (HM) in which His18 and
Met80 are the axial ligands, (2) a bis-histidine form (HH) where Met80 is replaced by
103 His26 or His33, (3) a histidine-water form (HW) where Met80 is replaced by a water
molecule, and (4) a five coordinated form (5C) where both the Met80- and His18- iron
bonds are broken and, instead, a water molecule is ligated to the heme as the single axial
ligand.
3.1.2.1.2 Fingerprint Region
The bands between 300 and 800 cm-1 originate from modes which include considerable contributions from vibrations involving the peripheral substituents of the porphyrin.29,31,35-37 Hence they are very sensitive to the polypeptide conformation near the
heme.28,40,41 In the native protein, the heme is highly ruffled42 due to the tension exerted on it by the surrounding polypeptide. As the result, the low-frequency spectra of heme displays rich resonance Raman spectra, congested with heme-peripheral substituent modes, porphyrin out-of-plane modes and so on. When the protein is unfolded by strong denaturants, the heme adopts a planar structure due to the relaxation of the tertiary
structure associated with the disruption of the heme-ligand interactions. Consequently,
the spectrum becomes rather simple with only a few features. Among these many
vibrational modes, the Raman lines at 397 and 394 cm-1 in the ferric and ferrous states, respectively, are especially useful, and can be used as marker lines for the formation of the native tertiary structure, since they involve motion of the covalent linkage between the heme and the polypeptide. The lines have been assigned as bending modes involving the sulfur atom of Cys14/17, the β-carbon atom of the heme, and the atom bridging these two.28
3.1.2.2 Hemoglobin (Hb) and Its Resonance Raman Spectra
104 Hemoglobin is the protein that carries oxygen from the lungs to the tissues and
carries carbon dioxide from the tissues back to the lungs. In order to function most
efficiently, hemoglobin needs to bind to oxygen tightly in the oxygen-rich atmosphere of
the lungs and be able to release oxygen rapidly in the relatively oxygen-poor environment
of the tissues. It does this in a most elegant and intricately coordinated way. The story of
hemoglobin is the prototype example of the relationship between structure and function
of a protein molecule.43
Hemoglobin is a tetramer composed of 4 globin molecules; 2 alpha globins and 2 beta globins. The alpha globin chain is composed of 141 amino acids and the beta globin chain is composed of 146 amino acids.44 Both alpha and beta globin proteins share similar secondary and tertiary structures, each with 8 helical segments (labeled helix A-
G). Each globin chain also contains one heme molecule. The heme molecule is composed of a porphyrin ring, which contains 4 pyrrole molecules cyclically linked together, and an iron ion ligand bound in the center. The heme molecule is located between helix E and helix F of the globin protein. The alpha and beta subunits of the globin chains exist in two
dimers which are bonded together strongly.
Since most of the previous research focused on hemoglobin as tetramer, we tried
to study the folding-unfolding behaviors of subunits of hemoglobin in this work. So the
tetramer hemoglobin was separated first before encapsulated in silica matrix with
controlled pores. In this way, the subunits were isolated from each other and could not
recombine. Then the silica matrix with encapsulated Hb could be placed in different
environment to examine the folding unfolding process of Hb subunit by RR spectroscopy.
3.1.2.3 Myoglobin (Mb) and Its Resonance Raman Spectra
105 Myoglobin and hemoglobin are heme proteins whose physiological importance is
principally related to their ability to bind molecular oxygen. Unlike hemoglobin,
myoglobin is a monomeric heme protein found mainly in muscle tissue, where it serves
as an intracellular storage site for oxygen. During periods of oxygen deprivation
oxymyoglobin releases its bound oxygen, which is then used for metabolic purposes.45
The tertiary structure of myoglobin is that of a typical water soluble globular
protein. Its secondary structure is unusual in that it contains a very high proportion (75%)
of a-helical secondary structure. A myoglobin polypeptide comprises of eight separate
right handed α-helices, designated A through H, which are connected by short non-helical regions. Amino acid R-groups packed into the interior of the molecule are predominantly hydrophobic in character, while those exposed on the surface of the molecule are generally hydrophilic, thus making the molecule relatively water soluble. 46
Each myoglobin molecule contains one heme prosthetic group inserted into a
hydrophobic cleft in the protein. Each heme residue contains one central coordinately
bound iron atom that is normally in the Fe2+, or ferrous, oxidation state. The oxygen carried by heme proteins is bound directly to the ferrous iron atom of the heme prosthetic group. Oxidation of the iron to the Fe3+, ferric, oxidation state renders the molecule incapable of normal oxygen binding. Hydrophobic interactions between the tetrapyrrole ring and hydrophobic amino acid R groups on the interior of the cleft in the protein strongly stabilize the heme protein conjugate. In addition, a nitrogen atom from a histidine R group located above the plane of the heme ring is coordinated with the iron atom further stabilizing the interaction between the heme and the protein. In
106 oxymyoglobin the remaining bonding site on the iron atom (the 6th coordinate position)
is occupied by the oxygen, whose binding is stabilized by a second histidine residue.47-49
Carbon monoxide also binds coordinately to heme iron atoms in a manner similar
to that of oxygen, but the binding of carbon monoxide to heme is much stronger than that
of oxygen. The preferential binding of carbon monoxide to heme iron is largely
responsible for the asphyxiation that results from carbon monoxide poisoning.46
J. M. Friedman and his colleagues50 has showed that acid induced cleavage of the
iron proximal histidine (Fe-His) in deoxymyoglobin (deoxyMb) can be retarded by sol-
gel encapsulation. Acid unfolding of deoxyMb in solution51-56 has been shown to proceed through two intermediates both of which exhibit a loss of the Fe-His bond. Between pH
4.5 and 3.5, intermediate I’ is formed. In this intermediate, the heme remains five-
coordinate, but water replaces the proximal histidine as the fifth ligand. Between pH 3.5
and 2.5, a second spectroscopically distinguishable intermediate, U’, is formed. In this
intermediate, the heme becomes four-coordinate but appears to remain within the heme
pocket. At the low end of this pH regime, further unfolding and associated heme losses
become the dominant processes.
But in his work a soft hydro silica gel without further aging was used to
encapsulate deoxyMb, therefore the porous structure of silica matrix can not be precisely obtained, so the effect of space size on deoxymyoglobin unfolding is still unknown. In this project, we tried to use our nonsurfactant sol-gel technique to encapsulate deoxyMb in silica matrix with controlled pores to repeat J. M. Friedman’s work to further investigate the extent to which confined space imposed stability influences the acid- induced formation of the deoxyMb unfolding intermediates.
107 In resonance Raman studies of various myoglobins (Mb) and hemoglobins (Hb),
the vibrational stretching frequency of the Fe- His bond (νFe-His) is seen in the 200-230
cm-1 region.57,58 So, the shift and intensity changes of this band would be used as an
indication of unfolding intermediate of deoxyMb in this project.
3.1.2.4 Protein Encapsulation
The novel nonsurfactant templated sol-gel method was used to encapsulate heme
proteins in silica matrix with controlled pore size.59-62 Generally, inorganic precursors such as tetraethyl orthosilicate (TEOS) or tetramethyl orthosilicate (TMOS) are first hydrolyzed with hydrochloric acid as catalyst followed by mixing with nonsurfactant molecules solution. Upon gelation and drying, the transparent template-silica composites can be obtained. At this time, they are still nonporous, with removal of template by water extraction, the porosity of the materials increases, leading to the final mesoporous materials after complete removal of the templates.
The porosity of the materials can be adjustable to a certain extent. Generally, the
values of these porous parameters increase with the template weight percentage. When
the weight percentage is between 30% and 50%, mesoporosity can be achieved.
One of the most important advantages of this technique is its biocompatibility.
The whole synthesis and encapsulation process can be readily accomplished at room
temperature and near neutral pH. Nonsurfactants such as sugar compounds are highly
biocompatible. Furthermore unlike surfactant molecules, which are tightly adhered to
silica matrix and difficult to remove, those nonsurfactant molecules can be easily
removed out by room temperature water extraction avoiding calcinations at high
temperature. These enabled us to develop a novel biotechnology: one-step direct
108 encapsulation of bioactive substances, such as proteins, polypeptides, cells, etc., inside
nanoporous matrices. Adjusting pH to desired value and partial removal of organic
byproducts (e. g. methanol or ethanol) can help the encapsulated bioactive substances to
maintain their bioactivities.
3.2 Experimental
The encapsulation of heme proteins in silica matrix with controlled pore size was
achieved by nonsurfactant templated sol-gel process which was developed by our group.
In this work, urea, fructose or glucose was used as both template molecules. The
characterization of silica matrix was carried by BET test and the investigation of heme
protein was done mainly through resonance Raman spectra.
3.2.1 Materials
Tetramethyl orthosilicate (TMOS, 98%, Aldrich), urea (99%, Sigma), D-(+)- glucose (ACS reagent, Batch # 014K0118, Sigma), D-(-)-fructose (SigmaUltra, Lot
47H03842, Sigma), cytochrome c (from horse heart, 95%, Sigma), myoglobin ( from horse skeletal muscle, 95-100%, lot 013K7020, Sigma), hemoglobin (from human adults, from Princeton University), sodium phosphate (monobasic anhydrous, minimum 99.0%,
Sigma), sodium phosphate dibasic heptahydrate (minimum 99.0%, Sigma), hydrochloric acid (HCl, Fisher), sodium chloride (NaCl, Sigma), sodium dithionite (Na2S2O4, Sigma), were all used as received without further purification.
3.2.2 Encapsulation of Cc into Silica Matrix by Using Urea as Template
Silica sol doped with cytochrome c and template, urea, was made by using the standard protocol. Typically, 3.1 g TMOS (Aldrich, 95%), 0.7 g deionized H2O and 0.03
109 ml HCl solution (40 mM) were mixed at room temperature for 1-2 h under stirring. Upon
mixing, the organic phase (TMOS) and inorganic phase (H2O) separated, so the mixture
was translucent at that time. After stirring vigorously for less than 5 minutes, a lot of heat
was generated during the exothermic hydrolysis reactions and the whole solution would
turn suddenly transparent. Then the mixture was cooled to 0 oC in an ice-water bath
followed by addition of appropriate amount of 9 M urea solution containing 10 mg of
horse heart cytochrome c. Amounts of 9 M urea were designed to yield 0-50% by weight
of templates in the final dry gel products (sample code: Ccu plus a number denoting
percentage of urea). The reaction was then sealed with a parafilm. Upon gelation of the
system within a few hours at room temperature, 12-15 holes were pinned in the parafilm
with a hypodermic syringe needle to allow for the evaporation of solvents and reaction
by-products. After 24 h, the system was placed in a vacuum oven and dried to reach a
constant weight at room temperature in about 6 days. Thus the silica biogel samples
containing urea and Cc were obtained as deep red, transparent, dry, brittle, glassy
monoliths. Then the samples were grinded into fine powders by hand with porcelain
pestle and mortar and kept in sealed vials at –15 oC. For each sample, the grinding time was maintained at around five minutes in order to keep the silica powders in uniform size.
According to SEM picture taken afterward, the average size of particles were from several micrometer to 20 micrometers.
3.2.3 Encapsulation of Cc into Silica Matrix by Using Glucose as Template
This encapsulation procedure was almost same to the procedure in section 3.2.2 except glucose was used as template instead of urea. Instead of dissolving Cc in 9M urea solution, it was dissolved in phosphate buffer (0.01M pH 7.0) at concentration of 1
110 mg/ml. Template glucose was added into the sol in the form of 50 wt% glucose solution
in deionized distillated water. The sample codes were correspondingly assigned as Ccg
plus a number denoting the percentage of glucose. Because glucose is biocompatible, Cc
can be encapsulated in silica matrices in its native state.
In this experiment, we tried to investigate Cc unfolding and refolding behavior in
the confined space.
3.2.4 Encapsulation of Hb into Silica Matrix by Using Glucose as Template
The encapsulation of Hb in silica matrix was achieved by following the standard
protocol with some modifications. The Hb sample was received from Prof. Thomas G
Spiro’s group in Princeton University. According to Dr. Gurusamy Balakrishnan who
prepared the sample, the concentration of Hb tetramer is 0.6 mM (2.4 mM as monomer)
and the buffer solution is 0.01M phosphate buffer (pH 7.4). As received, the solution
sample was kept frozen at -70 oC. Upon encapsulation, the frozen sample was thawed in ice-water mixture to avoid thermal denaturing of protein during the melting process. This
Hb sample from human adults was tetramer, which was made of four subunits, in order to study the single subunit encapsulated in silica matrix the tetramer Hb should be dissociated to monomer in the first step. Since high concentrated NaCl solution can lead to the dissociation of Hb tetramer, NaCl crystals were added into Hb solution to let the final concentration of NaCl to 1.0 M. After incubation at 0 oC for two hours, the Hb solution was ready for encapsulation.
The encapsulation procedure was done as a standard protocol. Typically, 3.1 g
TMOS, 0.7 g deionized H2O and 0.03 ml HCl solution (40 mM) were mixed at room temperature for 1-2 h under stirring. Upon mixing, the organic phase (TMOS) and
111 inorganic phase (H2O) was separated, so it looked translucent. After stirring vigorously for less than 5 minutes, a lot of heat was generated during the hydrolysis reactions and the whole solution would turn to transparent suddenly. Then the mixture was cooled to 0
oC in an ice-water bath. Appropriate amount of 50 wt% glucose solution was then added
into the sol with 1.5 ml prepared Hb solution (1.0 M NaCl, 0.6 M tetramer, .01M
phosphate buffer, pH 7.4). Amounts of 50 wt% glucose solution were designed to yield
0-50% by weight of templates in the final dry gel products (sample code: Hbg plus a number denoting percentage of glucose). The reaction was then sealed in a glass vial with a parafilm. Upon gelation of the system within a few hours at room temperature, 12-15 holes were pinned in the parafilm with a hypodermic syringe needle to allow for the evaporation of solvents and reaction by-products. After 24 h, the system was placed in a vacuum oven and dried to reach a constant weight at room temperature in about 6 days.
Thus, the silica biogel samples containing glucose and Hb were obtained as deep red, transparent, dry, brittle, glassy monoliths. Then, the samples were grinded into fine powders by hand with conventional porcelain pestle and mortar and kept in sealed vials in a –15 oC freezer. For each sample, the grinding time was maintained at around five minutes in order to keep the silica powders in uniform size. According to SEM picture taken afterward, the average size of particles were from several to 20 micrometers.
3.2.5 Encapsulation of DeoxyMb into Silica Matrix by Using Glucose as Template
To prepare 7.5 mg/ml Mb solution, appropriate amount of Mb powder as received was dissolved in phosphate buffer (0.01M, pH 7.4). DeoxyMb samples were prepared from nitrogen-purged solutions of Mb to which a slight excess of sodium dithionite
112 (solution) was added. All solutions and gels were prepared in and stored under anaerobic
conditions.
Same with the encapsulation of Hb, the encapsulation of deoxyMb also followed
the standard protocol. 3.1 g TMOS, 0.7 g deionized H2O and 0.03 ml HCl solution (40 mM) were mixed at room temperature for 1-2 h under stirring. Then the mixture was cooled to 0 oC in an ice-water bath. Appropriate amount of 50 wt% glucose solution was then added into the sol with 2.0 ml deoxyMb solution. Amounts of 50 wt% glucose solution were designed to yield 0-60% by weight of templates in the final dry gel products (sample code: deoxyMb plus a number denoting percentage of fructose). The reaction was then sealed with a parafilm. Upon gelation of the system within a few hours at room temperature, 12-15 holes were pinned in the parafilm with a hypodermic syringe needle to allow for the evaporation of solvents and reaction by-products. After 24 h, the system was placed in a vacuum oven and dried to reach a constant weight at room temperature in about 6 days. Thus the silica biogel samples containing fructose and deoxyMb were obtained as deep red, transparent, dry, brittle, glassy monoliths. Then the samples were grinded into fine powders by hands with conventional porcelain pestle and
mortar and kept in sealed vials in a –15 oC freezer. For each sample, the grinding time was maintained at around five minutes in order to keep the silica powders in uniform size.
3.2.6 Resonance Raman Spectroscopy
Resonance Raman spectra were acquired on Chromex single monochromator. The excitation source was a krypton ion laser at 413 nm. Samples contained in a 5-mm NMR tube were kept spinning to avoid local heating during laser illumination with the power of
10mW. Suspensions of biogel silica powder were used as samples in the whole project.
113 The RR spectrum of unfolded encapsulated Cc in Ccu series were obtained
immersing in 9M urea solution with template urea inside. Then the biogel silica powders
were separated from urea solution by centrifuging and washed by phosphate buffer
(0.01M, pH7.4). The detailed washing procedure was same to the description in chapter 2,
section 2.2.3. After removal of templates, those biogel silica powders were immersed
again in phosphate buffer (0.01M, pH7.4). At this time, the encapsulated unfolded Cc
was in a neutral environment and might refold back to its native state. Then another set of
RR spectrum of refolded Cc was taken. In addition, RR spectrum of refolded Cc in
elevated temperature and low pH environments were also recorded.
For the encapsulated native Cc samples, Ccg series, three sets of RR spectrum of
them were taken: native state when template glocuse was washed out and the samples
were incubated in buffer solution, unfolded state when they were immersed in 9M urea
solution and refolded state when they were separated from urea and reincubated in buffer
solution. All the separation processes were done by centrifuging.
Hbg and deoxyMb series samples were already sent to Princeton University, but
the results haven’t come out yet.
3.2.7 Characterizations of Nanoporous Silica Matrix
The characterization of the nanoporous structure parameters of the silica matrix
was carried out with N2 on a Micromeritics 2010 system (Micromeritics, Inc., Norcross,
GA) after the removal of template by water extraction. Prior to the measurements, the samples were degassed at 100 oC under vacuum for 6-7 h. For each measurement, it took
about 0.1-0.2 gram sample.
3.2.8 Fourier Transform Infrared Spectroscopy (FTIR) of Silica Matrix
114 FTIR was used to investigate the removal of template in silica matrix. FTIR spectrums of Ccu and Ccg series samples were taken before and after extraction of templates. To prepare the powder sample after water extraction, the powder samples were put into high vacuum oven in room temperature after washing for 5 days. After vacuuming for 2-3 days, the wetted powder became totally dry which could be verified by weighing.
3.3 Results and Discussion
Heme protein cytochrome c was encapsulated in silica matrix with controlled pore size. Resonance Raman spectroscopy was used to reveal the folding unfolding behaviors of encapsulated Cc in confined space. It was found that the size of pores, in which protein molecules are immobilized, had great effects on the folding unfolding process.
This conclusion was also supported by pH and temperature changing experiments.
3.3.1 Characterization of Silica Matrix
The characterization of the porous silica matrix was carried out on a
Micromeritics 2010 system (Norcross, GA) after the removal of templates by water extraction.
3.3.1.1 Ccg series
Figure 3-3 shows a representative set of N2 adsorption-desorption isotherms at -
196 ºC for the biogels after the removal of glucose. The isotherms evolved from Type I, typical of microporous materials (e.g., Ccg0), to Type IV with Type H2 hysteresis loops, typical of mesoporous materials (e.g., Ccg50) with the increase of glucose content. The
BJH pore size distribution of the biogels were obtained by plotting the differential
115 volume of the desorption branch of N2 sorption isotherms as a function of a pore size as
shown in Figure 3-4. The pore diameter tends to increase with weight percentage of
glucose. When glucose accounts for 40-60 wt%, the materials possess narrowly
distributed pore sizes centered at 33.2-44.5 Å with width at half peak height of about 5 Å.
Previous researches suggested that the aggregates of non-surfactant molecules instead of individual molecules might be responsible for the mesophase formation.62 As the
sequence, with the increasing of templates amount from 0 wt% to 60 wt%, the extent of
aggregation of template molecules form larger pores.
3.3.1.2 Ccu series
The analysis of porous structure of Ccu series have already been done in chapter 2.
In fact, there was not much difference in porous structure between Ccu and Ccg series.
This phenomenon indicated the mechanisms of pore forming by urea and glucose were
almost same and the size of pore depended mostly on weight percentage of templates in
final products.
Figure 3-5 shows a representative set of N2 adsorption-desorption isotherms at –
196 ºC for the biogels after the removal of urea. As the urea content is increased, the
isotherm evolved from Type I, typical of microporous materials (e.g., Ccu0), to Type IV
with Type H2 hysteresis loops, typical of mesoporous materials (e.g., Ccu50). The BJH
pore size distributions of the biogels were obtained by plotting the differential volume of
the desorption branch of N2 sorption isotherms as a function of a pore size as shown in
Figure 3-6. The pore diameter tends to increase with urea concentration. When urea accounts for 40-50 wt%, the materials possess narrowly distributed pore sizes centered at
33.2-34.2 Å with width at half peak height of about 5 Å.
116 3.3.2 Resonance Raman Spectroscopy of Ccu series
Figure 3-7 shows RR spectrums of Ccu series and free cytochrome c in phosphate
buffer with pH 7.4. It is known that tertiary structure of heme protein can be probed by
the low frequency region of resonance Raman spectrum (300-800 cm-1), which is very
sensitive to the polypeptide conformation near heme.25 In general, the complicacy of
spectrum in this region indicates folding extent of heme protein because the heme
distortion imposed by surrounding peptide chain.63 So the more complicated the spectrum is, the more native structure the protein remains. Compare to RR spectra of native free Cc on the bottom, we can see the tendency of becoming simplicity with amount of template going down, which means after extraction of urea, proteins entrapped in large pores can refold back more than those in small pores. In order to demonstrate this tendency clearly, we use peaks at 397 cm-1 and 567 cm-1 as markers of tertiary structure to do the plot of
intensity over host pore diameter. Peak 397 cm-1 can be used as marker line for formation of native tertiary structure, since it is a mode involving motion of the thioether linkage between the heme and the polypeptide.25 Another mode that is suitable for a marker of
-1 formation of tertiary structure is at 567 cm . This band is assigned to γ21 mode and is known to disappear upon unfolding of the protein when the heme geometry is relaxed.63
In order to remove the effect of concentration of cytochrome c solution on intensity of
RR spectra, we used peak at 418 cm-1 as internal standard, which is assigned to vibration
63 mode δ(CβCαCd)2,4 that is not relevant to changing of tertiary structure. According to
Figure 3-8 and Figure 3-9, intensity of those two peaks increases with pore size following a nearly linear relationship indicating more tertiary structure were remained for proteins in large pores than in small pores.
117 Among five samples in this series, we picked two extreme situations, Ccu0 with
smallest pore size and Ccu50 with the largest size for further stability studies. As the first
step, two samples were immersed in 9M urea for 24 hours followed by washing with
buffer at pH 7 several times over two days with the purposes of removing urea and giving
the entrapped proteins a chance to refold back. As the second step, resonance Raman
spectrums were taken when Ccu0 and Ccu50 were incubated in different pH buffers and
temperatures. The results are shown in Figure 3-10 and Figure 3-11. As shown in these
graphs, peak at 397cm-1 clearly seen for Ccu50 in pH 7 buffer disappeared in pH 3.5 buffer. On the contrary, this peak could not be found for Ccu0 in either pH 7 or pH 3.5 buffer. The same phenomenon can also be found in high temperature denaturation. When temperature increases from 25 oC to the denature temperature, 70 oC, the peak 397cm-1 degenerates from a single peak to a shoulder peak at 60 oC and finally disappeared at 70
oC for Ccu50. But for Ccu0 this peak remained undetectable regardless of temperature changing.
To explain those results, the structure of silica matrix should be known. Instead of pores with uniform size, the silica matrix we prepared consisted of interconnected channels pores with a narrow distribution of pore size. Several functional groups, siloxane (Si-O-Si), silaketone (Si=O), silanol (Si-OH) and siloxide (Si-O-) exist on the inner surface of those pores, to give a net negative charge of about 6- at pH7.0. On the other hand, cytochrome c bears net charge of 7+ at pH7.0.64 As the result, there should be electrostatic interaction between the protein and silica wall and this interaction will become weaker and weaker with increasing of distance between them. In the small pores, due to the limited space between unfolded protein and inner surface of silica pore, the
118 electrostatic interaction should be strong, therefore, even after urea was washed away,
this interaction and limited space can prevent the unfolded peptide chain to refold,
especially when the peptide chain extends into those pore channels, which connect silica
pores. That is why in Ccu0 sample, protein was shown in its unfolded state even in mild environment. On the other hand, in the large pores, ample space between proteins and silica surface leads to decrease of the electrostatic interaction since the electrostatic force is inversely proportional to distance square, according to Coulomb’s law. Consequently, the unfolded cytochrome c can easily refold because of smaller interactions with silica surface due to large enough space. However, even though the interaction is much weaker than in a small pore, it is still finite to prevent Cc refolding to its natural state. So in the figure, we can see that the two marker peaks recover to a smaller degree than that of the native state. The Cc50 sample at pH 7.4 buffer and room temperature could be in some sort of intermediate states, the details of which remain to be determined.
3.3.3 Resonance Raman Spectroscopy of Ccg series
In the first step, resonance Raman spectra of this series samples were taken in neutral buffer solution after removing templates. The results were shown in Figure 3-12.
Since high frequency region in RR spectra provides information about axial coordination, spin state of the iron at the center of the heme and polypeptide conformation near heme,25,28,41 the folding states of encapsulated Cc can be assigned. As shown in Figure 3-
12, the intensity of peaks at 1502 cm-1 and 1635 cm-1decreased from Ccg60 to Ccg15, on the contrary, the intensity of peak 1493 cm-1 increased. Meanwhile, the peak shifted from
1584 cm-1 for Ccg60 to 1580 cm-1 for Ccg15. According to deconvoluted resonance
Raman spectra of heme coordinated forms of cyt c in litrature,25 we found that Ccg60 was
119 in the native coordination form (HM) in which His18 and Met80 are the axial ligands,
and Ccg15 was clearly in a five coordination form (5C) where both the Met80- and
His18- iron bonds were broken instead a water molecules is ligated the heme as the single
axial ligand. In the low frequency region where the tertiary intrachain interactions can be
probed, we could see the same tendency from Ccg60 to Ccg15. For example, band at 397
cm-1 can be used as a maker line for the formation of native tertiary structure because it
reflects motion of the covalent linkage between the heme and the polypeptides. Though
we could still see the peak 397 cm-1 at Ccg15 sample, the intensity clearly showed the trend of decreasing from Ccg60 to Ccg15. As described above, a conclusion might be drawn that Cc entrapped in large pores remained in native structure but Cc entrapped in small pores might be denatured.
Then all the samples were immersed in 9M urea overnight followed by taking resonance Raman spectra again. This time, as shown in Figure 3-13, no difference was observed among the series of samples. Obviously, Cc was refolded by highly concentrated denaturant urea solution at this time. In high frequency region, all the samples were at 5C state with the characteristic peak at 1493cm-1 and the shifts of band at
1584 cm-1 and 1636 cm-1 towards low frequency, especially for large porous samples
since in small porous samples Cc were already at 5C state even before denatured by urea.
The same trend can be observed from low frequency region: the peak at 397 cm-1 disappeared from all the samples and the intensity of peak 567 cm-1 also decreased in 9M
urea.
In the last step, all samples were removed from 9M urea solution and kept in
phosphate buffer solution (0.01 M, pH 7.4) over night at 4 oC. Then resonance Raman
120 spectra were taken to monitor the refolding of encapsulated Cc. As shown in Fig 3-14,
the characteristic peak of native HM state did not show for all the samples except for
Ccg60, though only a shoulder peak. On the contrary, the peak, which characterizes in 5C
unfolded state at 1495 cm-1, exhibited a trend of increasing intensity from Ccg60 to
Ccg15. Another band of HM native state at 1584 cm-1 showed a movement towards low frequency from large porous sample to small porous sample and this movement was believed to indicate the transition from HM state to 5C state. In the low frequency region, peak at 397 cm-1 showed a trend of intensity decreasing from Ccg50 to Ccg15 which indicate more encapsulated Cc refolded back in large pores than in small pores.
As discussed before, since the peak at 1503 cm-1 can be seen as a characteristic peak of HM native state while peak at 1494 cm-1 as the indicator of 5C nonnative state, so the ratio of intensity of the two peaks can be considered as indication of population of
Cc in native state among all encapsulated Cc. In Figure 3-15, the ratios of the series samples in different conditions were shown. We can see at the beginning when all samples were immersed in neutral buffer solution, the ratios increased from Ccg15 to
Ccg60, which meant more Cc in native state in large pores than in small pores; when immersed in denaturant solution, the ratios of all samples were almost in a same level where was much lower than in water, at this moment, encapsulated Cc in all samples were in nonnative state; after urea was washed out and samples were kept in water overnight to let Cc to refold back, the ratios showed the increasing trend form Ccg15 to
Ccg60 while they were all lower than before immersed in 9M urea, that meant more Cc could refold back in large pores than in small pores but even in the largest porous sample, not all encapsulated Cc could refold back.
121 To explain those phenomena, the structure of silica matrix should be discussed first. From pore size distribution curve, we found the pore size was not uniform and the pore diameter of those matrix measured by BET was only an average value, which can be considered as the pore size that the majority pores had. Besides those pores with average size, there were still some pores with smaller or larger size. Meanwhile those pores were connected with each other by small channels which can be seen from TEM picture. So even most of Cc were encapsulated in pores with average sizes, some Cc may be entrapped in pores whose sizes were larger or smaller than average value.
During hydrolysis process, by-products like MeOH, can serve as a denaturant to
Cc, furthermore, the pH value of sol was around 2-3, even when Cc was dissolved in buffer solution, the mixture of sol and Cc solution might still be in acidic condition.
Consider in these effects, most Cc may already be in certain nonnative state when encapsulated in silica matrix. But after the samples were placed in buffer, encapsulated
Cc in large pores has enough space to refold back, on the contrary, due to confined space, encapsulated Cc in small pore even in native condition could not fold back. That is the reason why the ratio of native Cc to denatured Cc in Ccg15 was lower than in Ccg60.
After immersing in urea, encapsulated Cc in large porous samples could unfold without any hindrance and even in small porous sample like Ccg15 a little amount of Cc might reside in pores with above average sizes and could be unfolded. That might explain why even in Ccg15, the peak intensity ratio in urea were lower than in water too. After giving encapsulated Cc a chance to refold by replacing the samples in natural environment, Cc in large pores had more opportunities to fold back than in small pores. But since there were still some below average sized pores and channels inside large porous material, the
122 unfolded Cc may extended its chains to small pores and channels then lost the ability to refold back. That may be the reason why the refolded ratios were all lower than the first time in water.
3.4 Conclusions
In summary, the encapsulation of heme protein in silica matrix with controlled pore size has enabled us to study folding unfolding behaviors of protein in confined space.
Three kinds of heme proteins were used in this project, cytochrome c, hemoglobin and myoglobin.
The study of encapsulated Cc was performed in two approaches: encapsulation in unfolded state and folded state. Then by changing of environments for encapsulated Cc, the effect of cavity size on protein folding, unfolding could be studied. We found that the unfolded proteins showed different abilities of refolding according to the pore size where they were encapsulated; the refolding extent of Cc in large pores was larger than in smaller pores, which can be interpreted by interaction between protein and silica pore inner surface. The same conclusion could also be drawn from encapsulation of folded protein: small pores could prevent protein’s folding unfolding motion more effectively than in large pores.
In this work, resonance Raman spectroscopy was used as the major analytical method. It proved to be very effective, especially for the encapsulated Cc. There are several reasons: first, the sensitivity RR spectroscopy is high enough to detect the low concentration of encapsulated Cc in suspension; second, in RR spectrum the light scattering of silica powder could be easily identified and removed.
123 Our results demonstrate that this new method of protein encapsulation in silica matrix with controlled pore size has the potential to trap intermediate states of protein refolding process, thus may be useful in promoting our understanding of protein folding and unfolding processes.
3.5 Acknowledgements
I want to thank Dr. Tom G. Spiro and Dr. Gurusamy Balakrishnan at Department of Physics of Princeton University for their help and valuable discussions with resonance
Raman spectroscopy. I also want to thank Dr. Jian-Min Yuan of Department of Physics at
Drexel University for his assistance with resonance Raman spectroscopy analysis.
3.6 References
1. Smith, K., Silvernail, N. J., Rodgers, K. R., Elgren, T. E., Castro, M. & Parker, R. M. Sol-gel encapsulated horseradish peroxidase: A catalytic material for peroxidation. Journal of the American Chemical Society 124, 4247-4252 (2002). 2. Ray, A., Friedman, B. A. & Friedman, J. M. Trehalose glass-facilitated thermal reduction of metmyoglobin and methemoglobin. Journal of the American Chemical Society 124, 7270-7271 (2002). 3. Gottfried, D. S., Kagan, A., Hoffman, B. M. & Friedman, J. M. Impeded rotation of a protein in a sol-gel matrix. Journal of Physical Chemistry B 103, 2803-2807 (1999). 4. Campanini, B., Bologna, S., Cannone, F., Chirico, G., Mozzarelli, A. & Bettati, S. Unfolding of Green Fluorescent Protein mut2 in wet nanoporous silica gels. Protein Science 14, 1125-1133 (2005). 5. Shiu, Y. J., Su, C., Yeh, Y. L., Liang, K. K., Hayashi, M., Mo, Y., Yan, Y. J. & Lin, S. H. Experimental and theoretical studies of protein folding-unfolding. Journal of the Chinese Chemical Society 51, 1161-1173 (2004). 6. Eggers, D. K. & Valentine, J. S. Crowding and hydration effects on protein conformation: A study with sol-gel encapsulated proteins. Journal of Molecular Biology 314, 911-922 (2001). 7. Eggers, D. K. & Valentine, J. S. Molecular confinement influences protein structure and enhances thermal protein stability. Protein Science 10, 250-261 (2001).
124 8. Samuni, U., Dantsker, D., Juszczak, L. J., Bettati, S., Ronda, L., Mozzarelli, A. & Friedman, J. M. Spectroscopic and functional characterization of T state hemoglobin conformations encapsulated in silica gels. Biochemistry 43, 13674- 13682 (2004). 9. Navati, M. S., Samuni, U., Aisen, P. & Friedman, J. M. Binding and release of iron by gel-encapsulated human transferrin: Evidence for a conformational search. Proceedings of the National Academy of Sciences of the United States of America 100, 3832-3837 (2003). 10. Das, T. K., Khan, I., Rousseau, D. L. & Friedman, J. M. Temperature dependent quaternary state relaxation in sol-gel encapsulated hemoglobin. Biospectroscopy 5, S64-S70 (1999). 11. Long, D. A. Raman Spectroscopy (1976). 12. Asher, S. A. Uv Resonance Raman-spectroscopy for analytical, physical, and biophysical chemistry .2. Analytical Chemistry 65, A201-A210 (1993). 13. Asher, S. A. Uv Resonance Raman-spectroscopy for analytical, physical, and biophysical chemistry .1. Analytical Chemistry 65, A59-A66 (1993). 14. Spiro, T. G. & Strekas, T. C. Resonance Raman spectra of hemoglobin and cytochrome c: inverse polarization and vibronic scattering. Proceedings of the National Academy of Sciences of the United States of America 69, 2622-6 (1972). 15. Asher, S. A. 203 pp (Lawrence Berkeley Lab.,Univ. California,Berkeley,CA,USA., 1976). 16. Strekas, T. C. & Spiro, T. G. Hemoglobin resonance Raman excitation profiles with a tunable dye laser. Journal of Raman Spectroscopy 1, 387-92 (1973). 17. Strekas, T. C. & Spiro, T. G. Hemoglobin. Resonance Raman spectra. Biochimica et Biophysica Acta 263, 830-3 (1972). 18. Strekas, T. C. & Spiro, T. G. Hemoglobin: resonance Raman spectra. Biochimica et biophysica acta 263, 830-3 (1972). 19. Ibrahim, M., Denisov, I. G., Makris, T. M., Kincaid, J. R. & Sligar, S. G. Resonance Raman spectroscopic studies of hydroperoxo-myoglobin at cryogenic temperatures. Journal of the American Chemical Society 125, 13714-13718 (2003). 20. Mizutani, Y. & Kitagawa, T. Ultrafast dynamics of myoglobin probed by time- resolved resonance Raman spectroscopy. Chemical Record 1, 258-275 (2001). 21. Chottard, G., Mahy, J. P., Battioni, P. & Mansuy, D. Resonance Raman study of myoglobin derivatives formed by reaction with monosubstituted hydrazines. Spectrosc. Biol. Mol., Proc. Eur. Conf., 1st, 229-30 (1985). 22. Bangcharoenpaurpong, O., Schomacker, K. T. & Champion, P. M. Resonance Raman investigation of myoglobin and hemoglobin. Journal of the American Chemical Society 106, 5688-98 (1984). 23. Asher, S. Resonance Raman spectroscopy of hemoglobin. Methods in enzymology 76, 371-413 (1981). 24. Scott, R. A., Mauk, A. G. Cytochrome c: A Multidisciplinary Approach University Science Books, Sausalito, Calif. (1996). 25. Yeh, S. R., Han, S. W. & Rousseau, D. L. Cytochrome c folding and unfolding: A biphasic mechanism. Accounts of Chemical Research 31, 727-736 (1998).
125 26. Jordan, T., Eads, J. C. & Spiro, T. G. Secondary and tertiary Structure of the a- state of cytochrome-c from resonance Raman-spectroscopy. Protein Science 4, 716-728 (1995). 27. Boffi, A., Takahashi, S., Spagnuolo, C., Rousseau, D. L. & Chiancone, E. Structural characterization of oxidized dimeric scapharca-inaequivalvis hemoglobin by resonance Raman-spectroscopy. Journal of Biological Chemistry 269, 20437-20440 (1994). 28. Hu, S. Z., Morris, I. K., Singh, J. P., Smith, K. M. & Spiro, T. G. Complete assignment of cytochrome-c resonance Raman-spectra via enzymatic reconstitution with isotopically labeled hemes. Journal of the American Chemical Society 115, 12446-12458 (1993). 29. Abe, M., Kitagawa, T. & Kyogoku, Y. Resonance Raman spectra of octaethylporphyrinatonickel(II) and meso-deuterated and nitrogen-15 substituted derivatives. II. A normal coordinate analysis. Journal of Chemical Physics 69, 4526-34 (1978). 30. Kitagawa, T., Abe, M. & Ogoshi, H. Resonance Raman spectra of octaethylporphyrinatonickel(II) and meso-deuterated and nitrogen-15 and substituted derivatives. I. Observation and assignments of nonfundamental Raman lines. Journal of Chemical Physics 69, 4516-25 (1978). 31. Lee, H., Kitagawa, T., Abe, M., Pandey, R. K., Leung, H. K. & Smith, K. M. Characterization of low frequency resonance Raman bands of metallo- protoporphyrin. IX. Observation of isotope shifts and normal coordinate treatments. Journal of Molecular Structure 146, 329-47 (1986). 32. Gladkov, L. L. & Solov'ev, K. N. Normal coordinate analysis of porphine and its derivatives based on the solution of the inverse spectral problem for porphine and copper porphine-III. Interpretation of vibrational spectra of metal complexes of octamethylporphine and octaethylporphine. Spectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy 42A, 1-10 (1986). 33. Gladkov, L. L. & Solov'ev, K. N. The normal coordinate analysis of porphine and its derivatives based on the solution of the inverse spectral problem for porphine and copper porphine.- II. A valance force field for in-plane vibrations of the copper porphine molecule. Spectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy 41A, 1443-8 (1985). 34. Gladkov, L. L. & Solov'ev, K. N. The normal coordinate analysis of porphine and its derivatives based on the solution of the inverse spectral problem for porphine and copper porphine-I. A valence force field for in-plane vibrations of the porphine molecule. Spectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy 41A, 1437-42 (1985). 35. Li, X. Y., Czernuszewicz, R. S., Kincaid, J. R., Stein, P. & Spiro, T. G. Consistent porphyrin force field. 2. Nickel octaethylporphyrin skeletal and substituent mode assignments from nitrogen-15, meso-d4, and methylene-d16 Raman and infrared isotope shifts. Journal of Physical Chemistry 94, 47-61 (1990). 36. Li, X. Y., Czernuszewicz, R. S., Kincaid, J. R., Su, Y. O. & Spiro, T. G. Consistent porphyrin force field. 1. Normal-mode analysis for nickel porphine and nickel tetraphenylporphine from resonance Raman and infrared spectra and isotope shifts. Journal of Physical Chemistry 94, 31-47 (1990).
126 37. Li, X. Y., Czernuszewicz, R. S., Kincaid, J. R. & Spiro, T. G. Consistent porphyrin force field. 3. Out-of-plane modes in the resonance Raman spectra of planar and ruffled nickel octaethylporphyrin. Journal of the American Chemical Society 111, 7012-23 (1989). 38. Yeh, S. R. & Rousseau, D. L. Folding intermediates in cytochrome c. Nature Structural Biology 5, 222-228 (1998). 39. Takahashi, S., Yeh, S. R., Das, T. K., Chan, C. K., Gottfried, D. S. & Rousseau, D. L. Folding of cytochrome c initiated by submillisecond mixing. Nature Structural Biology 4, 44-50 (1997). 40. Yeh, S. R., Takahashi, S., Fan, B. C. & Rousseau, D. L. Ligand exchange during cytochrome c folding. Nature Structural Biology 4, 51-56 (1997). 41. Jordan, T., Eads, J. C. & Spiro, T. G. Secondary and tertiary structure of the A- state of cytochrome c from resonance Raman spectroscopy. Protein science : a publication of the Protein Society 4, 716-28 (1995). 42. Bushnell, G. W., Louie, G. V. & Brayer, G. D. High-resolution 3-Dimensional structure of horse heart cytochrome-c. Journal of Molecular Biology 214, 585-595 (1990). 43. Braunitzer, G., Hilse, K., Rudloff, V. & Hilschmann, N. The Hemoglobins. Advances in protein chemistry 19, 1-71 (1964). 44. Perutz, M. F. Hemoglobin structure and respiratory transport. Scientific American 239, 92-3, 95-6, 98, 103-6, 109-10, 112, 116, 119, 122, 125 (1978). 45. Hargrove, M. S., Krzywda, S., Wilkinson, A. J., Dou, Y., Ikedasaito, M. & Olson, J. S. Stability of myoglobin - a model for the folding of heme-proteins. Biochemistry 33, 11767-11775 (1994). 46. Springer, B. A., Sligar, S. G., Olson, J. S. & Phillips, G. N. Mechanisms of ligand recognition in myoglobin. Chemical Reviews 94, 699-714 (1994). 47. Pinker, R. J., Lin, L., Rose, G. D. & Kallenbach, N. R. Effects of alanine substitutions in alpha-helices of sperm whale myoglobin on protein stability. Protein Science 2, 1099-1105 (1993). 48. Barrick, D. & Baldwin, R. L. The molten globule intermediate of apomyoglobin and the process of protein folding. Protein Science 2, 869-876 (1993). 49. Barrick, D. & Baldwin, R. L. 3-State analysis of sperm whale apomyoglobin folding. Biochemistry 32, 3790-3796 (1993). 50. Das, T. K., Khan, I., Rousseau, D. L. & Friedman, J. M. Preservation of the native structure in myoglobin at low pH by sol-gel encapsulation. Journal of the American Chemical Society 120, 10268-10269 (1998). 51. Tang, Q., Kalsbeck, W. A., Olson, J. S. & Bocian, D. F. Disruption of the heme iron-proximal histidine bond requires unfolding of deoxymyoglobin. Biochemistry 37, 7047-7056 (1998). 52. Tang, Q., Kalsbeck, W. A. & Bocian, D. F. Acid-induced transformations of myoglobin .2. Effect of ionic strength on the free energy and formation rate of the 426-nm absorbing deoxyheme intermediate. Biospectroscopy 3, 17-29 (1997). 53. Palaniappan, V. & Bocian, D. F. Acid-induced transformations of myoglobin - characterization of a new equilibrium heme-pocket intermediate. Biochemistry 33, 14264-14274 (1994).
127 54. Postnikova, G. B., Komarov, Y. E. & Yumakova, E. M. Fluorescence study of the conformational properties of myoglobin structure .2. pH-induced and ligand- induced conformational-changes in ferricmyoglobins and ferrousmyoglobins. European Journal of Biochemistry 198, 233-239 (1991). 55. Sage, J. T., Morikis, D. & Champion, P. M. Spectroscopic studies of myoglobin at low pH - Heme structure and ligation. Biochemistry 30, 1227-1237 (1991). 56. Han, S., Rousseau, D. L., Giacometti, G. & Brunori, M. Metastable intermediates in myoglobin at low pH. Proceedings of the National Academy of Sciences of the United States of America 87, 205-209 (1990). 57. Friedman, J. M. in Hemoglobins, Pt C 205-231 (ACADEMIC PRESS INC, San Diego, 1994). 58. Spiro, T. G. Biological Applications of Raman Spectroscopy, Vol. 3: Resonance Raman Spectra of Heme and Metalloproteins John Wiley and Sons, New York, N. Y. (1988). 59. Wei, Y., Xu, J. G., Feng, Q. W., Lin, M. D., Dong, H., Zhang, W. J. & Wang, C. A novel method for enzyme immobilization: Direct encapsulation of acid phosphatase in nanoporous silica host materials. Journal of Nanoscience and Nanotechnology 1, 83-93 (2001). 60. Wei, Y., Xu, J. G., Feng, Q. W., Dong, H. & Lin, M. D. Encapsulation of enzymes in mesoporous host materials via the nonsurfactant-templated sol-gel process. Materials Letters 44, 6-11 (2000). 61. Wei, Y., Xu, J. G., Dong, H., Dong, J. H., Qiu, K. Y. & Jansen-Varnum, S. A. Preparation and physisorption characterization of D-glucose-templated mesoporous silica sol-gel materials. Chemistry of Materials 11, 2023-2029 (1999). 62. Wei, Y., Jin, D. L., Ding, T. Z., Shih, W. H., Liu, X. H., Cheng, S. Z. D. & Fu, Q. A non-surfactant templating route to mesoporous silica materials. Advanced Materials 10, 313-316 (1998). 63. Hu, S. Z., Smith, K. M. & Spiro, T. G. Assignment of protoheme Resonance Raman spectrum by heme labeling in myoglobin. Journal of the American Chemical Society 118, 12638-12646 (1996). 64. Shen, C. & Kostic, N. M. Kinetics of Photoinduced electron-transfer reactions within sol-gel silica glass doped with zinc cytochrome c. study of electrostatic effects in confined liquids. J. Am. Chem. Soc. 119, 1304-1312 (1997).
128
Table 3- 1 Pore parameters of water-extracted Ccu series prepared at various urea concentrations
Average pore Micropore data diameter Sample BET Single BET/nma BJH/nmb Surface Pore code surface point area volumec area/m2g-1 pore /m2g-1 /cm3g-1 volume /cm3g-1 Ccu0 530 0.315 2.38 2.56 120 0.067 Ccu15 294 0.249 2.53 2.72 83.8 0.047 Ccu30 524 0.341 2.60 2.61 8.74 0.002 Ccu40 494 0.443 3.58 3.07 ------Ccu50 667 0.610 3.66 3.12 ------
a b The average pore diameters calculated from 4 V/SBET by the BET method. Determined from the maxima of the BJH desorption pore size distribution curves with the Halsey equation. c Values determined from the t-plot analysis.
129
Table 3- 2 Pore parameters of water-extracted Ccg series prepared at various urea concentrations.
Average pore diameter Micropore data
Sample BET Single BET/nm a BJH/nm b Surface Pore Code surface point pore area volume area/m2g-1 volume /m2g-1 / cm3g-1 c /cm3g-1 Ccg0 591 0.38 2.6 2.7 107 0.06
Ccg15 627 0.41 2.6 2.6 47 0.02
Ccg30 940 0.63 2.7 2.6 ______
Ccg40 747 0.80 4.1 3.4 ______
Ccg50 699 0.89 5.0 3.9 ______
Ccg60 693 0.62 3.5 3.2 ______
a b The average pore diameters calculated from 4 V/SBET by the BET method. Determined from the maxima of the BJH desorption pore size distribution curves with the Halsey equation. c Values determined from the t-plot analysis.
130
Figure 3- 1 Diagram of a typical heme group.
131
Figure 3- 2 Deconvoluted resonance Raman spectra of the various heme coordinated forms of Cc.25
132
600 Ccg0 Ccg15 Ccg30 500 Ccg40 STP)
-1 Ccg50 g 3 400 Ccg60 (cm d
e 300 b sor
Ab 200 e m lu 100 Vo
0 0.00.20.40.60.81.0 Relative pressure(P/P ) 0
Figure 3- 3 N2 adsorption-desorption isotherm of Ccg at –196°C.
133
0.13 0.12 0.11 Ccg0 0.10 Ccg15 Ccg30 0.09 Ccg40 )
-1 0.08 Ccg50 A
-1 0.07 Ccg60 g 3
m 0.06 c
( 0.05 D d
/ 0.04
dV 0.03 0.02 0.01 0.00 -0.01 20 30 40 50 60 70 80 Pore Diameter (A)
Figure 3- 4 BJH pore size distributions for Ccg samples synthesized in the presence of 0- 60 wt% of glucose.
134
ccu50 ccu40 400 ccu30 350 ccu15 ccu0 STP)
-1 300 g 3
(cm 250
200
150
100 lume Absorbed o V 50
0.0 0.2 0.4 0.6 0.8 1.0
Relative Pressure (P/P0)
Figure 3- 5 N2 adsorption-desorption isotherm of Ccu at –196°C.
135
ccu50 ccu40 0.12 ccu30 ccu15 0.10 ccu0
0.08 ) -1 A -1
g 0.06 3
0.04 D (cm d
dV/ 0.02
0.00
20 30 40 50 60
Pore Diameter(A)
Figure 3- 6 BJH pore size distributions for the Ccu samples synthesized in the presence of 0-50% wt% of urea.
136
397cm-1 567cm-1
Ccu50
Ccu40
Ccu30
Ccu15
Ccu0
Free Cc
300 350 400 450 500 550 600 Raman Shift (cm-1)
Figure 3- 7 Low frequency region of Resonance Raman Spectra of Ccu series and native cytochrome c samples at pH 7.4.
137
0.7 0.65 0.6 0.55 0.5 418
/I 0.45 567 I 0.4 0.35 0.3 0.25 0.2 1.522.533.54 Average pore diameter (nm)
Figure 3- 8 Relationship between relative intensity (I567/I418) and BJH average pore diameter. For Ccu0 (wt%=0), pore diameter was taken as 1.7 nm.
138
0.75 0.73 0.71 0.69 0.67 418
/I 0.65 397 I 0.63 0.61 0.59 0.57 0.55 1.522.533.54 Average pore diameter (nm)
Figure 3- 9 Relationship between relative intensity (I397/I418) and BJH average pore diameter. For Ccu0 (wt%=0), pore diameter was taken as 1.7 nm.
139
-1 -1 397cm 397cm
Ccu50 in pH7.0 Ccu0 in pH7.0
Ccu50 in pH3.5 Ccu in pH3.5
300 350 400 450 500 300 350 400 450 500 -1 Raman Shift(cm-1) Raman Shift(cm )
Figure 3- 10 Resonance Raman spectra of Ccu50 and Ccu0 at pH 7.0 and pH 3.5.
140
-1 -1 397cm 397cm
Ccu50 at 70oC o Ccu0 in 70 C
o Ccu50 at 60oC Ccu0 at 60 C
o o Ccu0 at 25 C Ccu50 at 25 C 300 350 400 450 500 300 350 400 450 500 -1 -1 Raman Shift(cm ) Raman Shift(cm )
Figure 3- 11 Resonance Raman spectra of Ccu50 and Ccu0 at different temperatures.
(a)
141 3.0 2.8 1584 2.6 1502 1635 1317 2.4 1493 2.2 Ccg60w 2.0 1.8 u.) Ccg50w .
a 1.6 ( y
t 1.4 1.2 Ccg40w
tensi 1.0 n I 0.8 Ccg30w 0.6 0.4 0.2 Ccg15w 0.0
1300 1350 1400 1450 1500 1550 1600 1650 1700 Raman Shift (cm-1)
(b)
347 413 397
0.50 Ccg60w 0.45
0.40
0.35 Ccg50w
0.30
y 0.25 Ccg40w t
0.20 tensi n I 0.15 Ccg30w
0.10 416 Ccg15w 0.05 344
0.00
-0.05 250 300 350 400 450 500 550 600 650 Raman Shift (cm-1)
Figure 3- 12 Resonance Raman spectra of Ccg series samples when first immersed in water. (a: in high frequency region; b: in low frequency region)
142 (a)
1580 3.0 2.8 1493 2.6 1632 2.4 1317 2.2 Ccg60u 2.0 ) . 1.8 Ccg50u u
. 1.6 1.4 y (a it
s 1.2 Ccg40u n
te 1.0
In 0.8 Ccg30u 0.6 0.4 0.2 Ccg15u 0.0 -0.2 1300 1350 1400 1450 1500 1550 1600 1650 1700 Raman Shift (cm-1)
(b)
0.50 Ccg60u 0.45
0.40
0.35 Ccg50u
0.30 ) u. 0.25 Ccg40u (a. 0.20
ensity 0.15 Ccg30u Int 0.10
0.05 Ccg15u
0.00
-0.05 250 300 350 400 450 500 550 600 650 Raman Shift (cm-1)
Figure 3- 13 Resonance Raman spectra of Ccg series samples when immersed in 9M urea solution. (a: in high frequency region; b: in low frequency region)
143
(a)
3.2 1502 3.0 2.8 1494 1584 2.6 1317 1636 2.4 Ccg60re 2.2 2.0
u.) 1.8
. Ccg50re a 1.6 ( 1.4
nsity 1.2 Ccg40re 1.0 Inte 0.8 Ccg30re 0.6 0.4 0.2 Ccg15re 0.0
1300 1350 1400 1450 1500 1550 1600 1650 1700 Raman Shift (cm-1)
(b)
568 399 0.55
0.50
0.45 Ccg50re 0.40
0.35
) 0.30 Ccg40re . u.
a 0.25 ( y t 0.20 Ccg30re
tensi 0.15 n I 0.10 Ccg15re 0.05
0.00
-0.05
250 300 350 400 450 500 550 600 650 Raman Shift (cm-1)
Figure 3- 14 Resonance Raman spectra of Ccg series samples when washed out urea and reimmersed in water for 24 hours. (a: in high frequency region; b: in low frequency region)
144
1.8 1.6 in water in 9M urea 1.4 back in water 1.2
1494 1 I 0.8 1503/ I 0.6 0.4 0.2 0 Ccg15 Ccg30 Ccg40 Ccg50 Ccg60 Sample Code
Figure 3- 15 Ratio of intensity of peak 1503 cm-1 over peak 1494 cm-1 for Ccg series samples in different conditions.
145 Chapter 4: A Novel Method to Study Aggregation of Amyloid β1-42 - A Key Peptide
Associated with Alzheimer’s Disease
4.1 Introduction
Alzheimer’s disease has been one of the common causes of neurodegenerative
death in the world. It is associated with aggregation of insoluble amyloid β-peptide (Aβ)
fibrillar forms deposited in the brain.1-16 Aβ peptides are cleaved from the 695-770 amino
acid long amyloid precursor protein (APP) by various proteolytic steps, resulting in
heterogeneous ensembles of variant polypeptides. The heterogeneity occurs in both non-
terminal sequences, as well as at chain ends and the latter one is of particular importance
to the origin of Alzheimer’s disease. About 90% of the molecules end at position 40
[Aβ(1-40)] and 10% at position at position 42 [Aβ (1-42)]. Because Aβ (1-42) is more
hydrophobic and may produce the pathogenic seed in the development of the disease,14,17-
19 Aβ (1-42) was selected as the target molecule in this research.
4.1.1 Alzheimer’s Disease and Amyloid β
Alzheimer’s disease is characterized by two events: the accumulation of insoluble fibrillar aggregates of Aβ peptides and the degeneration, as well as death of neurons in the brain regions concerned with learning and memory processes. Unfortunately the relationship of these two events is still not clear.20 Some believe that that Aβ accumulation may cause the neuronal loss of Alzheimer’s disease.18,19 They suggest that
Aβ deposition is a normal part of aging, but in Alzheimer’s patients’ brains those
deposits can not be cleared for some reason. The growth-promoting activity of the Aβ
peptide might then contribute to plaque formation by fostering the outgrowth of the
neurites around the deposits. This may account for the profusion of degenerating
146 terminals in the plaques. It was found that exogenous Aβ causes neuronal degeneration in
primary cultured neurons and when injected into the adult rat brain.21-23 Synthetic Aβ
peptides show either trophic24,25 or toxic responses on neurons in culture.24,26-28 The
reasons for these deviations are believed to arise in part from differences in the
aggregation states and the solution structures of the Aβ peptides.17 It was found that aged
aggregated Aβ exhibited neurotoxicity, whereas newly dissolved Aβ did not.29 Though
Aβ is produced in normal individuals, as well as AD patients,17 amyloid formation is seen only in AD brains (and partially in normal aged brains). Talafous et al.30suggested that
one possibility is that the monomeric α-helical conformation is the neurotrophic species,
and when an α-helix (monomeric) to β-sheet (oligmeric or polymeric) transformation
occurs, the Aβ peptide becomes neurotoxic.
In another theory, Aβ peptides deposition may be the result of the neuronal
degeneration in Alzheimer’s brains. Neurons that fail to maintain their connections
usually degenerate, and this could lead to the release of the Aβ from their membranes.
Those Aβ peptides, once they are free, can readily associate with one another to form
polymeric, insoluble fibrillar deposits.31
Regardless the relationship between Aβ deposits formation and neuronal
degeneration, before the aggregates forms, the Aβ peptide must first be cut out of the
precursor protein, which is call amyloid precursor protein (APP). Though the detailed
mechanism of Aβ production from APP, the exact localization of the proteases involved,
and the functions of Aβ and APP are still not well understood, it has been accepted that
two major types of Aβ peptides can be generated from APP, one is Aβ40 with 40 amino
147 acids and the other one is Aβ42 with 42 amino acids. Aβ40 is the predominant form that
is produced during the metabolism of APP. Under pathological conditions the production
of Aβ42, normally a minor product, is enhanced, which makes Aβ42 an important target
for the AD study.
4.1.2 The Aggregation of Aβ peptides
As discussed in the above sections, the aggregation forms of Aβ peptide plays an
important role in Alzheimer’s disease. Therefore, to understand how the monomeric Aβ
peptides aggregate is very interesting and important research direction, which may lead
us the knowledge how the disease is initiated.
It is necessary to note that Aβ peptide may form various aggregates, ranging from
oligmeric species to large polymers as amyloid fibrils.32 Some of aggregation pathways have been worked out. For example, in one proposed pathway, the monomeric form of amyloid peptide is unstable in solution and prone to aggregate. It may be found in one of two states relevant for aggregation,33 which is shown in Figure 4-1: One of them is an
unfolded state characterized by the lack of a regular and stable order that follows the non-
ordered aggregation pathway (left, gray line): The other is the folded state characterized
by a β-sheet rich conformation, which is the basis for amyloid protofibril formation
following the ordered aggregation pathway (right, black line). The former pathway
should operate normally under physiological conditions whereas, the latter does operate
in disease. In the non-ordered aggregation pathway, the monomeric peptide is
incorporated into pseudospheres that readily reach a critical size upon which they are
unstabilized, hence exposing the peptide molecules to the environment as a lax meshwork
referred to as the amorphous aggregate, which may be degraded by proteases.
148 Alternatively, the ordered aggregation pathway proceeds when a critical concentration of
folded amyloid peptide is found, readily forming protofibrils. These species may
associate and grow in a reversible manner producing amyloid fibrils.
It is now believed that external factors such as the pH, ionic strength, metal ions
and solvent hydrophobicity can affect the relative proportions of the random coil, α-helix,
and β-sheet solution structures and especially can modulate the aggregation behaviors of
Aβ.34-41 It have been known that in highly acidic and basic environments, the Aβ peptide
is mostly in random coil or α-helix forms in aqueous solution. At pH 4-7, the peptide
gains aggregated β-sheet structures.42,43 In this study, we focused our study on the pH effects on Aβ aggregation in confined space using our nonsurfactant templated sol-gel bioencapsulation technique.
4.1.3 Detection of Aβ aggregation
Since the Aβ aggregates play such an important role in Alzheimer’s disease, it is necessary to find reliable methods in detect those aggregates both in vivo and in vitro.
Here, several analytical techniques, which have been well developed and used in this project, are introduced in the following sections:
4.1.3.1 The Thioflavine T Fluorescence Assay
Thioflavine T (ThT), whose structure is shown in Fig. 4-2, associates rapidly with aggregated fibrils of Aβ peptides, giving rise to a new excitation (ex) maximum at 450 nm and enhanced emission (em) at 482 nm, as opposed to the 385 nm (ex) and 445 nm
(em) of the free ThT. The fluorescence enhancement of ThT depends on the structure of the aggregated state of the Aβ. ThT is thought to interact with β-sheet, which is a common structural motif in Aβ fibril, in some as yet undefined way. It is likely that both
149 the β-sheet structure and the aggregated state provide the environment to stabilize the
long-wavelength ThT fluorescent complex.43-45
4.1.3.2 The Congo Red Birefringence Assay
Congo Red birefringence is a primary criterion for amyloid fibril identification both in vivo and in vitro. The mechanism of interaction between Congo Red and amyloid fibrils is not well understood. It has been suggested that the interaction may be from an oligomer of Congo Red molecules not a single monomer.46-49 Congo Red has several functional groups that could potentially interact with amyloid fibrils by: (1) hydrogen bonding with the primary amino groups acting as H-bond donors, (2) ionic interactions via the sulfonate, (3) hydrophobic interactions between the aromatic rings and the fibril,
(4) steric intercalation of the dye between β sheets, or (5) a combination of these interactions.
In this assay, the sample is stained by saturated Congo Red solution and then examined by polarized light microscopy. If the polarizers are aligned, the material stained with Congo Red will appear reddish pink (the affinity for Congo Red is known as congophilia). If the polarizers are crossed at a 90o angle to each other, the background of the sample will turn black. Any bright spots that appear are a result of birefringence (the sample bends the light in such a way that it can pass through the upper polarizer to reach eyes). The detection of yellow/green birefringence is considered a positive result for the presence of amyloid. The absence of such birefringence is a negative result. The birefringence under crossed polarizers should match the areas of Congo Red staining observed under visible light.
4.1.3.3 Negative staining of amyloid fibrils for TEM
150 Transmission electron microscopy (TEM) can also be used to detect the presence
of fibrils. Prior to the detection, the sample must be stained by with a 2% solution of
uranyl acetate (UA) in water.37
4.1.4 Bioencapsulation of Aβ peptide in silica matrix with controlled pore size
Though extensive research work has been done on Aβ aggregates, some considerations about aggregation assays make it difficult to extrapolate the value of the data to the physiological condition. First, because aggregation experiments are usually performed by dissolving micromolar concentrations of Aβ peptides, whereas the in vivo condition involves depositing nanomolar or picomolar concentrations of Aβ onto the brain parenchyma, which is closer to a solid-phase rather than a solution. Second, the local environment in which Aβ peptides are deposited in vivo may influence plaque growth but it is very difficult to reproduce in the assay tube.20
In this study, bioencapsulation of Aβ peptide by nonsurfactant templated sol-gel method was used to mimic the circumstance in vivo to examine the space confinement effect on Aβ peptide aggregation behavior. This novel nonsurfactant templated sol-gel method was developed in our group, which can be used widely in encapsulation of biological substances, such as protein, enzyme, peptide and cell, without destroying their bioactivities.50-56 In general, inorganic precursors such as tetraethyl orthosilicate (TEOS)
or tetramethyl orthosilicate (TMOS) are first hydrolyzed with hydrochloric acid as
catalyst followed by mixing with the aqueous solution of nonsurfactant template
compound solution and bio-substances. Upon gelation and drying, the transparent
template-silica composites can be obtained. At this time, they are still nonporous, with
removal of template by water extraction, the porosity of the materials increases, leading
151 to the final mesoporous materials after complete removal of the templates. Unlike most of sol-gel immobilizations in the literature (so called wet gels), the final materials thus obtained in our work are rigid, glassy and mechanically strong.
The porosity of the materials can be adjustable to a certain extent. Generally, the porous parameters, such as pore size, area and volume increase with the template weight percentage. When the weight percentage is between 30% and 50%, mesoporosity can be usually achieved.
Biocompatibility is the most significant advantage of this method. The whole synthesis and encapsulation process can be readily accomplished at room temperature and near neutral pH. Nonsurfactants, such as sugar compounds, are highly biocompatible.
Furthermore, unlike surfactant molecules, which are tightly adhered to silica matrix and difficult to remove, those nonsurfactant molecules can be easily removed out by room temperature water extraction avoiding calcinations at high temperature.
Generally, in this project, Aβ1-42 peptides were encapsulated into silica matrix in their monomeric state. Then the biogel was put into buffer solution with different pH values. So the aggregation behaviors of encapsulated Aβ1-42 peptides in confined space under different pH environments were monitored by fluorescence spectroscopy, polarized light microscope and so on.
The key idea is that if we give more space to the Aβ peptides, higher degree if aggregation might be achieved. At a certain an aggregation number, the aggregation might become irreversible that may be indicative of the onset of Alzheimer disease.
4.2 Experimental
4.2.1 Materials
152 Tetramethyl orthosilicate (TMOS, 98%, Aldrich), dimethylamine hydrochloride
(99%, Batch #: 17407DB, Aldrich), amyloid β (1-42) acetate (99.0%, counter ion: TFA,
Lot #: 2020442A, rPeptide, Atlanta, GA), amyloid β (1-28) acetate (>97%, Lot #:
30804128, rPeptide), ethyl alcohol (ACS/USP grade, Pharmco, Athens, GA), congo red
(>75%, Lot #: 073K3520, Sigma), Thioflavin T (Dye content approx. 75%, Lot #
043K3506 Sigma), uranyl acetate (ACS grade, Lot #: 0214B28, Amresco, Solon, OH),
sodium hydroxide 1N solution (Certified, Lot #: 946638-24, Fisher Chemical), sodium
phosphate (monobasic anhydrous, minimum 99.0%, Sigma), sodium phosphate dibasic
heptahydrate (minimum 99.0%, Sigma), 1, 1, 1, 3, 3, 3- Hexafluoro-2-propanol (HFIP,
99+%, Batch #: 17503MB, Aldrich), hydrochloric acid (HCl, Fisher), Gel/Mount
(permanent aqueous mounting medium with anti-fading agents, Biomeda Corp. Forster
City, CA), sodium chloride (NaCl, Sigma), sodium dithionite (Na2S2O4, Sigma), were all used as received without further purification.
4.2.2 Redissolving of Aβ peptide in their monomeric state
Both Aβ1-42 and Aβ1-28 peptides were received as white lyophilized powder.
According to the description from the provider, most of peptides are in their monomeric states. Prior to encapsulation, peptide powder must be redissolved and kept in monomeric state. We also studied Aβ 1-28 because it has been used widely as model peptides for spectroscopic studies.
Mary Jo LaDu and et al. found HFIP pretreatment can produce uniform, unaggregated Aβ peptides.57 In their protocol, lyophilized Aβ peptides were directly
dissolved in 100% HFIP and characterized by AFM. This treatment resulted in a dense,
homogenous unaggregated peptide immediately after dissolving. After 24-h incubation at
153 room temperature, no aggregates, fibrils, or protofibrils were detected in these HFIP Aβ
solutions.
The same protocol was applied in this project to achieve monomeric Aβ peptide
solution. As received, the lyophilized peptide was stored sealed glass vials with 1 mg in
each one. Prior to dissolution, each vial was allowed to equilibrate to room temperature
for 30 min sealed to avoid condensation upon opening the vial. Then 1 ml HFIP was
added into one vial to form Aβ 1 mg/ml solution.
4.2.3 Bioencapsulation of Aβ1-42 in silica matrix with controlled pore size
The encapsulation was followed the standard method with some modifications.
The largest difference arose from HFIP Aβ solution, which could not be easily dissolved in the silica sol to form homogeneous mixture. Thus, co-solvent ethyl alcohol (EtOH) and another template dimethylaimine hydrochloride (DMA) were used instead of sugar template, such as fructose and glucose. Typically, 0.634 g TMOS (Aldrich, 95%), 0.225 g deionized H2O and 6.25 µl HCl solution (40 mM) were mixed at room temperature for 1-
2 h under stirring. Upon mixing, the organic phase (TMOS) and inorganic phase (H2O) was separated, so it looked translucent. After stirring vigorously for less than 5 minutes, a lot of heat was generated during the hydrolysis reactions and the whole solution turned transparent suddenly. Then the mixture was cooled to 0 oC in an ice-water bath followed
by addition of appropriate amount of 50 wt% DMA solution and 0.2 g EtOH, which
served as co-solvent. Amounts of 50 wt% DMA solution were designed to yield 0, 30 and
50% by weight of templates in the final dry gel products (sample code: Abeta plus a
number denoting weight percentage of DMA). 200 µl HFIP Aβ solution (1 mg/ml) was added in the final step. To avoid phase separation during the mixing process, the 200 µl
154 HFIP Aβ solution was added drop by drop under vigorous stirring. The reaction was then sealed with a parafilm. Upon gelation of the system within a few hours at room temperature, 12-15 holes were pinned in the parafilm with a hypodermic syringe needle to allow for the evaporation of solvents and reaction by-products. After 24 h, the system was placed in a vacuum oven and dried to reach a constant weight at room temperature in about 6 days. Thus the silica biogel samples containing DMA and Aβ peptide were obtained as colorless, transparent, dry, brittle, glassy monoliths. Then the samples were ground into fine powders and kept in sealed vials in a –15 oC freezer. Conventional porcelain pestle and mortar were used for grinding and the whole grinding process was done manually. For each sample, the grinding time was maintained at around five minutes in order to keep the silica powders in uniform size consistently for all the samples.
4.2.4 pH changing of suspension by adding NaOH
The purpose of this project is to examine aggregation behavior of Aβ peptide in confined space with changing pH, so the samples must be suspended in buffers with different pH values. Though the silica biogel powder can be separated from suspension by centrifuging and resuspended in another buffer solution with a different pH value, the loss of powder would not be avoided during this process. So an alternated way was used to changing pH value by adding a predetermined amount of NaOH solution into the initial suspension with the lowest pH value. With adding NaOH, the pH value of suspension could change from low to high step by step as desired.
Because the sample holder in fluorescence spectroscopy, a 1 cm cuvette, was not convenient for pH testing especially during the fluorescence experiment, we
155 predetermined the amount of NaOH solution for pH changing from 2.3 to 3.0, 3.5, 4.7,
5.6, 6.3, 7.0, 8.1 and 8.9 before the fluorescence spectral measurements.
4.2.5 Fluorescence Spectroscopy Study
4.2.5.1 Steady State Fluorescence Spectroscopy
The aggregation behavior of Aβ1-42 peptides was monitored with fluorescence spectroscopy using a Jobin Yvon FluoroMax-3 spectrofluorometer. A 4-nm slit width setting and a cuvette with 1-cm pathlength was used throughout the whole experiment.
The excitation wavelength was chosen at 460 nm and the maximum of emission peak was at near 480 nm. In order to get reliable results, magnetic stirring was kept on a constant rate and approximate same amounts of entrapped Aβ1-42 peptides were used when recording fluorescence spectra. Since the weight percentages of template in a series of samples were different, the different amounts of silica biogel were used to keep the amount of Aβ peptide in suspension at a constant number. For example, for 0 wt% sample, Abeta42-0, 0.015 g of the biogel was used, but for 50 wt% sample, Abeta42-50, we used 0.030 g of the biogel in suspension.
In the beginning of the experiment, powder samples were put into a fluorescence cuvette with 3 ml 5 µM ThT phosphate buffer (pH 2.3, 0.05M) together, then by adding predetermined amount of NaOH (1M) step by step, the increase of pH value of ThT phosphate buffer from 2.3 to 3.0, 3.5, 4.7, 5.6, 6.3, 7.0, 8.1 and 8.9 was achieved. The fluorescence intensity at 480 nm was recorded at different pH conditions. The scanning increment was 0.5 nm and integration time was 2 seconds.
156 Since diffusion into silica matrix was slow, we found that fluorescence signals required about 30 min to be stable. So for each sample, we took measurements every 2 min around 20 times. The data we used to plot were all from the measurements at 30 min.
4.2.5.2 Time Scale Fluorescence Spectroscopy
To examine the aggregation process of Aβ peptides in confined space over time, we monitored the fluorescence intensity changing upon pH at 2.3 and 7.1.
In this experiment, samples containing about 0.012 µg of Aβ were put into 3 ml 5
µM ThT phosphate buffer (pH 2.3, 0.05M) in fluorescence cuvette. Fluorescence spectra were taken until signals went stable. Then 0.29 ml 1N NaOH was added into the cuvette to give the pH jump from 2.3 to 7.1 directly. Once NaOH solution was added in, fluorescence spectra was recorded about every 5 min until the signal became stable.
4.2.6 Congo Red Birefringence Assay
4.2.6.1 Preparation of the Staining Solution
To a solution of 80% EtOH: 20% distilled deionized water, a saturating amount of
NaCl was added. This solution was then stirred for a few minutes followed by filtering to remove the excess NaCl. An excess amount of Congo Red was added under stirring followed by filtration to obtain the final working solution. This staining solution was be used on the day of preparation.
4.2.6.2 Incubation of Silica Biogel Samples in Buffer Solution
ThT Buffer solutions with pH values of 2.3 and 7.1 were used to incubate encapsulated Aβ peptides. ThT concentration in the phosphate buffer solution was 5 µM and the phosphate ion concentration was remained as 0.05 M.
157 Abeta42-0 and Abeta42-50 were used in this assay because these two samples had
the smallest and largest pore sizes to give two distinct results. For each sample, about
0.01 gram biogel powder was incubated in 3 ml ThT buffer solution and the incubation
time was all kept one hour.
4.2.6.3 Mounting Silica Biogel Samples on Glass Slides
After incubation, the sediment of silica biogel was taken out by a pipette. Then
one drop of this suspension was placed on a piece of glass slide and allowed to be air-
dried in about 5 min.
About 200–400 µL of the staining solution was dropped onto the dried silica biogel powder sample. After a few seconds, the excess solution was blotted away with a lint-free Kimwipe, being careful not to touch the sample. The stained sample was allowed to be dry at room temperature in air. But the sample should not be totally dry, when most of solution was evaporated out and the surface of silica powder was still moisture, one or two drops of Gel/Mount were placed on the slides to cover the biogel powder sample.
Then touch the slide with one edge of the coverslip, gently lower the coverslip onto the slide, taking care not to create any bubbles. Seal the edges of the coverslip with nail polish. After that, the sample was ready for microscope observation.
The stained sample was examined by using a polarized light microscope (PLM)
(BX-51, Olympus, Japan) equipped with digital camera.
4.2.7 Negative staining of amyloid fibrils for TEM
The staining solution is 2% uranyl acetate in distilled deionized water. This solution can be stored for several weeks at 4 oC. 3 µl suspension sample was placed in the copper grid (carbon coated Cu grid, Lot #: 1050724, Spi supplies, West Chester, PA).
158 After 3 min, a piece of filter paper was placed at the edge of the grid to absorb the remaining liquid. Immediately (i.e., do not let the sample dry on the grid), place 3 µL of staining solution on the grid. After 3 min, excess solution was wicked away followed by drying in air.
The prepared Cu grid was stored in an EM grid case and ready for TEM examination.
TEM pictures have not been taken in this thesis work. The sample for TEM was prepared as described above. It will be carried out later in Wei laboratory by another researcher.
4.3 Result and Discussion
4.3.1 Characterization of Silica Matrix
The characterization of the porous silica matrix was carried out on a
Micromeritics 2010 ASAP system (Norcross, GA) after the removal of DMA template by water extraction. Table 4-1 summarized the porous parameters of Aβ series samples.
With the template weight percentage from 0 wt% to 50 wt%, both average pore size and pore volume increased. Figure 4-3 showed the average pore size was almost in the linear relationship with template content. Previous researches suggested that the aggregates of non-surfactant molecules instead of individual molecules might be responsible for directing the mesophase formation.58 As the sequence, with the increasing of templates amount from 0 wt% to 50 wt%, the aggregation extent of template molecules rise up to form larger pores.
4.3.2 Steady State Fluorescence Spectroscopy
159 As described before in experimental section, the changing of pH value of buffer
solution was achieved by adding predetermined amounts of NaOH solution step by step.
Thus the total volume of buffer solution inside the cuvette was not a constant, then the
concentration of the powder in suspension was changing. In the following calculation, the
fluorescence intensity was normalized according to concentration. Furthermore, in order
to eliminate the light scattering from silica powder, another set of control sample without
encapsulated Aβ peptide was done in the same time. So the final fluorescence intensity
data used in graphs were all recalculated according to the following equation:
I I I = S − C ms mc Vs Vc
Where,
I: calculated intensity
IS: intensity of sample from experiment
ms: mass of encapsulated Aβ peptide
Vs: volume of sample suspension
Ic: intensity of control sample
mc: mass of control sample
Vc: volume of control sample suspension.
4.3.2.1 Abata42 Series Samples
In Figure 4-4, the ratio of fluorescence intensity of entrapped Aβ1-42 peptides at different pH value over the intensity at pH 2.3 was used as y axis and the pH value as x axis. As indicated in this plot, as the pH value of ThT buffer solution was increased, the fluorescence intensity of encapsulated Aβ in silica matrix with three different pore sizes
160 were all increasing. Because only when ThT dye molecules combined with the amyloid
fibrils the fluorescence emission at 480 nm can be generated, the increase in fluorescence
intensity indicated increasing aggregation of Aβ1-42 peptide with rising pH of the buffer
solution. This observation was consistent with previous studies.42,43,59
In fact, the most important phenomenon in this plot is that the difference in the intensity ratio of these three samples with different template contents. In 0 wt% sample, the enhancement of fluorescence intensity was very small. With the template weight percentage increased to 30% and 50%, the intensity increased. According to the relationship between the template contents and porous structure of silica matrix as shown in Fig. 4-3, both average pore size and volume were increasing with template weight percentage. Therefore, the conclusion could be drawn that Aβ1-42 peptide encapsulated in
the silica matrix with larger pore size were more likely aggregated to fibril forms than in
the matrix with smaller pores as the pH value of buffer solution was increased.
The unique pore structure of our sol-gel silica material could be used to explain
the above phenomenon. Unlike MCM-41 family mesoporous materials, our materials do
not have long ordered range of nanoporous structure, discernable packing or orientation
of the nanopores/channels. The porosity of our samples is made of interconnected
channels of regular diameters. Such a worm-hole like structure were verified by the TEM
images. In this project, Aβ1-42 was encapsulated in these pores and channels. Compare to the amount of silica matrix (0.25 g), the amount of Aβ1-42 peptide was very small (200
µg). When the peptide HFIP solution was homogenously dispersed in silica sol, the peptide would be encapsulated separately inside the silica matrix. The purpose of using
HFIP as solvent was to dissociate Aβ1-42 peptide to monomeric state during the
161 encapsulation process. HFIP was found to be an excellent solvent to do this job.57 It was found that after treatment of HFIP, CD studies indicated that dissolving Aβ1-42 peptide in
100% HFIP removed any preexisting β-sheet secondary structure, yielding predominately
α-helix and random coil.
Hence we believe that in the silica matrix, encapsulated Aβ1-42 peptides were
distributed separately in their monomeric state or lower oligomeric state without large aggregation forms. When the silica biogel was immersed in an acidic buffer solution, the encapsulated peptides could remain in their monomeric state because the highly acidic
environment prevented aggregation. Thus, the fluorescence intensity was low at this time.
With the pH value increasing, the basic environment stimulated encapsulated peptides to
aggregate. The movement of peptides in large pores was easier than in small pores: in
large pores peptides could move around and meet other peptides nearby to form
aggregates; but in small pores most of peptides were fixed in their positions and did not
have enough freedom to move and meet other peptides. Thus more aggregates could be
formed in large pores than in small pores, then appeared in fluorescence spectroscopy,
the intensity of encapsulated Aβ1-42 in large pores were higher than in small pores.
4.3.3 Time Scale Fluorescence Spectroscopy
In Figure 4-5, the change in fluorescence peak intensity at 480 nm is plotted
against time upon adding NaOH solution to increase the pH value of buffer. The ratio of
fluorescence intensity over the initial intensity was used as y axis. Three trajectories
represent three samples with different pore size.
A same tendency could be observed from all three trajectories. The intensity
increased rapidly in the initial 10 min, then the increment became less and less and finally
162 the intensity reached a constant number. We believed that there were two reasons behind
the phenomenon. First, diffusion of small molecules into silica matrix may play an
important role. Unlike all the previous research in which Aβ1-42 peptide were in free
solution, the peptides were encapsulated in porous solid matrix in this study. To reach the
peptides inside porous materials, small molecules or ions must take some time to diffuse
through the porous structure. Second, aggregation is not a single molecule’s behavior, it
must involve more than one peptide. But in the silica matrix, all the peptides were
encapsulated quite separately. In order to form aggregates, those separated peptides must
travel through the matrix to reach to each other. Thus, movements of peptides should also
take some time.
According to previous researches, a time lag was observed during the aggregation
process.39 It was believed that small, but not necessarily detectable, amounts of prenucleus oligomers sequentially build up during the lag time (Figure 4-6). The rate of nucleus formation is slow, owing primarily to the unfavorability of the preceding association equilibria rather than to the intrinsically slow association rates.60 The length of the lag time can be extremely sensitive to protein concentration, depending on the oligomer size of the nucleus and other mechanistic details that are unclear yet in the case of Aβ (30–33). 60,61 But in our case, the lag is not detected. Even though we do not know the exact mechanism yet, we think that the difference must come from the porous silica matrix where the encapsulated peptides reside in. One of the possible explanations is that because encapsulated peptides may adhere to the silica matrix inner wall, the silica matrix may serve as prenucleus seeds. This will be tested with organically modified nanoporous
163 matrices in which the strength of interactions between peptides and wall of matrix
materials can be controlled.
It was supposed that small pore size should slow the diffusion rate and then the
peptide would need more time to aggregate, appeared on the graph, the intensity might
take longer to reach a constant. But there wasn’t much difference among these three
samples’ trajectories (Fig. 4-5), which indicated the pore size had no effect on
aggregation rate of encapsulated peptides. We believe that is because even compare to the
smallest pore size of silica matrix (around 3 nm), the size of Aβ1-42 peptide which only have 42 amino acids is so small that the difference of diffusion rates of peptides in large and small pores is not large enough to be appeared in the plot (i.e. Fig. 4-5).
Another possibility is the time interval between recorded data was too large to conceal the difference in diffusion rates, especially for the first 5 min. So in the future work, we suggest redesigning the experiment to monitor the fluorescence intensity change continuously in the first 5 min upon adding NaOH solution.
4.3.4 Congo Red Birefringence Assay
We tried to obtain the images of encapsulated peptide fibril in silica matrix.
Unfortunately, it turned out that Congo Red birefringence assay did not give good results.
As shown in microscope images in Figure 4-7 and Figure 4-8, silica particles could be
found but the characteristic yellow/green bright spots for amyloid fibril were absent.
We believe that there may be several reasons that prevent Congo Red birefringence assay to be a good method in this study. First, the magnification of optical light microscope used in Congo Red birefringence assay was not enough to detect amyloid fibril in porous silica materials. In fact, the average pore size in silica matrix is
164 below 5 nm, which is already beyond the limit of optical microscope. Amyloid fibrils
were supposed to be formed inside those pores, thus the fibrils light spots may be too small to be detected by the optical microscope. Second, the shape of silica particles was irregular. Since the refraction index of the mounting gel we used was not exactly same as the silica materials, light would be refracted in the interfaces. As the result, in the polarized microscope pictures, we can see some the light regions on silica particles, but clearly these light regions were not due to amyloid fibril but the light refraction at the interfaces. The true light spots from amyloid fibrils may be covered by those light regions.
We believe that the most appropriated method to see the fibril directly might be
TEM picture of negative staining amyloid fibrils. The detection limit of TEM can go down to nanometer level, which is consistent with the scale of pore size of silica matrix.
4.4 Conclusion
Aβ1-42 peptides in their monomeric or lower oligomeric state were encapsulated in
silica matrices with controlled pore size via non-surfactant templated sol-gel pathway to
form silica biogel. By changing the pH value of solution where the silica biogel powders
were immersed in, aggregation behavior of the encapsulated Aβ1-42 was monitored by
ThT fluorescence spectroscopy. Congo Red birefringence microscope assay was also used to try and failed to get the picture of encapsulated amyloid fibrils.
The important finding is that pore size had a significant effect on the peptide aggregation behavior. Peptides encapsulated in large pore have more chances to form fibrils than those in small pores. It is easy to be understood since in large pores peptides have more freedom to interact to form aggregate. In contrast, in small pores, peptides are
165 more likely fixed in pores and channels in silica matrix and can not move about to form
large aggregates.
In most of the previous researches on Aβ1-42 aggregation, free Aβ1-42 solution was
used in experiments. However, in living organism, the environments where the peptides
are very crowed and filled with a lot of other cellular substances. Hence in this project, we provided a new platform, silica matrix with controlled pore size, to mimic the true environments to study amyloid peptide aggregation. We believe further investigations based on this platform will provide us new insights into amyloid peptides aggregation.
Furthermore, the successful encapsulation Aβ1-42 inside the silica porous materials without losing its bioactivity proves that this encapsulation method can be used in various applications, such as biosensor materials, biocatalyst etc.
4.5 Acknowledgements
I want to thank Dr. Patrick J. Loll in the Department of Biochemistry &
Molecular Biology, College of Medicine at Drexel University for his help and valuable discussions on CD measurements. I also thank Dr. Alexander V. Mazin in the
Department of Biochemistry & Molecular Biology, College of Medicine at Drexel
University for his help and valuable discussions on fluorescence measurements. I also want to thank Dr. Christopher Li and his student, Lingyu Li in the Department of
Materials Science and Engineering at Drexel University for their help on polarized light microscopy.
This work was done in collaboration with Prof. Jian-Min Yuan and Prof. Reinhard
Schweitzer-Stenner of Department of Chemistry of Drexel University.
166 4.6 References
1. Tabaton, M. & Piccini, A. Role of water-soluble amyloid-beta in the pathogenesis of Alzheimer's disease. International Journal of Experimental Pathology 86, 139- 145 (2005). 2. LeVine, H. The Amyloid Hypothesis and the clearance and degradation of Alzheimer's beta-peptide. Journal of Alzheimers Disease 6, 303-314 (2004). 3. Parihar, M. S. & Hemnani, T. Alzheimer's disease pathogenesis and therapeutic interventions. Journal of Clinical Neuroscience 11, 456-467 (2004). 4. Agorogiannis, E. I., Agorogiannis, G. I., Papadimitriou, A. & Hadjigeorgiou, G. M. Protein misfolding in neurodegenerative diseases. Neuropathology and Applied Neurobiology 30, 215-224 (2004). 5. Hashimoto, M., Rockenstein, E., Crews, L. & Masliah, E. Role of protein aggregation in mitochondrial dysfunction and neurodegeneration in Alzheimer's and Parkinson's diseases. Neuromolecular Medicine 4, 21-35 (2003). 6. De Felice, F. G. & Ferreira, S. T. beta-amyloid production, aggregation, and clearance as targets for therapy in Alzheimer's disease. Cellular and Molecular Neurobiology 22, 545-563 (2002). 7. Thompson, A. J. & Barrow, C. J. Protein conformational misfolding and amyloid formation: Characteristics of a new class of disorders that include Alzheimer's and prion diseases. Current Medicinal Chemistry 9, 1751-1762 (2002). 8. Murphy, R. M. Peptide aggregation in neurodegenerative disease. Annual Review of Biomedical Engineering 4, 155-174 (2002). 9. Clippingdale, A. B., Wade, J. D. & Barrow, C. J. The amyloid-beta peptide and its role in Alzheimer's disease. Journal of Peptide Science 7, 227-249 (2001). 10. Dumery, L., Bourdel, F., Soussan, Y., Fialkowsky, A., Viale, S., Nicolas, P. & Reboud-Ravaux, M. beta-amyloid protein aggregation: its implication in the physiopathology of Alzheimer's disease. Pathologie Biologie 49, 72-85 (2001). 11. Harkany, T., Abraham, I., Konya, C., Nyakas, C., Zarandi, M., Penke, B. & Luiten, P. G. M. Mechanisms of beta-amyloid neurotoxicity: Perspectives of pharmacotherapy. Reviews in the Neurosciences 11, 329-382 (2000). 12. McLaurin, J., Yang, D. S., Yip, C. M. & Fraser, P. E. Review: Modulating factors in amyloid-beta fibril formation. Journal of Structural Biology 130, 259-270 (2000). 13. Barrow, C. J. Synthesis, structure and neurotoxicity of the A beta peptide of Alzheimer's disease. Protein and Peptide Letters 6, 271-279 (1999). 14. Storey, E. & Cappai, R. The amyloid precursor protein of Alzheimer's disease and the A beta peptide. Neuropathology and Applied Neurobiology 25, 81-97 (1999). 15. Evin, G., Beyreuther, K. & Masters, C. L. Alzheimers-disease amyloid precursor protein (a-Beta-Pp) - proteolytic processing, secretases and beta-a4 amyloid production. Amyloid-International Journal of Experimental and Clinical Investigation 1, 263-280 (1994). 16. Wisniewski, T., Ghiso, J. & Frangione, B. Alzheimers-disease and soluble a-beta. Neurobiology of Aging 15, 143-152 (1994).
167 17. Selkoe, D. J. Alzheimer's disease: Genes, proteins, and therapy. Physiological Reviews 81, 741-766 (2001). 18. Selkoe, D. J. Amyloid beta-protein and the genetics of Alzheimer's disease. Journal of Biological Chemistry 271, 18295-18298 (1996). 19. Selkoe, D. J. Neuroscience - Alzheimer's disease: Genotypes, phenotype, and treatments. Science 275, 630-631 (1997). 20. Marx, J. L. Brain protein yields clues to Alzheimer's disease. Science 243, 1664-6 (1989). 21. Reyes, A. E., Chacon, M. A., Dinamarca, M. C., Cerpa, W., Morgan, C. & Inestrosa, N. C. Acetylcholinesterase-A beta complexes are more toxic than A beta fibrils in rat hippocampus - Effect on rat beta-amyloid aggregation, laminin expression, reactive astrocytosis, and neuronal cell loss. American Journal of Pathology 164, 2163-2174 (2004). 22. Inestrosa, N. C., De Ferrari, G. V., Garrido, J. L., Alvarez, A., Olivares, G. H., Barria, M. I., Bronfman, M. & Chacon, M. A. Wnt signaling involvement in beta- amyloid-dependent neurodegeneration. Neurochemistry International 41, 341-344 (2002). 23. Kowall, N. W., Beal, M. F., Busciglio, J., Duffy, L. K. & Yankner, B. A. An invivo model for the neurodegenerative efects of beta-amyloid and protection by substance-P. Proceedings of the National Academy of Sciences of the United States of America 88, 7247-7251 (1991). 24. Yankner, B. A., Duffy, L. K. & Kirschner, D. A. Neurotrophic and neurotoxic effects of amyloid beta-protein - Reversal by tachykinin neuropeptides. Science 250, 279-282 (1990). 25. Whitson, J. S., Selkoe, D. J. & Cotman, C. W. Amyloid b protein enhances the survival of hippocampal neurons in vitro. Science (Washington, DC, United States) 243, 1488-90 (1989). 26. Lorenzo, A. & Yankner, B. A. Beta-amyloid neurotoxicity requires fibril formation and is inhibited by congo red. Proceedings of the National Academy of Sciences of the United States of America 91, 12243-12247 (1994). 27. Mattson, M. P., Cheng, B., Davis, D., Bryant, K., Lieberburg, I. & Rydel, R. E. Beta-amyloid peptides destabilize calcium homeostasis and render human cortical-neurons vulnerable to excitotoxicity. Journal of Neuroscience 12, 376- 389 (1992). 28. Koh, J. Y., Yang, L. L. & Cotman, C. W. Beta-amyloid protein increases the vulnerability of cultured cortical-neurons to excitotoxic damage. Brain Research 533, 315-320 (1990). 29. Pike, C. J., Burdick, D., Walencewicz, A. J., Glabe, C. G. & Cotman, C. W. Neurodegeneration induced by beta-amyloid peptides invitro - the role of peptide assembly state. Journal of Neuroscience 13, 1676-1687 (1993). 30. Talafous, J., Marcinowski, K. J., Klopman, G. & Zagorski, M. G. Solution structure of residues-1-28 of the amyloid beta-peptide. Biochemistry 33, 7788- 7796 (1994). 31. Sisodia, S. S., Koo, E. H., Beyreuther, K., Unterbeck, A. & Price, D. L. Evidence that beta-amyloid protein in Alzheimers-Disease is not derived by normal Processing. Science 248, 492-495 (1990).
168 32. Roher, A. E. & Kuo, Y. M. in Amyloid, Prions, and Other Protein Aggregates 58- 67 (ACADEMIC PRESS INC, San Diego, 1999). 33. Morgan, C., Colombres, M., Nunez, M. T. & Inestrosa, N. C. Structure and function of amyloid in Alzheimer's disease. Progress in Neurobiology 74, 323- 349 (2004). 34. Bush, A. I., Masters, C. L. & Tanzi, R. E. Copper, beta-amyloid, and Alzheimer's disease: Tapping a sensitive connection. Proceedings of the National Academy of Sciences of the United States of America 100, 11193-11194 (2003). 35. Bush, A. I., Pettingell, W. H., Multhaup, G., Paradis, M. D., Vonsattel, J. P., Gusella, J. F., Beyreuther, K., Masters, C. L. & Tanzi, R. E. Rapid induction of Alzheimer a-beta amyloid formation by Zinc. Science 265, 1464-1467 (1994). 36. Mantyh, P. W., Ghilardi, J. R., Rogers, S., Demaster, E., Allen, C. J., Stimson, E. R. & Maggio, J. E. Aluminum, iron, and zinc ions promote aggregation of physiological concentrations of beta-amyloid peptide. Journal of Neurochemistry 61, 1171-1174 (1993). 37. Nilsson, M. R. & Raleigh, D. P. Analysis of amylin cleavage products provides new insights into the amyloidogenic region of human amylin. Journal of Molecular Biology 294, 1375-1385 (1999). 38. Dong, A., Matsuura, J., Manning, M. C. & Carpenter, J. F. Intermolecular beta- sheet results from trifluoroethanol-induced nonnative alpha-helical structure in beta-sheet predominant proteins: Infrared and circular dichroism spectroscopic study. Archives of Biochemistry and Biophysics 355, 275-281 (1998). 39. Harper, J. D. & Lansbury, P. T. Models of amyloid seeding in Alzheimier's disease and scrapie: Mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annual Review of Biochemistry 66, 385-407 (1997). 40. Orlando, R., Kenny, P. T. M. & Zagorski, M. G. Covalent modification of Alzheimers amyloid beta-peptide in formic-acid solutions. Biochemical and Biophysical Research Communications 184, 686-691 (1992). 41. Jackson, M. & Mantsch, H. H. Beware of proteins in DMSO. Biochimica Et Biophysica Acta 1078, 231-235 (1991). 42. Levine, H. Thioflavine-T interaction with synthetic Alzheimers-Disease beta- amyloid peptides - Detection of amyloid aggregation in solution. Protein Science 2, 404-410 (1993). 43. Levine, H. Thioflavine-T interaction with amyloid beta-sheet structures. Amyloid- International Journal of Experimental and Clinical Investigation 2, 1-6 (1995). 44. Westermark, G. T., Gebre-Medhin, S., Steiner, D. F. & Westermark, P. Islet amyloid development in a mouse strain lacking endogenous islet amyloid polypeptide (IAPP) but expressing human IAPP. Molecular Medicine 6, 998-1007 (2000). 45. Bonifacio, M. J., Sakaki, Y. & Saraiva, M. J. 'In vitro' amyloid fibril formation from transthyretin: The influence of ions and the amyloidogenicity of TTR variants. Biochimica Et Biophysica Acta-Molecular Basis of Disease 1316, 35-42 (1996). 46. Jin, L. W., Claborn, K. A., Kurimoto, M., Geday, M. A., Maezawa, I., Sohraby, F., Estrada, M., Kaminsky, W. & Kahr, B. Imaging linear birefringence and
169 dichroism in cerebral amyloid pathologies. Proceedings of the National Academy of Sciences of the United States of America 100, 15294-15298 (2003). 47. Khurana, R., Uversky, V. N., Nielsen, L. & Fink, A. L. Is Congo red an amyloid- specific dye? Journal of Biological Chemistry 276, 22715-22721 (2001). 48. Skowronek, M., Stopa, B., Konieczny, L., Rybarska, J., Piekarska, B., Szneler, E., Bakalarski, G. & Roterman, I. Self-assembly of Congo Red - A theoretical and experimental approach to identify its supramolecular organization in water and salt solutions. Biopolymers 46, 267-281 (1998). 49. Ashburn, T. T., Han, H., McGuinness, B. F. & Lansbury, P. T. Amyloid probes based on Congo Red distinguish between fibrils comprising different peptides. Chemistry & Biology 3, 351-358 (1996). 50. Wei, Y., Dong, H., Xu, J. G. & Feng, Q. W. Simultaneous immobilization of horseradish peroxidase and glucose oxidase in mesoporous sol-gel host materials. Chemphyschem 3, 802-+ (2002). 51. Wei, Y., Xu, J. G., Feng, Q. W., Lin, M. D., Dong, H., Zhang, W. J. & Wang, C. A novel method for enzyme immobilization: Direct encapsulation of acid phosphatase in nanoporous silica host materials. Journal of Nanoscience and Nanotechnology 1, 83-93 (2001). 52. Feng, Q. W., Xu, J. G., Lin, M. D., Dong, H. & Wei, Y. One-step direct immobilization of acid phosphatase in mesoporous silica sol-gel materials. Abstracts of Papers of the American Chemical Society 220, U364-U364 (2000). 53. Dong, H., Xu, J. G., Feng, Q. W. & Wei, Y. Simultaneous immobilization of oxidase/peroxidase in the mesoporous sol-gel silicate matrix. Abstracts of Papers of the American Chemical Society 220, U364-U364 (2000). 54. Xu, J. G., Dong, H., Feng, Q. W. & Wei, Y. Direct immobilization of horseradish peroxidase in mesoporous hybrid sol-gel materials. Abstracts of Papers of the American Chemical Society 219, U421-U422 (2000). 55. Xu, J. G., Dong, H., Feng, Q. W. & Wei, Y. Immobilization and activin assay of horseradish peroxidase in mesoporous silica sol-gel materials. Abstracts of Papers of the American Chemical Society 219, U458-U459 (2000). 56. Wei, Y., Xu, J. G., Feng, Q. W., Dong, H. & Lin, M. D. Encapsulation of enzymes in mesoporous host materials via the nonsurfactant-templated sol-gel process. Materials Letters 44, 6-11 (2000). 57. Dahlgren, K. N., Manelli, A. M., Stine, W. B., Baker, L. K., Krafft, G. A. & LaDu, M. J. Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability. Journal of Biological Chemistry 277, 32046-32053 (2002). 58. Wei, Y., Jin, D. L., Ding, T. Z., Shih, W. H., Liu, X. H., Cheng, S. Z. D. & Fu, Q. A non-surfactant templating route to mesoporous silica materials. Advanced Materials (Weinheim, Germany) 10, 313-316 (1998). 59. LeVine, H. Stopped-flow kinetics reveal multiple phases of thioflavin T binding to Alzheimer beta(1-40) amyloid fibrils. Archives of Biochemistry and Biophysics 342, 306-316 (1997). 60. Jarrett, J. T. & Lansbury, P. T. Seeding one-dimensional crystallization of amyloid - a pathogenic mechanism in Alzheimers-Disease and scrapie. Cell 73, 1055-1058 (1993).
170 61. Eaton, W. A. & Hofrichter, J. The biophysics of sickle-cell hydroxyurea therapy. Science 268, 1142-1143 (1995).
171 Table 4- 1 Summary of porous parameters of Abeta series samples after removel of templates.
3 2 Sample Pore Size(BET, Pore Size(BJH, Vsp(cm /g) SBET(m /g) Relative Å) Å) Fluorescence Intensity at pH 7.02 0% Template 31.1 33.6 0.28 364 1.1
30% Template 38.7 33.3 0.29 299 1.5
50% Template 75.8 62.2 0.91 482 2.8
Free solution ______18
172
Table 4- 2 Relative fluorescence intensity of immobilized Amyloid β 1-42 and free at difference pH.
pH Relative Fluorescence Intensity
0% 30% 50% Free
2.35 1 1 1 1 2.97 1.1 1.1 1.1 1.1 3.49 1.1 1.1 1.1 4.2 4.74 1.2 1.4 1.4 6.6 5.57 1.3 1.6 2.6 12.4 6.3 1.2 1.7 3.3 17.5 7.02 1.1 1.5 2.8 17.9 8.05 1.3 1.8 2.8 18.9 8.9 1.6 2.1 2.5 19.6
173
Figure 4- 1 Model of alternate aggregation pathways.33
174
H C 3 S
N(CH3)2
N Cl
CH3
Figure 4- 2 Molecular structure of Thioflavin T.
175
)
A 80 70 er ( 60 amet 50 40 re Di o 30 P e 20 10 erag Av 0 0 102030405060 Weight Percentage of Template
Figure 4- 3 Relationship between average pore diameter and amount of DMA template used in the synthesis.
176 4.5
nce 4 3.5 0% Template 3 30% Template 2.5 50% Template 2 Intensity ve Fluoresce 1.5 1 0.5 Relati 0 2.35 2.97 3.49 4.74 5.57 6.3 7.02 8.05 8.9 pH
Figure 4- 4 (a) Relative fluorescence intensity of immobilized Amyloid β 1-42 at different pH.
25
Free solution 20
15
10
5 Relative Fluorescence Intensity 0 2.35 2.97 3.49 4.74 5.57 6.3 7.02 8.05 8.9 pH
Figure 4-4(b). Relative fluorescence intensity of free Amyloid β 1-42 at different pH.
177 Abeta42-0
y 5 t i
s Abeta42-30
n 4.5
te Abeta42-50 4 In
e 3.5 c n
e 3 c s 2.5 re o
u 2 l
F 1.5 e v
ti 1 a l
e 0.5 R 0 0 5 10 15 20 25 30 Time (min)
Figure 4- 5 Time scale fluorescence study of Abeta42 series samples when the pH value jumping from 2.35 to 7.02.
178
Figure 4- 6 The aggregation of Aβ1-42 peptides into amyloid fibrils typically begins with a “lag phase” in which no aggregation is observed. During this time, the entropically unfavorable process of initial association occurs. Once the aggregation process begins and a critical nucleus is formed, the aggregation proceeds rapidly into amyloid fibrils (solid line). The lag phase, however, can be overcome (dotted line) by the addition of a pre-formed nucleus (i.e., an aliquot of solution containing pre-formed fibrils). This schematic represents the “nucleation–polymerization” kinetics for amyloid fibril formation.39
179
(a)
(b)
Figure 4- 7 Microscope images of control silica samples without Aβ1-42 inside: (a) under ordinary light (b) under polarized light.
180
(a)
(b)
Figure 4- 8 Microscope images of Abeta42-50 samples with encapsulated Aβ1-42 inside after incubation in pH 7.1 buffer for 2 hours: (a) under ordinary light (b) under polarized light.
181 Chapter 5: Fabrication of Poly (2-hydroxyethyl methacrylate)-Silica Nanoparticle
Hybrid Nanofibers via Electrospinning
5.1 Introduction
In the last decade a new class of hybrid organic-inorganic nanocomposite material has drawn more attention. In these materials, organic and inorganic phases can be covalently bond with each other and the domain size of one of the two phases is below
100 nm. The resulting materials are more homogenous than conventional composite materials. They can carry both the advantages of organic materials like flexibility, light weight and the advantages of inorganic materials such as high strength, thermal and chemical stablity. 1-16
Besides the hybrid materials, another interesting topic in materials science is nanomaterials. When the size of materials can be reduced to the nanometer scale, some new characteristics can often be found.17-26 For example, as the diameters of fibrous materials reduce from micrometers to nanometers, some appealing characteristics may arise, such as extremely large surface area, various surface functionalities and excellent mechanical strength, etc.27
In this project, the synthesis of a new kind of organic-inorganic hybrid material with nanostructure was achieved by electrospinning of nanocomposite organic-inorganic hybrid materials. This new material may combine the advantages of both hybrid material and nano fibrous materials and give us some interesting properties.
5.1.1 Organic-Inorganic Nanocomposite: Opportunities to Advanced Materials
There are two important concerns in synthesis of organic-inorganic hybrid materials: (1) the interface between the organic and inorganic phase; (2) domain sizes of
182 these two phases. The final properties of hybrid materials depend strongly on these two factors. Normally, strong interactions, like covalent bonding between two phases can result in excellent mechanical properties. The smaller the domain sizes are, the more homogenous the materials will be. Traditional composite materials are frequently prepared by the mechanical blending of polymers and glass fibers or other inorganic filler materials. Hence the two phases are only physically mixed and domain sizes are in millimeter or micrometer scales. Due to the organic-inorganic thermodynamic incompatibility, mechanical failure occurs often at macroscopic interfaces between the inorganic filler and organic matrices. In addition, optical properties such as transparency of either inorganic component or organic polymers may not be maintained due to the large domain size.
The new nanocomposite materials provide a solution by reducing at least one of the phase domain sizes to 100 nm and smaller. Because reduced domain size results in enlargement of interfacial area between organic and inorganic phases, nanometer scale phase domains provide materials with increased interfacial interactions. Thus, the materials have a high degree of interpenetration between the two dissimilar phases. As a result of this high degree of mixing, the ordinarily sharp interfacial zone will be blurred and a continuum of the structure and the properties of organic and inorganic components could be achieved. The other interesting consequence of such a fine morphology is the optical transparency of these composites as long as their primary constituents are transparent. Those new properties enable the new nanocomposites to be used in wide variety of applications, such as scratch and abrasive-resistant hard coating, electrical and non-linear optical materials, contact lens materials, catalysis materials etc.28-30
183 There have been various synthetic routes to synthesize these nanocomposites. The organic component can be introduced as (1) a monomer or an oligomer which can be polymerized afterward;31-34 (2) a preformed polymer in molten, solution or emulsion states;30,35-38 (3) a preformed polymer network into which the future inorganic components can penetrate.39-41 In the same way, the inorganic component can be introduced as (1) a monomer;30,31,34-42 (2) presynthesized inorganic particles, whiskers, or platelets32,36,41,43-45 and (3) an existing nanoporous structure.46-49 In this present study, we use a monomer (specifically, 2-hydroxyethyl methacrylate) as the organic resource and preformed vinyl modified silica nanoparticles as the inorganic species to prepare nanocomposite with covalent bonding between the organic and inorganic phases. After the synthesis, electrospinning was used to fabricate this nanocomposite to nanofibrous material. In the following part, we are going to briefly introduce the electrospinning technique.
5.1.2 Electrospinning
The origin of electrospinning is from Formalas,50-56 who received a series of patents from 1934 to 1944, describing an experimental setup for the production of polymer filaments using an electrostatic force. Since the 1980s and especially in recent years, the electrospinning process has gained more attention probably due in part to surging interest in nanotechnology.57 Unlike the other fiber spinning techniques (wet spinning, dry spinning, melt spinning, gel spinning) to produce polymer fibers with diameters in the micrometer range, electrospinning can fabricate polymer fibers in the nanometer range.58 The high specific surface area and small diameter of electrospun nanofibers make them interesting candidates for a wide variety of applications including
184 multifunctional membrane, biomedical structural elements (scaffolding used in tissue engineering, wound dressing, drug delivery, artificial organs, vascular grafts),59-65 protective shields in special fabrics,66 filter media for submicron particles in separation industry,67-69 composite reinforcement,70,71 and sensor materials,72-74 etc.
A schematic diagram to describe electrospinning of polymer nanofibers is shown in Figure 5-1. Generally speaking, three components can build up an electrospinning system: a high voltage supplier, a capillary tube with a pipette or a needle of small diameter and a metal collecting screen.27 The electrospinning process is achieved by applying a high voltage between the capillary which is filled with the polymer fluid and the collector screen. In this process, the high voltage electric field induces a charge on the surface of the polymer fluid. Mutual charge repulsion and the contraction of the surface charges to the counter electrode cause a force directly opposite to the surface tension.75
With the intensity of the electric field increasing, the hemispherical surface of the fluid at the tip of the capillary tube first elongates to form a conical shape known as the Taylor cone. After a critical value at which the repulsive electrostatic force overcomes the surface tension is attained, the charged jet of fluid is ejected from the tip of the Taylor cone.76 The discharged polymer solution jet undergoes an elongation process, which allows the jet to be very long and thin. During the polymer solution’s flying process to the collector screen, the solvent evaporates and polymer fibers are collected on the screen.
The morphology of electrospun nanofibers is controlled by the following parameters77: (a) System parameters, like molecular weight, molecular weight distribution and architecture (branched, linear etc.) of the polymer or viscosity, elasticity, conductivity and surface tension of the solution; (b) Process parameters such as electric
185 voltage, flow rate, concentration, distance and angle between the capillary and collection screen. Among them one of the most significant parameters influencing the fiber diameter is the viscosity of the solution. Normally high viscosity means large diameter of electrospun nanofibers. Because the viscosity of a polymer solution is always proportional to the polymer concentration and molecular weight of the polymers, the higher the polymer concentration the larger resulting nanofiber diameters will be. On the other hand, the viscosity of a polymer solution should not be too high; otherwise the viscosity may severely hinder the motion of the polymer solution induced by the electric field. Another parameter, which affects the fiber diameter to a remarkable extent is the applied electrical voltage. In general, a higher applied voltage ejects more fluid in a jet, resulting in a larger fiber diameter.78
One of the biggest problems encountered in electrospinning is that defects, such as beads and pores, may occur in polymer nanofibers. Beads can be considered as capillary break-up of the jets by surface tension. A lot of parameters, such as charge density carried by the jet, surface tension and viscoelastic properties of the solution can all affect the morphology of electrospun fibers. For example, it was pointed out that by reducing surface tension of a polymer solution, fibers could be obtained without beads and the higher the solution viscosity is, the fewer beads are formed. 58,77
5.2 Experimental
5.2.1 Materials
Highlink OG100-31, silica nanoparticles with surface modification of 2- hydroxylethyl methacrylate (HEMA), was provided by Clariant Corporation. Ethyl
186 alcohol (ACS/USP grade, Pharmco, Athens, GA), n-hexane (HPLC grade, 95+%,
Aldrich), dimethylformamide (DMF, 99%, Aldrich), phosphorus pentoxide (97%,
Aldrich) were all used as received without further purification. Benzoyl peroxide (BPO, minimum 75%, Sigma) was recrystalized before usage.
5.2.2 Synthesis of the Hybrid Material
The OG100-31 sample is a viscous, light yellow transparent liquid. According to the supplier, Clariant Corporation, the 70 wt% liquid component in the sample is HEMA monomers and the 30 wt% solid component is silica nanoparticles with diameters of 13 nm. Those silica nanoparticles are stably suspended in HEMA monomers without any precipitation over a long period. Moreover, as these nanoparticles are grafted by HEMA monomers on the surface, they can copolymerize with the free HEMA of the suspension medium and work as a crosslinker of high functionality. Thus, the inorganic component, silica nanoparticles, can be covalent bonded with the organic component, poly (2- hydroxylethyl methacrylate) (PHEMA).
Ethyl alcohol was used as a cosolvent and BPO as a thermal initiator during the synthesis. Typically, 11.3 g OG100-31 was dissolved in 33.8 g ethanol (about 25 wt %) in a three-neck round bottom flask. The resulting suspension was translucent and white.
Then 0.034 g BPO (0.3 wt %) was dissolved in the suspension as thermal initiator. Very interestingly, as the colorless BPO crystal was added into the white suspension, the color turned into deep green. Though this color changing mechanism is not clear yet, it may be due to the adsorption of BPO molecules on the surface of silica nanoparticles. Then under protection of nitrogen gas, the mixture was heated up to 65 oC with continuous stirring.
As the polymerization reaction proceeded on, the color of the mixture changed from deep
187 green to white with a little bit of pink and the viscosity of the liquid increased greatly.
After 12 hours, heating was stopped and the mixture was cooled down to room temperature. Then it was added dropwise into a large amount of n-hexane to precipitate the product. During the precipitation process, n-hexane was kept stirring. White floccules of hybrid material products were formed during the process. The floccules were then washed by n-hexane three times followed by separation through filtering.
The products were collected in the watch glass and put into a vacuum oven to dry.
For absorbing moisture and solvent from the products, phosphorus pentoxide was put into the vacuum oven as desiccant and changed every day. When the mass of the products became a constant of 9.4 gram, they were removed and ready for redissolution for electrospinning and characterizations. The drying procedure normally took about one week.
5.2.3 Set-up of Electrospinning Apparatus
The picture of the set-up of electrospinning apparatus is shown in Figure 5-2. The sample holder was a glass pipette. It was inserted into the syringe and fixed on an iron stand by a clamp. By adjusting the clamp, different angles between the pipette and collector screen could be achieved. Both the iron stand and the clamp were covered by insulated tapes to prevent short circuit. A thin metal needle, which was connected to the positive electrode was inserted into the glass pipette and touched the polymer solution. A piece of copper plate was used as collector screen, which is connected to the ground. It was fixed vertically to the ground and faced to the pipette. In the electrospinning process, the copper plate was always covered by a piece of aluminum foil. Then electrospun nanofibers could be formed on the foil and easily be removed for further
188 characterizations. Both collector screen and sample holder were covered by a transparent plastic cubic to prevent electric shocking. The high voltage is supplied by an ES30P-10W
HV Power Supply (GAMMA High Voltage Research, Ormond Beach, FL). The adjustable voltage range is up to 30 kV.
5.2.4 Electrospinning of PHEMA-Silica Hybrids
Because the PHEMA-silica hybrids were slightly crosslinked, to find a good solvent was a big problem. It was found that EtOH is a good solvent for PHEMA but not suitable for electrospinning. On the other hand, DMF is a good solvent for electrospinning but not a good solvent for the hybrid material. So mixtures of the two solvents were used to dissolve PHEMA-silica hybrids for electrospinning. To investigate the solvent effects on electrospun nanofibers, several co-solvents with different ratios of the two portions were used to dissolve the hybrid materials. The ratio was from 20:80 to
80:20 by volume.
Since the concentration of polymer solution is also an important factor on electrospun fibers, 15 wt%, 20 wt% and 25 wt% solutions were compared.
Typically, about 1 to 2 ml of solution was put into the pipette. Then a 20kV electric field was applied between the polymer solution and collector screen. Under the electric driven force, very thin sprays from the pipette tip could be observed traveling to the collector screen. After 5 min, a layer of white solid could be seen on the surface of the collector screen.
5.2.5. Instrumentation and Characterization
5.2.5.1 FTIR Spectroscopy
189 Samples were scratched from the collector screen. Infrared spectra of KBr powder-pressed pellets were recorded on a Perkin-Elmer Model 1600 FTIR spectrophotometer.
5.2.5.2 Thermal Gravimetric Analysis (TGA)
The tests were performed on a Q50 TGA instrument (Thermal Analysis
Instruments, DE) in air atmosphere for all the experiments. In a typical procedure, the temperature was ramped to 100 oC at the rate of 20 oC per min, then isothermal for 10 min to get rid of moisture in samples. Then the temperature was ramped from room temperature at the speed of 20 oC / min to 900 oC.
5.2.5.3 SEM and TEM
Scanning electron microscopy (SEM) was performed on an Environmental scanning electron microscope (ESEM, Philips XL30), with an accelerating voltage of 20 kV. The samples were gold-coated before the SEM characterization. Because the nanofibers were directly electrospun on aluminum foil, it was easy to cut one piece from the foil and attached it onto a SEM mount by double-side conductive scotch tape.
Transmission Electron Microscopy (TEM) was performed on a Transmission
Electron Microscope (TEM, JEOL 2000FX). Carbon coated Cu grid (Spi supplies, West
Chester, PA) was used as sample holder. In order to put a single layer of nanofibrous materials on the grid, Cu grids were attached on the center of collected screen and electrospun for only 10 seconds. In this way, single electrospun nanofiber could be observed under TEM.
5.3 Results and Discussion
190 The OG100-31 sample from Clariant Cooperation was used as initial reagent in this study. Unlike other silica nanoparticle suspensions, which are commercially available, the silica nanoparticles in this reagent are modified with a HEMA monomer on their surface. Though information about bonds between HEMA monomer and silica nanoparticles is not available, it was believed that the bond, Si-O-C, exist between the
HEMA monomer and silica surface or can be formed during the heating process. Because of the double bonds in the grafted HEMA vinyl group, silica nanoparticles can be polymerized with surrounding free HEMA monomers. Figure 5-3 shows FTIR spectra for the pure PHEMA and hybrid nanofiber. Both spectra show a broad band between 3100 and 3700 cm –1 related to the presence of (i) C-OH (from PHEMA) and Si-OH (from the silica particles), (ii) inter- and intramolecular hydrogen bonds; a band around 3000 cm –1 related to C-H and a single band at 1700 cm –1 due to C=O stretching. The difference between these two spectra is in the region between 1300 cm –1 and 1000 cm –1. In this region, the bands of PHEMA bending-groups were not clearly identifiable in the spectrum of hybrid material, because they overlap with the broad bands of Si-O-Si and
Si-O-C in hybrid material. This indicated that inorganic component silica were covalently bonded to the organic component PHEMA.30,31,79,80
Thermal stability of the material was examined by TGA under air atmosphere.
The TGA curves of pure PHEMA and our hybrid material are shown on Figure 5-4. For pure PHEMA, weight loss occurred between 200 oC and 400 oC and the rate of decomposition was the fastest around 290 oC. This may be due to the thermal depolymerization of the polyacrylate. PHEMA completely degraded at 500 oC. There are two stages in the TGA trace of the hybrid fiber: the first stage below 200 oC may be due
191 to breaking of bonds connecting silica particles with PHEMA chains; the second stage ended at 400 oC was thought to be depolymerization of PHEMA. The remaining weight percentage is close to 30% which is also consistent with the data provided by Clariant
Company.
Since more than one of the HEMA monomers was grafted on the surface of silica particles, the particles can functionalize as crosslinking reagents during polymerization.
So the hybrid material was crosslinked and the crosslinking extent depended on the concentration of initiator and reaction time. Because the hybrid material must be dissolved in a particular solvent prior to electrospinning, the crosslinking level must be minimized to allow the material dissolve. Reducing the concentration of initiator and reaction time can lower crosslinking extent. On the other hand, the molecular weight of a polymer should be high enough to be electrospun, so the appropriated reaction time and concentration of initiator should be determined.
In this study, reaction time was kept at about 12 hours and the weight percentage of initiator was 0.3 wt%. Because this material was crosslinked, and non-soluble silica nanoparticles were embedded inside, it was difficult to get information about molecular weight. But the molecular weight could be estimated by watching the viscosity of the solution. If the viscosity was too low, it meant that the molecular weight was not high enough and the solution could not be electrospun to nanofibers, so only some electrospun particles were shown in Figure 5-5. On the other hand, if the viscosity was too high, the high crosslinked resulting material could not be easily dissolved in solvent.
TEM was the best analytical method to observe the intrinsic structure of the nanofibers. As shown in Figure 5-6, hybrid nanofibers electrospun from DMF-EtOH
192 mixed solution (50:50) exhibited a different morphology compared to PHEMA nanofibers. Compared to the pure PHEMA electrospun fibers with uniform diameter and smooth surface, the hybrid fiber showed rather large range of diameter distribution and roughness on its surface. These unique characteristics were due to the nature of our hybrid material. As in the discussion above, more or less extents of crosslinking should exist in the hybrid material and result in broadening of molecular weight distribution.
Because the molecular weight of a polymer has an effect on electrospun fibers, the diameter’s non-uniformity of electrospun fibers was caused by the wide molecular weight distribution. The roughness of fiber surface was believed to result from the presence of silica nanoparticle aggregation which can be seen clearly in the TEM picture in Figure 5-
6(b). As expected, the black particles with diameters of about 13nm embedded in the fiber indicated that the silica nanoparticles and HEMA monomers were connected with each other inside the hybrid material.
The composition of the nanocomposite sample was analyzed with EDX. As shown in the last TEM picture, the distribution of silica nanoparticles inside nanofibers was not even. This was also proven by EDX analysis. In Figure 5-7, the detection spot was focused on a bead in a fiber where silicon components were believed enriched. The results showed that the silicon weight percentage was around 40 wt%. Because aluminum foil was used as substrate, when subtracting the contribution from aluminum, the weight percentage of silicon in that spot should be around 42 wt%, which was much higher than
30 wt% of silica in OG100-31. The enrichment of silica content in this region indicated that the beads of hybrid fiber might be the induced by aggregation of silica nanoparticles.
193 In this study, DMF/ EtOH mixed solvents were used for electrospinning the hybrid material. The effect of composition of mixed solvent on fiber forming was also examined. The hybrid material was dissolved in mixed solvent with volume ratio of
DMF:EtOH from 80:20 to 20:80 then electrospun with all the other conditions identical.
With the ratio of DMF to EtOH dropping down, more and more beads were formed and the average diameter of the fiber became smaller, which is shown in SEM picture of Fig.
5-8. The trends could be explained in the following two ways. First, the different resistivities of solutions had an effect on morphology of electrospun fibers. DMF is a dipolar aprotic solvent. It has a high dielectric constant (36.7 at 25 oC and dipole moment
(3.8D)), therefore, it can be seen as a polyelectrolyte. On the other hand, the dielectric constant and dipole moment of EtOH are 24.3 at 25 oC and 1.69 D. With the composition of DMF increasing in the mixed solvent, the resistivity of the solution becomes lower.
According to Fong,81 the net charge density is inversely proportional to the resistivity of the solution and the higher the net charge density a solvent carried, the more likely that a smooth fiber will be formed. So as the ratio of DMF to EtOH increased from 20:80 to
80:20, the net charge density of the co-solvent became higher, thus the resulting electrospun nanofibers turned out to be smoother. Second, viscosity of the solution also played an important role. EtOH is a better solvent for the organic component PHEMA than DMF, and the viscosity of the solution increased with percentage of DMF. Since high viscosity leads to less beads,77 we found the hybrid material also obeyed the rule in this experiment.
The concentration of solution was found to have a significant influence on the fiber morphology such as fiber diameter and size distribution. It has been know that
194 viscosity and surface tension of a polymer solution play important roles in determining the size of fibers.82 When a solid polymer is dissolved in solvent, the viscosity is proportional to the concentration. So the higher concentration of the solution the larger diameter of electrospun fiber will be. Furthermore defects like beads and pores in electrospun fibers depended on concentration of solution. Fong et al. pointed out that higher concentration resulted in fewer beads.81 These relationships can also be applied to our hybrid material. As shown in Figure 5-9, beads were formed more than fibesr when the concentration was 15%. When the concentration went up to 20%, the number of beads were reduced and the diameter of the fiber was around 50-100 nm. When the concentration kept increasing to 25%, though beads were still observed due to crosslinking effect, the diameters of fiber increased to around 100-200 nm as expected.
5.4 Conclusion
In this study, organic-inorganic hybrid nanofibers of PHEMA/silica nanoparticles have been successfully obtained by electrospinning technique. Because the silica nanoparticles were modified with HEMA monomers on the surface, those particles were able to be covalently bonded to PHEMA matrix during the polymerization process. TEM and SEM pictures showed that nanoparticles with 13 nm diameter were embedded inside the electrospun nanofibers, and the average diameter of fiber was below 200 nm. The effects of concentration of solution and composition of mixed solvent on the morphology of electrospun nanofibers were examined. Fibers with average diameters of 50-100 nm were also obtained.
This might be the first reported electrospun nanofibers that has an organic
PHEMA component as matrix and inorganic silica particles embedded inside and
195 covalently bonded to PHEMA. Because the inorganic domain size in this fibrous material was 13 nm and was covalent bonded to the organic component, the interactions between organic and inorganic phases were stronger than conventional composite materials.
Furthermore, by the means of electrospinning, nanofibers of this nanocomposite were achieved and the average diameter of fiber can be below 200 nm. Because of the high surface area of nanofibers and the excellent thermal and mechanical properties brought by the hybrid nanocomposite, we believe that nanofibrous material can be used in many applications.
5.5 Acknowledgements
I want to thank Ms. Alpa C. Patel in our group for setting up the electrospinning apparatus. I also thank Dr. Shuxi Li in our group for his help and valuable discussions with TGA measurements.
5.6 References
1. Wen, J. Y. & Wilkes, G. L. Organic/inorganic hybrid network materials by the sol-gel approach. Chemistry of Materials 8, 1667-1681 (1996). 2. Shanmugam, S., Viswanathan, B. & Varadarajan, T. K. Preparation, characterization and electrocatalytic activity of phosphomolybdic acid embedded organic-inorganic nanocomposites. Indian Journal of Chemistry Section a- Inorganic Bio-Inorganic Physical Theoretical & Analytical Chemistry 44, 994- 1000 (2005). 3. Stathatos, E. Organic-inorganic nanocomposite materials prepared by the sol-gel route as new ionic conductors in quasi solid state electrolytes. Ionics 11, 140-145 (2005). 4. Park, Y. I., Moon, J. & Kim, H. K. Proton conducting organic-inorganic nanocomposite membranes from MPTS and GPTS. Electrochemical and Solid State Letters 8, A191-A194 (2005).
196 5. Shchipunov, Y. A., Karpenko, T. Y. & Krekoten, A. V. Hybrid organic-inorganic nanocomposites fabricated with a novel biocompatible precursor using sol-gel processing. Composite Interfaces 11, 587-607 (2005). 6. Park, J. & Kim, E. in On the Convergence of Bio-Information-, Environmental-, Energy-, Space- and Nano-Technologies, Pts 1 and 2 1039-1043 (TRANS TECH PUBLICATIONS LTD, Zurich-Uetikon, 2005). 7. Huang, Y., Pan, Q. Y., Cheng, Z. X., Zhang, W. & Xia, W. Synthesis and photochroism of a novel organic-inorganic nanocomposite film entrapping tungstosilicate acid. Chemical Journal of Chinese Universities-Chinese 26, 204- 208 (2005). 8. Cho, J. D., Ju, H. T. & Hong, J. W. Photocuring kinetics of UV-initiated free- radical photopolymerizations with and without silica nanoparticles. Journal of Polymer Science Part a-Polymer Chemistry 43, 658-670 (2005). 9. Wang, Y., Ma, C. L., Sun, X. L. & Li, H. Preparation and photoluminescence properties of organic-inorganic nanocomposite with a mesolamellar nickel oxide. Microporous and Mesoporous Materials 71, 99-102 (2004). 10. Chiang, C. L., Ma, C. C. M., Wu, D. L. & Kuan, H. C. Preparation, characterization, and properties of Novolac type phenolic/SiO2 hybrid organic- inorganic nanocomposite materials by sol-gel method. Journal of Polymer Science Part a-Polymer Chemistry 41, 905-913 (2003). 11. Zhang, L. Z. & Peng, C. Recent advances in the study of the optical and electronic properties of organic-inorganic nanocomposite materials. Chinese Journal of Inorganic Chemistry 19, 7-14 (2003). 12. Sayari, A. & Hamoudi, S. Periodic mesoporous silica-based organic - Inorganic nanocomposite materials. Chemistry of Materials 13, 3151-3168 (2001). 13. Costa, R. O. R., Vasconcelos, W. L., Tamaki, R. & Laine, R. M. Organic/inorganic nanocomposite star polymers via atom transfer radical polymerization of methyl methacrylate using octafunctional silsesquioxane cores. Macromolecules 34, 5398-5407 (2001). 14. Honma, I., Takeda, Y. & Bae, J. M. Protonic conducting properties of sol-gel derived organic/inorganic nanocomposite membranes doped with acidic functional molecules. Solid State Ionics 120, 255-264 (1999). 15. Shouji, E. & Buttry, D. A. New organic-inorganic nanocomposite materials for energy storage applications. Langmuir 15, 669-673 (1999). 16. Chen, Y. F., Jin, L. M. & Xie, Y. S. Sol-gel processing of organic-inorganic nanocomposite protective coatings. Journal of Sol-Gel Science and Technology 13, 735-738 (1998). 17. Patzke, G. R., Krumeich, F. & Nesper, R. Oxidic nanotubes and nanorods - Anisotropic modules for a future nanotechnology. Angewandte Chemie- International Edition 41, 2446-2461 (2002). 18. Joo, S. H., Choi, S. J., Oh, I., Kwak, J., Liu, Z., Terasaki, O. & Ryoo, R. Ordered nanoporous arrays of carbon supporting high dispersions of platinum nanoparticles. Nature 412, 169-172 (2001). 19. Moriarty, P. Nanostructured materials. Reports on Progress in Physics 64, 297- 381 (2001).
197 20. Johnson, S. A., Ollivier, P. J. & Mallouk, T. E. Ordered mesoporous polymers of tunable pore size from colloidal silica templates. Science 283, 963-965 (1999). 21. Martin, C. R. & Mitchell, D. T. Nanomaterials in analytical chemistry. Analytical Chemistry 70, 322A-327A (1998). 22. Che, G., Lakshmi, B. B., Martin, C. R., Fisher, E. R. & Ruoff, R. S. Chemical vapor deposition based synthesis of carbon nanotubes and nanofibers using a template method. Chemistry of Materials 10, 260-267 (1998). 23. Lakshmi, B. B., Patrissi, C. J. & Martin, C. R. Sol-gel template synthesis of semiconductor oxide micro- and nanostructures. Chemistry of Materials 9, 2544- 2550 (1997). 24. Hulteen, J. C. & Martin, C. R. A general template-based method for the preparation of nanomaterials. Journal of Materials Chemistry 7, 1075-1087 (1997). 25. Decher, G., Lehr, B., Lowack, K., Lvov, Y. & Schmitt, J. New nanocomposite films for biosensors - layer-by-layer adsorbed films of polyelectrolytes, proteins or DNA. Biosensors & Bioelectronics 9, 677-684 (1994). 26. Martin, C. R. Nanomaterials - a membrane-based synthetic approach. Science 266, 1961-1966 (1994). 27. Huang, Z. M., Zhang, Y. Z., Kotaki, M. & Ramakrishna, S. A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Composites Science and Technology 63, 2223-2253 (2003). 28. Weissman, J. M., Sunkara, H. B., Tse, A. S. & Asher, S. A. Thermally switchable periodicities and diffraction from mesoscopically ordered materials. Science 274, 959-960 (1996). 29. Asher, S. A., Holtz, J., Liu, L. & Wu, Z. J. Self-assembly motif for creating submicron periodic materials - polymerized crystalline colloidal arrays. Journal of the American Chemical Society 116, 4997-4998 (1994). 30. Landry, C. J. T., Coltrain, B. K. & Brady, B. K. In situ polymerization of tetraethoxysilane in poly(methyl methacrylate) - morphology and dynamic mechanical-properties. Polymer 33, 1486-1495 (1992). 31. Bosch, P., DelMonte, F., Mateo, J. L. & Levy, D. Photopolymerization of hydroxyethylmethacrylate in the formation of organic-inorganic hybrid sol-gel matrices. Journal of Polymer Science Part a-Polymer Chemistry 34, 3289-3296 (1996). 32. BourgeatLami, E., Espiard, P., Guyot, A., Gauthier, C., David, L. & Vigier, G. Emulsion polymerization in the presence of colloidal silica particles - Application to the reinforcement of poly(ethyl acrylate) films. Angewandte Makromolekulare Chemie 242, 105-122 (1996). 33. Espiard, P. & Guyot, A. Poly(Ethyl Acrylate) Latexes encapsulating nanoparticles of silica .2. grafting process onto silica. Polymer 36, 4391-4395 (1995). 34. Ellsworth, M. W. & Novak, B. M. Mutually interpenetrating inorganic organic networks - new routes into nonshrinking sol-gel composite-materials. Journal of the American Chemical Society 113, 2756-2758 (1991). 35. Girardreydet, E., Lam, T. M. & Pascault, J. P. In-situ polymerization of tetraethoxysilane in poly(vinyl acetate). Macromolecular Chemistry and Physics 195, 149-158 (1994).
198 36. Landry, C. J. T., Coltrain, B. K., Landry, M. R., Fitzgerald, J. J. & Long, V. K. Poly(vinyl acetate) silica filled materials - material properties of in-situ vs fumed silica particles. Macromolecules 26, 3702-3712 (1993). 37. Moriya, Y., Sonoyama, M., Nishikawa, F. & Hino, R. Preparation of polymer hybrids of the sio2-pva or sio2-peg system and porous materials made from the hybrid gels. Nippon Seramikkusu Kyokai Gakujutsu Ronbunshi-Journal of the Ceramic Society of Japan 101, 518-521 (1993). 38. Landry, C. J. T., Coltrain, B. K., Wesson, J. A., Zumbulyadis, N. & Lippert, J. L. Insitu polymerization of tetraethoxysilane in polymers - chemical nature of the interactions. Polymer 33, 1496-1506 (1992). 39. Wen, J. A. & Mark, J. E. Synthesis, structure, and properties of poly(dimethylsiloxane) networks reinforced by in situ-precipitated silica-titania, silica-zirconia, and silica-alumina mixed oxides. Journal of Applied Polymer Science 58, 1135-1145 (1995). 40. Wang, S. H., Ahmad, Z. & Mark, J. E. A Polyamide-silica composite prepared by the sol-gel process. Polymer Bulletin 31, 323-330 (1993). 41. Garrido, L., Mark, J. E., Sun, C. C., Ackerman, J. L. & Chang, C. NMR characterization of elastomers reinforced with insitu precipitated silica. Macromolecules 24, 4067-4072 (1991). 42. Novak, B. M. & Ellsworth, M. W. Inverse organic-inorganic composite-materials - high glass content non-shrinking sol-gel composites. Materials Science and Engineering a-Structural Materials Properties Microstructure and Processing 162, 257-264 (1993). 43. Bourgeatlami, E., Espiard, P. & Guyot, A. Poly(ethyl acrylate) latexes encapsulating nanoparticles of silica .1. functionalization and dispersion of silica. Polymer 36, 4385-4389 (1995). 44. Espiard, P., Guyot, A., Perez, J., Vigier, G. & David, L. Poly(ethyl acrylate) latexes encapsulating nanoparticles of silica .3. morphology and mechanical- properties of reinforced films. Polymer 36, 4397-4403 (1995). 45. Tsubokawa, N., Saitoh, K. & Shirai, Y. Surface grafting of polymers onto ultrafine silica - cationic polymerization initiated by benzylium perchlorate groups introduced onto ultrafine silica surface. Polymer Bulletin 35, 399-406 (1995). 46. Pope, E. J. A. & Mackenzie, J. D. Porous and dense composites from sol-gel. Materials Science Research 20, 187-94 (1986). 47. Pope, E. J. A. & Mackenzie, J. D. Sol-gel processing of silica. II. The role of the catalyst. Journal of Non-Crystalline Solids 87, 185-98 (1986). 48. Pope, E. J. A., Asami, M. & Mackenzie, J. D. Transparent silica gel-PMMA composites. Journal of Materials Research 4, 1018-26 (1989). 49. Klein, L. C. & Abramoff, B. PMMA-impregnated silica gel. Polymer Preprints (American Chemical Society, Division of Polymer Chemistry) 32, 519-20 (1991). 50. Formhals, A. ((Alien Property Custodian). US2349950, 1944). 51. Formhals, A. ((Alien Property Custodian). US2323025, 1943). 52. Formhals, A. ((Schreiber-Gastell, Richard). US2077373, 1937). 53. Formhals, A. ((Schreiber-Gastell, Richard). US1975504, 1934). 54. Formhals, A. (DE, US584801, 1933).
199 55. Formhals, A. (GB, US364780, 1929). 56. Formhals, A. ((Verein fur chemische industrie A.-G.). FR, US707191, 1930). 57. Baumgarten, P. K. Electrostatic spinning of acrylic microfibers. Journal of Colloid and Interface Science 36, 71-79 (1971). 58. Frenot, A. & Chronakis, I. S. Polymer nanofibers assembled by electrospinning. Current Opinion in Colloid & Interface Science 8, 64-75 (2003). 59. Zussman, E., Yarin, A. L. & Weihs, D. A micro-aerodynamic decelerator based on permeable surfaces of nanofiber mats. Experiments in Fluids 33, 315-320 (2002). 60. Kenawy, E. R., Bowlin, G. L., Mansfield, K., Layman, J., Simpson, D. G., Sanders, E. H. & Wnek, G. E. Release of tetracycline hydrochloride from electrospun poly(ethylene-co-vinylacetate), poly(lactic acid), and a blend. Journal of Controlled Release 81, 57-64 (2002). 61. Fertala, A., Han, W. B. & Ko, F. K. Mapping critical sites in collagen II for rational design of gene-engineered proteins for cell-supporting materials. Journal of Biomedical Materials Research 57, 48-58 (2001). 62. Buchko, C. J., Kozloff, K. M. & Martin, D. C. Surface characterization of porous, biocompatible protein polymer thin films. Biomaterials 22, 1289-1300 (2001). 63. Huang, L., McMillan, R. A., Apkarian, R. P., Pourdeyhimi, B., Conticello, V. P. & Chaikof, E. L. Generation of synthetic elastin-mimetic small diameter fibers and fiber networks. Macromolecules 33, 2989-2997 (2000). 64. Buchko, C. J., Slattery, M. J., Kozloff, K. M. & Martin, D. C. Mechanical properties of biocompatible protein polymer thin films. Journal of Materials Research 15, 231-242 (2000). 65. Buchko, C. J., Chen, L. C., Shen, Y. & Martin, D. C. Processing and microstructural characterization of porous biocompatible protein polymer thin films. Polymer 40, 7397-7407 (1999). 66. Schreuder-Gibson, H., Gibson, P., Senecal, K., Sennett, M., Walker, J., Yeomans, W., Ziegler, D. & Tsai, P. P. Protective textile materials based on electrospun nanofibers. Journal of Advanced Materials 34, 44-55 (2002). 67. Groitzsch, D. & Fahrbach, E. 30 pp ((Freudenberg, Carl, K.-G., Fed. Rep. Ger.). Application: DE, 1986). 68. Schreuder-Gibson, H. L., Gibson, P. & Hsieh, Y.-L. Transport properties of electrospun nonwoven membranes. International Nonwovens Journal [online computer file] 11, 21-26 (2002). 69. Emig, D., Klimmek, A. & Raabe, E. ((Fibermark Gessner Gmbh & Co., Germany). Application: EP, 2000). 70. Maruyama, B. & Alam, H. Carbon nanotubes and nanofibers in composite materials. Sampe Journal 38, 59-70 (2002). 71. Thostenson, E. T., Ren, Z. F. & Chou, T. W. Advances in the science and technology of carbon nanotubes and their composites: a review. Composites Science and Technology 61, 1899-1912 (2001). 72. Norris, I. D., Shaker, M. M., Ko, F. K. & MacDiarmid, A. G. Electrostatic fabrication of ultrafine conducting fibers: polyaniline/polyethylene oxide blends. Synthetic Metals 114, 109-114 (2000).
200 73. Drew, C., Wang, X., Senecal, K., Schreuder-Gibson, H., He, J., Tripathy, S. & Samuelson, L. Electrospun nanofibers of electronic and photonic polymer systems. Annual Technical Conference - Society of Plastics Engineers 58th, 1477-1481 (2000). 74. Senecal, K. J., Ziegler, D. P., He, J., Mosurkal, R., Schreuder-Gibson, H. & Samuelson, L. A. Photoelectric response from nanofibrous membranes. Materials Research Society Symposium Proceedings 708, 285-289 (2002). 75. Fang, X. & Reneker, D. H. DNA fibers by electrospinning. Journal of Macromolecular Science-Physics B36, 169-173 (1997). 76. Taylor, G. Electrically Driven Jets. Proceedings of the Royal Society of London. Series A, Mathematical and Physical Sciences 313, 453-475 (1969). 77. Doshi, J. & Reneker, D. H. Electrospinning Process and Applications of Electrospun Fibers. Journal of Electrostatics 35, 151-160 (1995). 78. Demir, M. M., Yilgor, I., Yilgor, E. & Erman, B. Electrospinning of polyurethane fibers. Polymer 43, 3303-3309 (2002). 79. Hajji, P., David, L., Gerard, J. F., Pascault, J. P. & Vigier, G. Synthesis, structure, and morphology of polymer-silica hybrid nanocomposites based on hydroxyethyl methacrylate. Journal of Polymer Science Part B-Polymer Physics 37, 3172-3187 (1999). 80. Kaddami, H., Gerard, J. F., Hajji, P. & Pascault, J. P. Silica-filled poly(HEMA) from HEMA/grafted SiO2 nanoparticles: Polymerization kinetics and rheological changes. Journal of Applied Polymer Science 73, 2701-2713 (1999). 81. Fong, H., Chun, I. & Reneker, D. H. Beaded nanofibers formed during electrospinning. Polymer 40, 4585-4592 (1999). 82. Deitzel, J. M., Kleinmeyer, J., Harris, D. & Tan, N. C. B. The effect of processing variables on the morphology of electrospun nanofibers and textiles. Polymer 42, 261-272 (2001).
201
Figure 5- 1 Schematic diagram to show polymer nanofibers by electrospinning.
202
Figure 5- 2 The picture of the set-up of electrospinning apparatus in our group.
203
115 Hybrid fiber
95 PHEMA f i ber
75
Transmittance (a. u.) 55 4000 3500 3000 2500 2000 1500 1000 500
wavelength (nm)
Figure 5- 3 FTIR spectra of pure PHEMA electrospun fiber and hybrid electrospun fiber.
204
) 120
(% 100 ge
ta 80
n e
c 60 r e 40 Hybrid Fiber
20 ght P i Pur e PHEMA Fiber e 0 W 0 200 400 600 800
Temperature (0C)
Figure 5- 4 TGA spectra of pure PHEMA electrospun fiber and hybrid electrospun fiber.
205
Figure 5- 5 SEM picture of hybrid electrospun fiber when the molecular weight of hybrid material was not high enough.
(a)
206
(b)
Figure 5- 6 SEM (a) and TEM (b) pictures of hybrid electrospun fiber.
207
Element Wt% At% C K 19.35 29.56 O K 35.73 40.96 AlK 05.39 03.66 SiK 39.53 25.82
Figure 5- 7 EDX analysis of hybrid electrospun fiber. (The white cross in the SEM picture indicates the detection spot)
208
80:20 70:30
60:40
40:60 Ratio of DMF to EtOH
30:70
20:80
Figure 5- 8 SEM pictures of electrospun hybrid fiber under different composition of solvents.
209
25 wt%
20 wt% Increase of Concentration
15 wt%
Figure 5- 9 SEM pictures of electrospun hybrid fiber in solutions with different concentrations.
210 Chapter 6: Synthesis and Characterization of Dental Composite Containing
Nanoporous Silica as Fillers
6.1 Introduction
Dental resin composites are an important group of dental restorative materials and are of great importance in restorative dentistry.1,2 For the restoration of anterior lesions and the supply of smaller and medium-sized defects in the posterior region, direct composite filling materials are used. For larger defects, ceramic restorations are prefabricated before and then attached to the tooth structure with composite-based cements. Dental composites usually consist of several components: highly crosslinked organic polymer matrix and dispersed inorganic, reinforcing filler particles (silica, zirconia, etc.) and coupling agent.3
6.1.1 Resin Matrix
Usually, the organic polymer matrix is constructed based on methacrylate chemistry. Especially crosslinked dimethacrylates, like 2,2-bis(4-(2-hydroxy-3- methacryloxypropoxy)phenyl) propane (Bis-GMA), ethoxylated Bis-GMA (EBPDMA), urethane dimethacrylate (UDMA), dodecanediol dimethacrylate (D3MA) or triethyleneglycol dimethacrylate (TEGDMA) are used in dental composites. The fist dental resin used as the direct esthetic restoration of teeth was unreinforced methyl methacrylate (MMA) resin.4,5 Because of its serious limitations of MMA in term of the durability and physical properties, Bowen et. al. developed a new dental resin composites, which combined the advantages from both epoxy and methacrylate resins by using the products of reaction of bisphenol and glycidyl methacrylate, BisGMA, as the resin matrix.6,7 Because all the above composite monomers carry more than one functional
211 group, a three-dimensional network is formed by free-radical polymerization of the monomers. The selection of the monomers strongly influences the reactivity, viscosity and polymerization shrinkage of the composite paste, the mechanical properties, water uptake, and swelling by water uptake in the cured composite materials.
The BisGMA based resin composites have been proved to be a successful material in esthetic restorations, especially in the requirement of reactivity, stability, biocompatibility and mechanical properties. There is still a major shortcoming with
BisGMA resins, i.e. marginal leakage due to volume shrinkage during polymerization and insufficient abrasion resistance. Therefore some of new monomers such as ring- opening monomers,8-15 liquid crystalline,16-19 branched or dendritic monomers,20,21 ormocers22-25 etc., for restorative dental materials have also been studied extensively.
6.1.2 Filler System
Since pure resin matrix itself usually can not provide sufficient physical and mechanical strength, inorganic fillers need to be incorporated into the organic polymer matrix to reinforce the material.1 The fillers include fused quartz, colloidal amorphous silica, glass particles containing metals (Ba, Zn, Zr, Sr) for radiopacity, lithium aluminum silicates, short glass fibers metal oxides (Al2O3, TiO2, ZrO2), hydroxyapatite powder, glass fibers, prepolymerized organic particles, organic-inorganic hybrid sol-gel materials, nanoporous sol-gel silica, and silica-fused silicon nitride ceramic single-crystalline whiskers.
The filler volume fractions, particle shape, size or size distribution, index of refraction, radiopacity, hardness and toughness are all important factors in determining the properties and the clinical performance of the composite material.
212 6.1.3 Coupling Agent
Another important component in dental composite system is the coupling agent.26-
31 Simply mechanical mixing inorganic fillers into resin matrix is usually not enough to achieve desired mechanical strength because of phase separation of inorganic and organic phases, especially in an oral environment where water may penetrate along the filler-resin interface. Strong and durable resin matrix-filler bonding is needed to reinforce the composite by allowing the stress transfer from the more plastic polymer matrix to the stiffer particles. This bond between the organic and inorganic phases of composites is provided by a coupling agent. A properly applied coupling agent can improve physical and mechanical properties and provide hydrolytic stability by preventing water from penetrating along the filler-resin interface. The most frequently used coupling agent is an organosilane, for example, 3-methacryloxypropyl trimethoxysilane. After hydrolysis, the silanol groups on the silane can bond to silanols on the filler surface by forming a siloxane bond (Si-O-Si). The methacrylate groups of the organosilane compounds provide double bonds for covalent bonding with the resin during curing, thus completing the coupling between the organic and inorganic phases. Appropriated coupling, by means of the organosilanes, is of critical importance to the clinical performance of conventional resin composites.
6.1.4 Limitations of Coupling Agent
Though organosilane coupling agents have been proven to be a good method to connect inorganic and organic phase together, it has its own deficiencies, i.e. hydrolysis of silane coupling between the fillers and resin in the oral environment might significantly reduce the mechanical properties of dental composites over time.32 There is
213 a lot of evidence in literatures that siliceous filler and the silane coupling in dental composite resins are hydrolytically unstable and undergo dissolution in water.33-35
The debonding of filler and resin matrix can result in micro-separation of inorganic and organic phases thus induce filler exposure and filler loss, which are believed to be the main reason for wear resistance reduction.
6.1.5 Porous Filler without Coupling Agent
The limitations of silane coupling agents can be overcome by improving the interaction between filler particles and the matrix of dental composite through micro- mechanical interlocking of the two phases, which was first proposed by Bowen and
Reed.7 In the absence of chemical bonding, there is no concern about hydrolysis at the filler-polymer matrix interface. Micromechanical bonding can be achieved by introducing the polymer matrix into the pore channels of inorganic fillers. Silica – polymer nanocomposites with interpenetrating networks have been made before in a number of ways.36 For dental applications, one of the practical ways is to first synthesize the porous inorganic filler, then mix with resin monomers and polymerize the mixture in situ i.e. within the tooth cavity. Nanoporous fillers also possess the potential to have less volume shrinkage compared to the inorganic – organic hybrid nanocomposites made in situ by sol
– gel process.1,37
Porous silica fillers (average pore size: 1 – 2.5 nm) have been tested as potential reinforcing agents in dental composites and have been shown to improve wear resistance.38 Luo, et al. showed that the wear resistance depended on the nature of the nanoporous structure present in those fillers.38 Mesoporous materials, with pore sizes in the range of 1.5 – 10 nm,39 have been explored for a number of potential applications. In
214 this work, nanoporous silica particle fillers were synthesized via non-surfactant templated sol-gel process,40 followed by heat treatments at different temperature.
6.1.6 Non-surfactant Templated Sol-Gel Process
A novel non-surfactant pathway to synthesize nanoporous materials via sol-gel process has been developed by our group.40-44 In this method, instead of using surfactant molecules as template, we chose non-surfactant molecules, such as glucose, fructose and urea, etc, to direct the mesopore formation during the sol-gel process. This method turned out to be an effective way to synthesize nanoporous materials with high surface area ( e.g.
1000 m2/g) and pore volume ( e.g. 0.5-1.0 cm3/g) as well as pore size in the range of 2-12 nm with narrow size distributions.
Unlike M41S family mesoporous materials which are synthesized via surfactant template pathway, our materials do not have long ordered range of nanoporous structure, discernable packing or orientation of the nanopores/channels. In fact, the porous structures inside non-surfactant templated samples are made of interconnected channels of regular diameters. Indeed, such a worm-hole like structure can be observed in the TEM images of all nanoporous materials prepared via the non-surfactant templated pathway.
Though the long range ordered structure is missing from the nonsurfactant templated materials, it is interesting to point out that it may be advantageous in this dental applications because the nanoporosity is accessible in all directions. Thus, resin polymer matrix can interlock with the silica matrix three-dimensionally. Furthermore, the walls of silica matrix in M41S family materials are always too thin to be strong enough in dental applications. On the other hand, the silica matrix via non-surfactant templated pathway has much thicker walls, which can provide excellent mechanical properties.
215 6.2 Experimental
6.2.1 Materials
Ethanol (ACS/USP grade, Pharmco, Athens, GA), 3-methacryloxypropyl trimethoxysilane (MPS, 98%, Aldrich), tetraethyl orthosilicate (TEOS, 98%, Aldrich), bis-phenol-A bis-(2-hydroxypropyl)methacrylate (BisGMA, Esstech, Essington, PA), tri(ethylene glycol) dimethacrylate (TEGDMA, Esstech, Essington, PA), tertiary amine dimethylaminoethyl methacrylate (DMAEMA, Esstech, Essington, PA) and camphorquinone (CQ, Esstech, Essington, PA), Benedict Qualitative Solution (For detection of reducing sugars, Fisher Scientific Company, Fair Lawn, NJ) were all used as received.
6.2.2 Preparation of Nanoporous Silica Filler
Nanoporous silica fillers were prepared by hydrochloric acid-catalyzed sol-gel reactions of tetraethyl orthosilicate (TEOS) in the presence of fructose molecules, which served as the nonsurfactant template. In a typical procedure, 104 g TEOS, 36 g water, 69 g ethanol and 0.25 ml HCl solution (2M) were refluxed at 60 oC for half hour under stirring and was then cooled to room temperature. In the beginning, the mixture was translucent because of phase separation, with the hydrolysis reaction continued; the mixture became more and more homogenous and finally turned transparent. An appropriate amount of 50 wt% fructose solution was added to this mixture and on standing gelled within 12 h.( sample code: P plus a number denoting percentage of fructose) The gelation time can be shortened to several minutes by adding 3 ml of 0.25 M
NaOH solution under stirring drop by drop. Upon gelation, the container was covered with parafilm containing 12 – 15 pinholes to allow for slow evaporation of solvents and
216 reaction by-products. After 24 h, the silica gel system was placed in a vacuum oven at room temperature for drying until it reached a constant weight.
After drying, the monolithic silica was crushed into fine powder using a mortar and pestle by hand and the fructose templates were removed by water extraction. In a typical washing procedure, about 1-2 grams of silica sample was placed in a test tube with about 10-15 ml water, and then the test tube was fixed in an Aliquot mixer by scotch tapes. To avoid losing silica powder during washing steps, the test tubes were centrifuged at several thousand rpm in an IEC Spinette centrifuge (Damon/ IEC Division). After 5 minutes, most of silica powder settled down firmly to the bottom of the test tubes. Then the upper clear supernant can be discarded carefully without losing powder. In the first two days, washing water was changing every two hours. After that, the time could be extended to 4 hours. The washing process usually took about 4 to 5 days. Then
Benedict’s test was used to test for the presence of fructose in supernant. When the test showed no fructose in the supernant, one more day’s washing was needed to make sure that all fructose was washed out from silica nanoporous materials. Then all the silica powder was put in oven at 70-80 oC to let it dry.
To examine the heat treatment effect of silica filler on mechanical properties of this dental material, the dried silica powders were divided into three parts and heated at three different temperatures (i. e. 200, 500, and 800 oC) for 4 hours within a NEY 6-160A furnace (NEYTECH, Bloomfield, CT). (Sample code: code of filler plus temperature, e. g.
P40-200)
6.2.3 Characterization of Nanoporous Silica Filler
217 The Brunauer-Emmett-Teller (BET) surface area and pore volume were determined using a surface area and pore size analyzer (Micromeritics ASAP 2010,
Norcross, GA) at –196 °C. Prior to the measurements, the samples were degassed at 100 oC under vacuum for 6-7 h. For each measurement, 0.1 - 0.2 gram sample was used.
Nitrogen adsorption isotherms were determined before and after heat treatment of mesoporous silica.
6.2.4 Preparation of Dental Resin
Light curable dental resin was prepared by mixing 50 wt% bisphenyl A glycidyl dimethacrylate (BisGMA) and 50 weight % triethylene glycol dimethacrylate (TEGDMA) by a laboratory blender (Waring Model, 51BL30, CT). Because the viscosity of BisGMA is extremely high, TEGDMA was used to lower the viscosity and make the resin easy to handle. When a uniform consistency was achieved, camphorquinone (0.5 weight %) and
2 – (dimethylamino) ethyl methacrylate (DMAEMA) (0.5 weight %) were added to be used as photoinitiator and accelerator, respectively. All the procedures were performed under yellow light to prevent light initiation of polymerization. Then the resin was stored in a brown bottle and saved in dark for future use.
6.2.5 Silanization of Non-Porous Silica Particles
MPS was dissolved in acetone to obtain silane concentrations of 5 wt%. The silica particles were silanated by agitation for 2 h in this solution and were then separated using a centrifuge run at 10,000 rpm. The silanated silica particles were dried at room temperature (25 oC) under vacuum overnight and then additionally at 110 oC for 2 h at atmospheric pressure. The particles were further washed three times with methanol, then dried in vacuum overnight. The sample code was assigned as NP (non-porous).
218 6.2.6 Preparation of Dental Composite
Two different types of experimental composites were made 1) with mesoporous silica fillers without silane treatment and 2) with silane (MPS) treated silica fillers (1.5 micron). Because the resin solution was too viscous, resin and silica fillers were need to be mixed with a laboratory blender (Waring Model. 51BL30) for 5 min. (Sample code: filler name plus a number denoting weight percentage of filler in the dental material)
Removing entrapped air from the composite mixtures was an essential step before curing, because tiny air bubbles would significantly reduce the mechanical properties of dental material. In the case of mesoporous filler based composite mixtures, a combination of vacuum, vibration and ultrasonic treatment was applied to remove the entrapped air from the pores. Normally, the mixture was first placed in a ultrasonic bath (Bransonic ultrasonic cleaner, Branson 2510MT, Branson Ultrasonics Corporation, Danbury, CT) in degassing mode for 5 min. Then the mixture was rested on a vibrator (No. 1A Vibrator,
Buffalo Dental Mfg. Co., Inc, Syosset, NY) to let the air bubbles move up the surface.
These two steps could be repeated for several times until no air bubbles can be seen.
Finally, the mixture was put in a vacuum oven for 6 hours to remove the rest of the bubbles.
The composite mixtures were then carefully placed into cylindrical glass molds.
During the transfer process, some air bubbles might be generated again, so the above steps to remove air bubbles were redone. The composite mixtures were then polymerized in a light-curing unit (Triad II, Dentsply International, York, PA) for six minutes. The composite cylinders were removed from the glass molds and post-cured for 24 h at 37 oC.
The resulting material is translucent with a slightly yellow color.
219 6.2.7 Evaluation of Mechanical Properties
Experimental composites containing mesoporous and silica fillers were tested in compressive mode to find the compressive strength and modulus. The compression test specimens were cut according to the ASTM standard (length to diameter ratio = 2:1) using a diamond saw (IsoMet® Low Speed Saw, Buehler Ltd., Lake Bluff, IL).
Compressive tests were performed on post-cured specimens using a servo-hydraulic machine (MTS Mini-Bionix 2, Eden Prairie, MN). A crosshead speed of 1 mm/min was used. The compressive strengths were calculated using the following equation:
F σ = A
Where σ = Compressive strength, F = Maximum Failure Load and A = Cross- sectional area of the specimen
Compressive modulus of the specimens was determined from the slope of a straight line fit to the initial linear portion of the stress-strain curve. The slope was calculated using peak slope method.
6.2.8 Aging Test
Experimental dental composites containing 35 wt% mesoporous silica fillers
(heat-treated at 200 oC for 4 h) were aged in water at 37 oC for 14 days and 35 days.
Some of these composites were not soaked in water and were used as controls. After drying (by using tissue paper) the specimens immediately after each aging interval, compression testing was performed on the specimens using a servo-hydraulic machine.
The results were then compared with in house aging studies conducted on experimental composites containing similar weight percentage silane treated SiO2 as fillers.
6.3 Results and Discussion
220 6.3.1 BET Analysis
As shown in Table 6-1, the surface area and pore volume of mesoporous silica increased with an increase in weight percent of fructose template. As the fructose template weight percent from 30 to 50 wt%, the surface area of mesoporous fillers varied from 232 m2/g to 345 m2/g and the pore volume increased from 0.47 cm3/g to 0.71 cm3/g.
The pore size of the mesoporous fillers was approximately 8 nm.
The summary of porous parameters of nanoporous silica particles after heat treatment at different temperatures was shown at Table 6-2. From that table, we can find that with temperature increase, the average pore size didn’t change much but surface area was reduced.
6.3.2 Compression Testing
6.3.2.1 Comparison of Non-Porous, Nanoporous and Neat Resin Materials
The effects of filler porosity on mechanical properties of dental composites were examined. The maximum mesoporous filler loading that can be achieved in these composites was approximately 40 wt%. Above this filler loading, the composite mixture becomes dry due to insufficient resin to wet its surfaces. For comparison purposes, the filler loading for all filled samples were 40 wt%. Three different dental composites were used: i. e. P40-200-40, in which 40 wt% P40-200 was used as filler; NP-40, in which 40 wt% silanized nonporous silica particles was used as filler; Neat Resin, which is pure dental resin without any filler inside.
The results were shown on Table 6-3. At the same filler loading (40 wt %), the composites made with nanoporous fillers had higher compressive modulus than composites made with silane treated SiO2 fillers. These results indicated that nanoporous
221 silica could provide better mechanical property than silanized non-porous silica filler. As expected, neat resin without filler had the lowest compressive modulus. However, the compressive strength of composites with SiO2 filler was higher than the composites with mesoporous filler. Mesoporous composites containing the maximum filler loading
(approximately 40 wt %) did not have a post yield region during compression testing.
This implied that mesoporous composites at maximum filler loading were brittle.
6.3.2.2 Comparison of Heat Treatment Temperature Effects on Nanoporous Filler
It is already known that heat treatment at high temperature will increase the mechanical strength of sol-gel silica. In the heat treatment process, especially above 300 to 400 oC, two phenomena could happen and contribute to densification of silica matrix.45
One is condensation reaction:
Si − OH + HO − Si → Si − O − Si + H 2O
This reaction can occur both within and on the surface of the inorganic skeleton and introduce more silanol bonds, and then enhance the strength of silica matrix.
Another one is structural relaxation, a process by which excess free volume is removed, allowing the structure to approach the configuration characteristic of the metastable liquid.45 In the process, the porosity of silica matrix changed. However, according to BET data we obtained, the change was not significant.
The increased mechanical property of silica filler was supposed to increase the mechanical property of final dental composite. In this study, four lots of nanoporous silica fillers with almost identical porous structure but heat treated at four different temperatures, 120, 200, 500, 800 oC were mixed with dental resin at 40 wt %. In Figure
6-1, it can be noticed that with the heated treatment temperature increased from 120 to
222 800 oC, the compressive modulus of dental materials increased correlating with temperatures.
6.3.2.3 Aging Test
Mesoporous filler based composites were soaked in water at 37 oC for up to 35 days. They showed no significant difference in compressive modulus between the aged sample and control. In case of composites with SiO2 fillers, the compressive modulus decreased as the sample aged. (Table 6-4)
These results demonstrated that mechanical properties of dental materials with nanoporous silica particles as fillers didn’t change much in the aging test, on the contrary, mechanical properties of dental materials with silanized non-porous silica particles reduced a lot in the test. This phenomenon can be explained by difference in structures of the two silica materials and interaction between filler and resin matrix. In conventional dental materials, silanized non-porous silica particles bond to resin matrix via silanol bonds. This bond between the coupling agent and silica content can be easily hydrolyzed especially in oral environments. In this aging test, we used water at pH 7 at 37 oC to mimic the oral environment. It was believed in this environment, the silanol bonds between filler and resin matrix were dissociated with time, in turn the connection between inorganic filler and organic matrix became loose, and leading to the reduction in mechanical properties of this dental material. On the other hand, for the novel dental material develop in our group by micro-mechanical interlocking nanoporous silica fillers with resin matrix physically, this hydrolysis problem did not exist. Because of three dimensionally interconnected porous structures inside the nanoporous filler, organic dental resin monomers can diffuse inside before polymerization. Upon photoinitiated
223 polymerization, organic phases can interlock with inorganic silica. Thus, in oral environment, even water molecules may diffuse inside the material, it would not destroy the interaction of organic and inorganic phases and the inorganic fillers do not have a chance to leak out of the material. That is the great advantage of nanoporous filler over non-porous filler.
6.4 Conclusion and Future Works
In this study, we developed a new type of nanoporous silica filler prepared via non-surfactant sol-gel pathway. This non-surfactant sol-gel method was first used in our group and provides us with a new nanoporous material with three-dimensional interconnected porous structures inside. The unique structural characteristic of the material might have the prospective for potential dental filler. When this filler is mixed with dental resin monomer before polymerization, the monomer can diffuse inside pores of the filler. Then after polymerization, dental resin matrix can interlock with nanoporous silica fillers, thus the organic and inorganic phases can tightly interact with each other via physical interaction.
Compared with conventional silanized non-porous silica filler, in which silanol bonds are used to connect with resin matrix, our new dental material demonstrated better mechanical properties. Most importantly, unlike non-porous silica filler, the new materials showed much better resistance to degradation in oral environment. The reason lies in the difference of interactions between organic and inorganic phases in these two types of materials. Non-porous silica fillers are bonded to resin matrix via silanol bonds, which can be easily hydrolyzed in oral environment, then phase separations can happen and result in lost filler and structure which are responsible for reduction of mechanical
224 strength of the material. But nanoporous silica fillers interlock with resin matrix physically, thus even water can penetrate into the interface of resin and silica filler, the interaction between these two phases will not lessen. Thus, the mechanical properties of the new dental material did not become worse in the aging test.
Effects of heat treatment at different temperatures on the mechanical properties of dental materials were also examined in this study. As expected, because the hardness of sol-gel silica increased with increase in heat treatment temperature from 200 oC to 800 oC.
The mechanical properties dental materials with these silica particles as filler also follow the same trend.
Though the new type of nanoporous silica filler has great potential as dental filler, there are some shortcomings with these fillers. Compared with commercial available nonporous fillers, one of shortcomings is low filler loading. The filler loading of commercial dental materials is usually 70 % (v/v), but the highest filler loading for nanoporous materials was merely 40 wt % (around 30 % (v/v)). Since the surface area of nanoporous particle is much larger than non-porous particles, wetting of silica particles by resin monomer is key problem, which prevents increasing filler loading.
In the future, we can try to improve the material using two approaches. The first one is solve the wetting problem by introducing some organic groups on the surface of nanoporous silica particles. Those organic groups, like methyl, propyl or phenyl, can be introduced in sol-gel process by mixing methyltrimethoxysilane, n- propyltrimethoxysilane, phenyltrimethoxysilane with TEOS during hydrolysis. With some organic groups, it is expected that wetting by resin monomer will be easier because the surface of inorganic phase is modified with some organic groups. Secondly, the effect
225 of porosity on dental material should be examined systematically. In this study, though the difference of nanoporosity and non-porosity has been observed, the pore size effects are still unknown. In the next steps, it is suggested that silica porous particles with distinctive difference should be synthesized and used to be dental fillers. Thus the most suitable porosity could be found out.
In conclusion, this new type of nanoporous silica dental filler has some particular advantages over conventional non-porous silica dental filler, such as resistance of degradation and compressive modulus and so on. Though it still has some deficiencies, we still believe it can be developed into a good dental filler with great potentials for dental applications.
6.5 Acknowledgements
I want to thank Dr. Solomon Praveen of Drexel University and Dr. George Baran of Temple University for their help in the synthesis and characterization of dental materials, especially in compressive tests.
6.6 References
1. Klapdohr, S. & Moszner, N. New inorganic components for dental filling composites. Monatshefte Fur Chemie 136, 21-45 (2005). 2. Ferracane, J. L. Current trends in dental composites. Critical Reviews in Oral Biology and Medicine 6, 302-318 (1995). 3. Moszner, N. & Salz, U. New developments of polymeric dental composites. Progress in Polymer Science 26, 535-576 (2001). 4. Bowen, R. L. 4 pp ; Division of U S 3,066,112 (CA 58, 6999a) ((U.S. Dept. of Commerce). Application: US US, 1965).
226 5. Craig, R. G., O'Brien, W. J. & Powers, J. M. Dental Materials: Properties and Manipulation (1975). 6. Antonucci, J. M. & Bowen, R. L. Dimethacrylates derived from hydroxybenzoic acids. Journal of Dental Research 55, 8-15 (1976). 7. Bowen, R. L. & Reed, L. E. Semiporous reinforcing fillers for composite resins: I. Preparation of provisional glass formulations. Journal of Dental Research 55, 738-47 (1976). 8. Moszner, N., Zeuner, F., Volkel, T. & Rheinberger, V. Synthesis and polymerization of vinylcyclopropanes. Macromolecular Chemistry and Physics 200, 2173-2187 (1999). 9. Moszner, N., Zeuner, F., Volkel, T., Fischer, U. K. & Rheinberger, V. Polymerization of cyclic monomers. VII. Synthesis and radical polymerization of 1,3-bis (1-alkoxycarbonyl-2-vinylcyclopropane-1-yl)carboxy benzenes. Journal of Applied Polymer Science 72, 1775-1782 (1999). 10. Moszner, N., Zeuner, F. & Rheinberger, V. Polymerization of cyclic monomers .4. Synthesis and radical polymerization of bi- and trifunctional 2- vinylcyclopropanes. Macromolecular Rapid Communications 18, 775-780 (1997). 11. Moszner, N., Volkel, T., Rheinberger, V. & Klemm, E. Polymerization of cyclic monomers .3. Synthesis, radical and cationic polymerization of bicyclic 2- methylene-1,3-dioxepanes. Macromolecular Chemistry and Physics 198, 749-762 (1997). 12. Zeuner, F., Moszner, N. & Rheinberger, V. Polymerization of cyclic monomers .2. Synthesis and radical polymerization of vinylcyclopropanes. Macromolecular Chemistry and Physics 197, 2745-2752 (1996). 13. Nuyken, O., Bohner, R. & Erdmann, C. Oxetane photopolymerization - A system with low volume shrinkage. Macromolecular Symposia 107, 125-138 (1996). 14. Moszner, N., Zeuner, F. & Rheinberger, V. Polymerization of Cyclic Monomers .1. Radical Polymerization of Unsaturated Spiro Orthocarbonates. Macromolecular Rapid Communications 16, 667-672 (1995). 15. Cho, I., Kim, B. G., Park, Y. C., Kim, C. B. & Gong, M. S. Photoinitiated Free- Radical Ring-Opening Polymerization of 2-Phenyl-4-Methylene-1,3-Dioxolane. Makromolekulare Chemie-Rapid Communications 12, 141-146 (1991). 16. Holter, D., Frey, H., Mulhaupt, R. & Klee, J. E. Ambient-temperature liquid- crystalline bismethacrylates based on cholesterol: Cholesteric and smectic thermosets. Advanced Materials 10, 864-+ (1998). 17. Holter, D., Frey, H., Mulhaupt, R. & Klee, J. E. Liquid crystalline thermosets based on branched bismethacrylates. Macromolecules 29, 7003-7011 (1996). 18. Doornkamp, A. T., Vanekenstein, G. & Tan, Y. Y. Kinetic-study of the photoinitiated polymerization of a liquid-crystalline diacrylate monomer by dsc in the isothermal mode. Polymer 33, 2863-2867 (1992). 19. Hikmet, R. A. M., Zwerver, B. H. & Broer, D. J. Anisotropic polymerization shrinkage behavior of liquid-crystalline diacrylates. Polymer 33, 89-95 (1992). 20. Klee, J. E., Neidhart, F., Flammersheim, H. J. & Mulhaupt, R. Monomers for low shrinking composites, 2 - Synthesis of branched methacrylates and their application in dental composites. Macromolecular Chemistry and Physics 200, 517-523 (1999).
227 21. Klee, J. E., Walz, U., Holter, D., Frey, H. & Mulhaupt, R. Branched macromonomers and their application in dental composites - Monomers for low- shrinking composites, 3. Angewandte Makromolekulare Chemie 260, 71-75 (1998). 22. Hickel, R., Dasch, W., Janda, R., Tyas, M. & Anusavice, K. New direct restorative materials. International Dental Journal 48, 3-16 (1998). 23. Sellinger, A., Laine, R. M., Chu, V. & Viney, C. Palladium-catalyzed and platinum-catalyzed coupling reactions of allyloxy aromatics with hydridosilanes and hydridosiloxanes - novel liquid-crystalline organosilane materials. Journal of Polymer Science Part a-Polymer Chemistry 32, 3069-3089 (1994). 24. Ellsworth, M. W. & Novak, B. M. Inverse organic-inorganic composite- materials .3. high glass content nonshrinking sol-gel composites via poly(silicic acid-esters). Chemistry of Materials 5, 839-844 (1993). 25. Wei, Y., Bakthavatchalam, R. & Whitecar, C. K. Synthesis of new organic inorganic hybrid glasses. Chemistry of Materials 2, 337-339 (1990). 26. Zhao, F. M. & Takeda, N. Effect of interfacial adhesion and statistical fiber strength on tensile strength of unidirectional glass fiber/epoxy composites. Part I: experiment results. Composites Part a-Applied Science and Manufacturing 31, 1203-1214 (2000). 27. Kessler, A. & Bledzki, A. Correlation between interphase-relevant tests and the impact-damage resistance of glass/epoxy laminates with different fibre surface treatments. Composites Science and Technology 60, 125-130 (2000). 28. Wang, J. Y. & Ploehn, H. J. Dynamic mechanical analysis of the effect of water on glass bead epoxy composites. Journal of Applied Polymer Science 59, 345-357 (1996). 29. Mohsen, N. M. & Craig, R. G. Effect of silanation of fillers on their dispersability by monomer systems. Journal of Oral Rehabilitation 22, 183-189 (1995). 30. Mohsen, N. M. & Craig, R. G. Hydrolytic stability of silanated zirconia-silica- urethane dimethacrylate composites. Journal of Oral Rehabilitation 22, 213-220 (1995). 31. Lin, C. T., Lee, S. Y., Keh, E. S., Dong, D. R., Huang, H. M. & Shih, Y. H. Influence of silanization and filler fraction on aged dental composites. Journal of Oral Rehabilitation 27, 919-926 (2000). 32. Sarkar, N. K., Karmaker, A., Prasad, A. & Shih, F. Simulation of in vivo degradation of dental composites. Journal of Materials Science Letters 18, 1749- 1752 (1999). 33. Pearson, G. J. Long term water sorption and solubility of composite filling materials. Journal of Dentistry 7, 64-68 (1979). 34. Lee, S.-Y., Greener, E. H., Mueller, H. J. & Chiu, C.-H. Effect of food and oral simulating fluids on dentine bond and composite strength. Journal of Dentistry 22, 352-359 (1994). 35. Lee, S.-Y., Greener, E. H. & Mueller, H. J. Effect of food and oral simulating fluids on structure of adhesive composite systems. Journal of Dentistry 23, 27-35 (1995).
228 36. Ji, X. L., Hampsey, J. E., Hu, Q. Y., He, J. B., Yang, Z. Z. & Lu, Y. F. Mesoporous silica-reinforced polymer nanocomposites. Chemistry of Materials 15, 3656-3662 (2003). 37. Wei, Y., Jin, D. L., Yang, C. C. & Wei, G. A fast convenient method to prepare hybrid sol-gel materials with low volume-shrinkages. Journal of Sol-Gel Science and Technology 7, 191-201 (1996). 38. Luo, J., Lannutti, J. J. & Seghi, R. R. Mechanical performance of inorganic/organic nanocomposites. Journal of Dental Research 77, 170-170 (1998). 39. Mou, C. Y. & Lin, H. P. Control of morphology in synthesizing mesoporous silica. Pure and Applied Chemistry 72, 137-146 (2000). 40. Wei, Y., Jin, D. L., Ding, T. Z., Shih, W. H., Liu, X. H., Cheng, S. Z. D. & Fu, Q. A non-surfactant templating route to mesoporous silica materials. Advanced Materials 10, 313-316 (1998). 41. Wei, Y., Xu, J., Dong, H., Dong, J. H., Qiu, K. & Jansen-Varnum, S. A. Preparation and physisorption characterization of d-glucose-templated mesoporous silica sol-gel materials. Chemistry of Materials 11, 2023-2029 (1999). 42. Pang, J.-B., Qiu, K.-Y., Xu, J., Wei, Y. & Chen, J. Synthesis of mesoporous silica materials via nonsurfactant urea-templated sol-gel reactions. Journal of Inorganic and Organometallic Polymers 10, 39-50 (2000). 43. Pang, J.-B., Qiu, K.-Y. & Wei, Y. A new nonsurfactant pathway to mesoporous silica materials based on tartaric acid in conjunction with metallic chloride. Chemistry of Materials 13, 2361-2365 (2001). 44. Zheng, J.-Y., Pang, J.-B., Qiu, K.-Y. & Wei, Y. Synthesis of mesoporous silica materials via nonsurfactant templated sol-gel route by using mixture of organic compounds as template. Journal of Sol-Gel Science and Technology 24, 81-88 (2002). 45. Brinker, C. & Scherer, G. Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing (1990).
229
Table 6- 1 BET analysis of mesoporous fillers at different fructose concentration.
Fructose weight percentage Surface Area(m2/g) Vsp (cm3/g) 30% 232 0.47 40% 259 0.51 50% 345 0.71
230
Table 6- 2 The pore parameters of the nanoporous silica fillers after template removal of fructose by water extraction and heat treatments at different temperatures.
Heat treatment Weight Surface Pore BET pore BJH pore Sample temperature percentage area(m2/g) Volume diameter (Å)b diameter (Å)c Codea (oC) (cm3/g) 100 0% 267 0.67 99.3 80.1 zs020805-3 500 0% 311 0.78 99.1 79.1 800 0% 168 0.44 102.9 79.5 zs020805-5 100 20% 260 0.74 113.4 92.9 100 30% 232 0.47 80.4 65.5 200 30% 255 0.51 78.5 65.6 zs110504-3 300 30% 228 0.45 78.5 63.8 500 30% 232 0.46 77.7 63.6 800 30% 115 0.29 100.7 68.7 100 40% 259 0.51 77.8 61.6 zs110504-4 400 40% 265 0.49 72.7 57.7 100 50% 345 0.71 80.9 64.5 400 50% 370 0.72 77 62.9 zs110504-5 500 50% 349 0.7 78.1 63.5 600 50% 323 0.67 80.1 63.1 800 50% 184 0.38 81.1 57.5 100 30% 276 0.47 67.1 53 zs110804-2 200 30% 275 0.46 65.7 52.8 300 30% 274 0.47 67.3 53.7 800 30% 123 0.26 83.9 50.3 400 40% 358 0.53 58.1 47.8 zs110804-3 500 40% 338 0.51 58.9 47.3 600 40% 308 0.46 57.6 45.7 800 40% 159 0.25 60 42.2 100 50% 327 0.69 82.9 66.3 zs110804-4 200 50% 352 0.71 79.6 63.2 300 50% 348 0.71 80.6 64.5 800 50% 235 0.46 76.2 58.1
a The sample codes were assigned by the preparation dates. b The average pore diameters c calculated from 4 V/SBET by the BET method. Determined from the maxima of the BJH desorption pore size distribution curves with the Halsey equation.
231
Table 6- 3 Comparison of compressive properties of composites with different types of fillers. The number of specimens tested is given in parenthesis.
Sample code Compressive
Modulus (GPa) Strength (MPa)
Neat Resin 2.7 ± 0.1 (6) 274 ± 35 (6)
NP-40 4.5 ± 0.3 (4) 260 ± 28 (4)
P40-200-40 5.4 ± 0.1 (3) 193 ± 9 (3)
232
Table 6- 4 The effect of aging (in water at 37o C) on the compressive properties of composites prepared using mesoporous and SiO2 fillers. The number of specimens tested is given in parenthesis.
Filler type Modulus (GPa) Compressive
Strength (MPa)
Mesoporous (35 wt %, Control) 5.1 ± 0.3 (5) 171 ± 9 (5)
Mesoporous (35 wt %, 14 days) 5.2 ± 0.6 (7) 126 ± 7 (7)
Mesoporous (35 wt %, 35 days) 5.0 ± 0.3 (4) 113 ± 8 (4)
SiO2 (40 wt %, Control) 4.5 ± 0.3 (4) 260 ± 28 (4)
SiO2 (40 wt %, 35 days) 3.5 ± 0.2 (4) 243 ± 32 (4)
233
Figure 6- 1 The effect of filler heat treatment on the compressive modulus of post cured composites.
6.5
6.1 a P M
us 5.7 odul M e v i
s 5.3 s e r p m o
C 4.9
4.5 0 100 200 300 400 500 600 700 800 900 Temperature C
234 Chapter 7: Dense Packing of Vinyl Modified Silica Nanoparticles and Its Potential
Application as Low Shrinkage Dental Materials
7.1 Introduction
Modern dental restorative procedures that are related to polymer science have been focused on the use of polymer resin composites in place of silver/mercury amalgams. For example, in filling cavities or other defects in the tooth’s surface, many dental professionals now use polymerizable resin compositions containing inorganic glass fillers to impart desired compressive strength in place of dental amalgams. Such filled polymerizable materials are easy to apply, can be colored and shaped to correspond to the original tooth surface, and often exhibit chemical adhesion to the tooth surface when polymerized as opposed to the metallic appearance and mechanical adhesion of metal amalgams.1
However, certain problems still exist due to the nature of the composites though filler polymerizable resin composites are in widely usage. For instance, most of resin molecules are monomers before they are applied to tooth surface. Thus polymerization reactions are needed to solidify the resin composites. But the majority of polymerization reactions result in volume shrinkage as particularly vinyl monomers change from their free liquid state into their denser, cross-linked polymerized state. This volume shrinkage can exert stresses in the adjacent tooth structure. The shrinkage stress occurs when the contraction is obstructed and the material is rigid enough to resist sufficient plastic flow to compensate for the original volume.
Such shrinkage and resultant stresses are often detrimental, especially in so-called
“Class V” type of dental restoration, wherein the restoration is affected at the dentin-
235 enamel junction at the cervival region of the tooth, and also in “Class I” restorations such as deep cavities involving restorations contacting opposing walls of the tooth.2,3 Those shrinkage and related stresses have been reported to cause separation of the restoration from at the dentin surface of the tooth, leading to marginal gaps at the interface between the restorative and the adjacent tooth surface and causing microleakage.4 Current commercial composites shrink from 2.2 to about 3.5 % by volume fraction.
To solve this problem, a lot of effort has been made to develop new composites and methods to reduce or eliminate shrinkage-related stresses and marginal gaps in dental restorations.1,5-10 Reported approaches include reliance on the “flow” of the composite during chemical self-curing, which proceeds slowly11 or by incremental insertion of composite in the restorative site.12 Others have proposed multi-step application procedures using low viscosity, unfilled resins to seal the marginal gaps directly after initial curing of the composite,13 or use of so-called “flexible” intermediate layers of unfilled resins or light-cured glass ionomer layers applied as a thin layer between the tooth surface and the composite.14 But these multi-step, multi-material approaches also introduce complexity into the dental restoration process, such as more steps, different materials, increase in the time spent and cost incurred by the dental professional and patient in the treatment process.
This study aimed at development of dental composite materials with a vinyl modified silica nanoparticle as filler to reduce the polymerization shrinkage and the risk of microleakage and which still can exhibit easy handling, good bonding to tooth and possess good tensile and compressive strength.
236 In this study, instead of using pure silica particles as inorganic fillers, silica nanoparticles modified with vinyl groups on the surface were used. We started with a commercial available material, OG100-31 from Clariant Corp. In this viscous, light yellow transparent liquid sample, the liquid component is HEMA monomers and the 30 wt% solid component is silica nanoparticles with diameter of 13 nm. Those silica nanoparticles are suspended in HEMA monomers to form a colloidal solution, which is stable over a long period of time. Moreover, as these nanoparticles are grafted by HEMA monomers on the surface, they can copolymerize with the free HEMA of the suspension medium and work as a crosslinker of high functionality. Thus, the inorganic component, silica nanoparticles, can be covalent bonded with the organic component, PHEMA.
Unfortunately, there are two limitations which prevent this material to be directly used as dental material. First, the organic component in this material, HEMA, can not be used as a good dental rein material because PHEMA is a water-soluble polymer which may dissolve in oral environment. Second, the weight percentage of inorganic component in this material is too low (i.e. 30% by weight). As the results, the volume shrinkage of polymerization will be too large and mechanical strength of the material would not be strong enough.
In order to develop this material for dental applications, two approaches are designed. First, HEMA monomers should be removed from the commercial sample and replaced by conventional dental resin monomer, such as BisGMA and TEDGMA. Since both BisGMA and TEDGMA are also vinyl monomers with C=C double bonds as their functional groups, during the polymerization process, the vinyl groups carried by silica nanoparticles on their surface can react with dental resin to incorporate into the dental
237 resin matrix and form a new type of dental material. Second, the percentage of silica nanoparticles needs to be increased to achieve strong mechanical strength and to reduce volume shrinkage in polymerization.
This new dental material can be classified as organic-inorganic hybrid nanocomposite.15-19 This material provides a solution to the thermodynamic incompatibility organic-inorganic phases by reducing the inorganic phase domain size to
13 nm.20,21 Because reduced domain size results in enlargement of interfacial area between organic and inorganic phases, nanometer scale phase domains provide materials with increased interfacial interactions. Thus the materials have a high degree of interpenetration between the two dissimilar but covalently bonded phases. As a result of this high degree of mixing, the ordinarily sharp interfacial zone will be blurred and a continuum of the structure and the properties of organic and inorganic components could be achieved.22 The other interesting consequence of such a fine morphology is the optical transparency of these composites as far as their primary constituents are transparent.
Those new properties enable the new nanocomposite a potential dental material as well as in wide applications, such as scratch and abrasive-resistant hard coating, electrical and non-linear optical materials, contact lens materials, catalysis materials and so on.
7.2 Experimental
7.2.1 Materials
HighlinkTM OG100-31, silica nanoparticles with surface modification of 2- hydroxylethyl methacrylate (HEMA), was provided by Clariant Corporation. Ethyl alcohol (ACS/USP grade, Pharmco, Athens, GA), bis-phenol-A bis-(2-
238 hydroxypropyl)methacrylate (BisGMA, Esstech, Essington, PA), tri(ethylene glycol) dimethacrylate (TEGDMA, Esstech, Essington, PA), tertiary amine dimethylaminoethyl methacrylate (DMAEMA, Esstech, Essington, PA) and camphorquinone (CQ, Esstech,
Essington, PA), were all used as received.
7.2.2 Separation of Nanoparticle from OG100-31
In order to remove HEMA monomer from the sample and concentrate silica nanoparticles, several methods have been used as described in the following sections.
7.2.2.1 Reduced Pressure Distillation
Because high temperature may initiate polymerization reaction between HEMA monomers, reduced pressure distillation was tried to separate HEMA monomers from
OG100-31 and thus to concentrate silica nanoparticles percentage. At a reduced pressure of 4 mmHg and temperature of 80-90 oC, the distillation was carried out for half an hour until the viscosity of mixture became too high to be stirred. The highest concentration of silica nanoparticles achieved was 50 wt%.
7.2.2.2 Ultracentrifuging
Colloidal silica suspension can be separated theoretically by ultracentrifuge at more than 20K rpm. In order to achieve the best separation results, several attempts were made.
Directly centrifuging: In this effort, about 8 ml OG100-31 sample was placed into a round bottom centrifuge tube (Nalgene 3110-0380 round centrifuge tube, PPCO). The ultracentrifuging was carried on a Sorval ultracentrifuge machine at 25 oC and 25K rpm for 30 min. After ultracentrifuging, previous homogenous OG100-31 colloidal was separated to two layers. It was difficult to observe the separation because both sediments
239 and supernant had the same color and almost same refractive index. But when the supernant was poured out from the top of the centrifuge tube, the solidified sediment could be observed. The sediment was hard and non-flowing. A spatula was used to remove the sediment from the bottom of the centrifuge tube.
Using EtOH to dilute OG100-31: Because of the high viscosity of the original
OG100-31, ultracentrifuging may not separate all silica nanoparticles from the colloidal suspension. EtOH was used to dilute OG100-31 to lower the viscosity. Typically, the volume ratio of EtOH and OG100-31 was 1:1. After EtOH was mixed with the sample, the transparent OG100-31 turned into white translucent suspension. After ultracentrifuging, two layers can be clearly observed. The bottom sediment was clear solid, and the supernant was also white translucent suspension. This sediment was denser than the sediment obtained by directly centrifuging and dried quickly as since EtOH is a volatile solvent.
Using Acetone to dilute OG100-31: In this approach, acetone was used as solvent instead of EtOH as in the last one, but all other conditions were kept identical with the last approach. The results showed that there is no difference between acetone and EtOH.
However acetone can dissolve the plastic centrifuge tube. This lead to cracks in the centrifuge tube with slight leakage.
Recentrifuging: To concentrate nanoparticles in sediments, the sediment was redispersed and recentrifuged. EtOH was used as solvent and was placed in ultrasonic bath (Bransonic ultrasonic cleaner, Branson 2510MT, Branson Ultrasonics Corporation,
Danbury, CT). Typically, the ultrasonic treatment took 2-3 hours. To prevent thermal- initiated polymerization during this process (the temperature will increase if the
240 ultrasonic treatment takes a longtime), ice-bath was used to keep the temperature down.
The redispersed suspension was a white translucent colloidal and was stable over months.
7.2.3 Atomic Force Microscopic (AFM) Measurements
Atomic force microscopy images were obtained on an AFM IIIa scanning probe microscope (Veeco Metrology Group, Digital Instrument) operated under tapping mode.
The sediments were prepared on a piece of mica. For mica, the top layer was peeled off with scotch tape to provide a fresh and smooth surface ready for the sample casting. The sample preparation was achieved by directly smearing the sediment paste on the fresh mica surface. The AFM probe used in this study was Veeco NanoProble tip (Model#
TESPWU).
7.2.4 Thermal Gravimetric Analysis (TGA)
The TGA tests were performed on a Q50 TGA instrument (Thermal Analysis
Instruments, DE) in air atmosphere for all the experiments. In a typical procedure, temperature was ramp to 100 oC at the rate of 20 oC per min, then isothermal for 10 min to get rid of moisture in samples. Then temperature was ramp 20 oC / min to 900 oC.
7.2.5 FTIR Spectroscopy
7.2.5.1 Solid Sample
Solid samples included centrifuge sediments and dried nanoparticles powders.
Both of these two species were first mixed with KBr powder. Infrared spectra of KBr powder-pressed pellets were recorded on a Perkin-Elmer Model 1600 FTIR spectrophotometer.
7.2.5.2 Liquid Sample
241 Liquid samples were dropped on the preformed pure KBr powder-pressed pellets, and the spectra were recorded on a Perkin-Elmer Model 1600 FTIR spectrophotometer.
7.2.6 Isolation of Silica Nanoparticles
Vinyl modified silica nanoparticles inside OG100-31 were isolated in their dry state. After centrifuging and redispersion twice, the sediments were dispersed in acetone again by ultrasonic bath (Bransonic ultrasonic cleaner, Branson 2510MT, Branson
Ultrasonics Corporation, Danbury, CT). Then, the white translucent suspension was distilled under high vacuum in room temperature to remove acetone completely. Finally, only white solid powders were left in the flask. We believed that the white powder was most likely made of vinyl modified silica nanoparticles.
7.2.7 Preparation of Dental Resin
Light curable dental resin was prepared by mixing 50 weight % bisphenyl A glycidyl dimethacrylate (BisGMA) and 50 weight % triethylene glycol dimethacrylate
(TEGDMA) by a laboratory blender (Waring Model. 51BL30, CT). Because the viscosity of BisGMA is extremely high, in fact the purpose of mixing with TEGDMA is to low the viscosity and make the resin easy to handle with. When a uniform consistency was achieved, camphorquinone (0.5 weight %) and 2 – (dimethylamino) ethyl methacrylate
(DMAEMA) (0.5 weight %) were used as photoinitiator and accelerator respectively. All the processes were done in the yellow light to prevent light initiation of polymerization.
Then the resin was stored in a brown bottle and in dark for future use.
7.2.8 Preparation of Dental Composite
Vinyl modified silica nanoparticles were mixed with dental resin, which was prepared in the last step. Because once the nanoparticles were added into the resin, the
242 viscosity of the mixture would increase dramatically; several methods were used to solve this problem and make the final material easy to handle.
Directly Mixing: In this approach, the sediments were directly put into dental resin. However, the dispersion of sediments reaching a homogenous mixture was not easily achieved because the viscosity of dental resin was too high. So both mechanical stirring and ultrasonic mixing were used in this process. First, large pieces of sediment were broken into small pieces by glass rod and magnetic stirring bar; then the mixture was put into ultrasonic bath with ice cubes to keep the temperature low. The ultrasonic mixing usually took 2-3 hours at temperature of about 0-5 oC.
Acetone as co-solvent: To lessen the problem brought by the high viscosity of dental resin, acetone was added into the mixture serving as a co-solvent to lower the viscosity. In fact, the acetone made the homogenous mixture of silica nanoparticles and dental resin was easily achieved by ultrasonic bath. Then high vacuum pumping was used to remove acetone from the mixture.
Ultracentrifuging nanoparticles: In order to get higher nanoparticle loading in the dental resin, a new method of centrifuging silica nanoparticle was developed. This method began with the mixture containing acetone as co-solvent. Instead of evaporating acetone under high vacuum, the mixture was ultracentrifuged at 20K rpm for 30 min.
Acetone and most of resin monomers consisted of supernant. The majorities of the sediment were silica nanoparticles and possibly some resin molecules. TGA was used to monitor the weight percentage of silica content in the sediment.
After mixing the silica nanoparticles with dental resin, the composite mixtures were then carefully placed in cylindrical glass molds. During the transfer process, some
243 air bubbles might be generated again, a combination of vacuum, vibration and ultrasonic treatments were applied to remove the entrapped air. Normally, the mixture was first put into an ultrasonic bath (Bransonic ultrasonic cleaner, Branson 2510MT, Branson
Ultrasonics Corporation, Danbury, CT) in degassing mode for 5 min, then placed on a vibrator (No. 1A Vibrator, Buffalo Dental Mfg. Co., Inc, Syosset, NY) to let the air bubbles move up the surface. These two steps were repeated several times until no air bubble could be seen. Finally, the mixture was placed in a vacuum oven for 6 hours to remove the rest of the bubbles.
The composite mixtures were then polymerized by exposing to a light-curing unit
(Triad II, Dentsply International, York, PA) for six minutes. The composite cylinders were removed from the glass molds and post-cured for 24 h at 37 oC. The resulting material is mostly transparent with a slight yellow color.
7.2.9 Evaluation of Mechanical Properties
Experimental composites containing silica nanofillers were tested for the compressive strength and modulus. The compression test specimens were cut according to the ASTM standard (length to diameter ratio = 2:1) using a diamond saw (IsoMet®
Low Speed Saw, Buehler Ltd., Lake Bluff, IL). Compressive tests were performed on post-cured specimens using a servo-hydraulic machine (MTS Mini-Bionix 2, Eden
Prairie, MN). A crosshead speed of 1 mm/min was used. The compressive strengths were calculated using the following equation:
F σ = A
Where σ = Compressive strength, F = Maximum Failure Load and A = Cross- sectional area of the specimen
244 Compressive modulus of the specimens was determined from the slope of a straight line fit to the initial linear portion of the stress-strain curve. The slope was calculated using peak slope method.
7.3 Results and Discussion
In this study, the purposes of FTIR spectra were to monitor vinyl groups on the surface of silica nanoparticles. Figure 7-1 shows the FTIR spectra of dry silica nanoparticles. In the IR spectra, the peaks at 1100-1200 cm-1 can be assigned to the Si-O bond which is contributed to silica content. The characteristic bands at 1720 cm-1 and
1635 cm-1 can be asigned to the C=O and C=C stretching vibrations, respectively. But these two peaks were very small in this spectrum compared with pure HEMA monomer’s
FTIR spectrum. It indicated that the HEMA monomer contents in dry silica nanoparticles was very small, which could be explained by the structure of silica nanoparticles. The silica nanoparticles are grafted on their surface with HEMA monomer functional groups with C=O and C=C bonds. Though we did not know how many HEMA monomers were grafted on one silica nanoparticle, this number should not be large because the size of the silica nanoparticle was only 13 nm in diameter. In the dry silica nanoparticles, the major part was silica which was shown in FTIR spectrum by the silica band in 1100-1200 cm-1; organic components provided by grafted HEMA monomer were little, thus the intensities from the two groups in HEMA monomer were low.
The FTIR spectra of sediments obtained acetone and EtOH as solvents were shown in Figure 7-2 respectively. Several differences could be observed from these two spectra and the spectrum of dry silica nanoparticles in Figure 7-1. The most important one is the difference of intensity of C=C peak at 1635 cm-1. Obviously, in the wet
245 sediment separated by either acetone or EtOH as solvent, the C=C double bond contents were higher than in dry silica nanoparticles. This phenomenon could be explained by two reasons: first, in wet sediments, some free HEMA monomer may still be left, which can contribute to the enhancement of intensity of C=C double bonds. Second, during the drying process, polymerization reactions may happen between particles because after the process silica nanoparticles were all packed together to form clear crystals. This polymerization reaction also may cause the intensity weakening of C=C double bonds.
Another FTIR spectrum shown in Figure 7-3 is the dental materials with those modified silica nanoparticles as inorganic fillers after polymerization. In this spectrum, carbonyl stretching band (C=O) could be clearly noticed at 1720 cm-1, which was assigned to C=O groups on BisGMA and TEGDMA. The largest difference between this spectrum and the spectra in Figure 7-2 lied in the absence of carbon double (C=C) at around 1635 cm-1, because those double bonds in vinyl groups were all involved in the polymerization reactions.
TGA spectra also provide information about the organic-inorganic composition.
Figure 7-4 shows two spectra of silica nanoparticle sediments after ultracentrifugation with either EtOH or acetone as solvent. Though the residue percentages of these two samples were not identical, we did not see much difference in the shapes of two curves.
Hence, we believed that the main components in the two sediments were almost same and the inorganic contents could be concentrated to more than 90 wt%, which indicated nearly all HEMA monomer could be removed by ultracentrifugation effectively. In
Figure 7-5, TGA curve of the new dental composite with vinyl modified silica nanoparticles as inorganic fillers is shown. The organic components were mixture of
246 BisGMA and TEGDMA and inorganic components were silica nanoparticles. The curve demonstrated the highest loading percentage we could get, i.e. more than 50 wt%.
Unfortunately, since the extremely high viscosity of the 50 wt% composite, the compressive test sample with uniform size could not be achieved, so the compressive modulus data was not available. But the picture of this sample was shown in Figure 7-6.
As it shown, this sample was homogenous and transparent, which meant most of the silica nanoparticles in this sample were distributed separately and evenly without large particle clusters and aggregates.
The dense packing of silica nanoparticles was illustrated by Atomic Force
Microscopy (AFM). In the Figure 7-7, AFM picture of wet sediment showed silica nanoparticles were packed tightly three dimensionally. All the particles touched with each other and no interparticle spaces could be seen in this image. Because the particles were so close to each other, it was believed that during the polymerization those particles could covalently bond with each other directly through the HEMA functional groups grafted on the particle surfaces.
The volume shrinkage of dental resin is due to polymerization reactions of vinyl monomers. But in the inorganic organic hybrid material, which consists of silica nanoparticles densely packed three dimensionally, the backbone was made up of inorganic nanoparticles, which would not shrink and the function of organic phase was only to connect the particles. Thus we believed that the volume shrinkage of this material should be smaller than conventional composite materials. The assumption was backed up by our preliminary experimental results. We found that when the silica content went up to
247 50 wt% in the dental material, the volume shrinkage was only about 2% which is lower than conventional dental materials with volume shrinkage is 3% to 5% .1
Due to the high viscosity if resin-nano silica composite mixtures, we could only make compression sample with 30 wt% nano silica fillers. The compressive modulus of
30 % composite was found to be 4 GPa. Compare with neat resin (2.7 GPa), this value was promising though still lower than commercial samples.
7.4 Conclusion and Future Work
In this study, OG100-31, a colloidal “solution” of vinyl modified silica nanoparticle (30 wt %) suspended in HEMA monomer, was used as starting raw material.
In order to use the vinyl modified silica nanoparticles as inorganic fillers in dental material, those particles needed to be separated from HEMA monomer first. Two methods were used to separate silica nanoparticles from HEMA in this study: distillation and ultracentrifuge. We found that distillation could only concentrate the weight percentage to 50 wt %. By using ultracentrifuge, we could obtain almost pure silica nanoparticles. In the next step after separating the nanoparticles, we tried to mix those nanoparticles with dental resin (mixture of BisGMA and TEGDMA) to get hybrid dental materials with vinyl modified silica nanoparticle dense packing as inorganic component.
Our preliminary results show that at 50 wt % of silica loading, the volume shrinkage can be reduced to only 2 % (v/v). We believe that the reduction of volume shrinkage was due to the dense packing and covalent bonding of vinyl modified silica nanoparticles in the polymerization of vinyl groups on the surface of silica nanoparticles.
Furthermore, because the size of nanoparticles is only 13 nm in diameter, which is far below wavelength of visible light and the distribution of those silica nanoparticles are
248 even inside the dental resin. Hence the resulting dental materials were transparent. The mechanical property of the new dental materials was found to be reasonably good.
Unfortunately, there are still some problems existing in this new dental material.
One is the poor handling of this material. When the vinyl modified silica nanoparticles were mixed with dental resin, the viscosity becomes extremely high, especially in high silica loading (>30 wt %). The high viscosity made removing air bubbles and filling the material in a container very difficult. That is one of reasons why limited data could be obtained for the mechanical tests of this dental material.
Thus, in the future, a new technique of mixing nanoparticles and dental resin needs to be discovered to solve the handling problem. We believe that if this problem could be solved, the new hybrid material would be a promising dental material with minimal volume shrinkage.
7.5 Acknowledgements
I am grateful to Dr. Margaret A. Wheatley and her students in Department of
Chemical and Biological Engineering at Drexel University fro their help on ultracentrifuge instrument. I thank Dr. Solomon Praveen of Drexel University and Dr.
George Baran of Temple University for their help in the synthesis and characterization of dental matetrials, especially on compressive tests. I also thank to Dr. Guoliang Yang and his students in Department of Physics at Drexel University for their discussion and help in AFM experiments.
249 7.6 References
1. Yin, R., Suh, B. I., Sharp, L. & Tiba, A. 37 pp ((Bisco, Inc., 2001-US43296). 2003). 2. Feilzer, A. J., De Gee, A. J. & Davidson, C. L. Setting stress in composite resin in relation to configuration of the restoration. Journal of Dental Research 66, 1636-9 (1987). 3. Davidson, C. L., de Gee, A. J. & Feilzer, A. The competition between the composite-dentin bond strength and the polymerization contraction stress. Journal of dental research 63, 1396-9 (1984). 4. Bausch, J. R., De Lange, K., Davidson, C. L., Peters, A. & De Gee, A. J. Clinical significance of polymerization shrinkage of composite resins. Journal of Prosthetic Dentistry 48, 59-67 (1982). 5. Cohen, G. M. & Huang, D. D.-J. 11 pp ((USA). Application: US2004-935943 2005). 6. Bowman, C. N., Lu, H. & Stansbury, J. W. 35 pp ((The Regents of the University of Colorado, USA, 2004-US34968). 7. Dauvillier, B. S. & Feilzer, A. J. Low-shrinkage dental restorative composite: modeling viscoelastic behavior during setting. Journal of Biomedical Materials Research, Part B: Applied Biomaterials 73B, 129-139 (2005). 8. Hwang, M. S., Kim, C. K. & Kim, O. Y. Changes in the volumetric shrinkage of the novel dental composites. PMSE Preprints 90, 537-538 (2004). 9. Angeletakis, C., Nguyen, M.-D. S. & Kobashigawa, A. I. 11 pp , Cont -in-part of U S Ser No 859,106 ((Kerr Corporation, USA). Application:US, 2003). 10. Dauvillier Bibi, S. & Feilzer Albert, J. Low-shrinkage dental restorative composite: modeling viscoelastic behavior during setting. J Biomed Mater Res B Appl Biomater 73, 129-39 (2005). 11. Davidson, C. L. & De Gee, A. J. Relaxation of polymerization contraction stresses by flow in dental composites. Journal of Dental Research 63, 146-8 (1984). 12. Davidson, C. L. Resisting the curing contraction with adhesive composites. Journal of prosthetic dentistry 55, 446-7 (1986). 13. Kemp-Scholte, C. M. & Davidson, C. L. Marginal sealing of curing contraction gaps in Class V composite resin restorations. Journal of dental research 67, 841-5 (1988). 14. Kemp-Scholte, C. M. & Davidson, C. L. Complete marginal seal of Class V resin composite restorations effected by increased flexibility. Journal of dental research 69, 1240-3 (1990). 15. Lebeau, B., Patarin, J. & Sanchez, C. Design and properties of hierarchically organized hybrid organic-inorganic nanocomposites (review). Advances in Technology of Materials and Materials Processing Journal 6, 298-307 (2004). 16. Bourgeat-Lami, E. Organic/inorganic nanocomposite colloids. Encyclopedia of Nanoscience and Nanotechnology 8, 305-332 (2004).
250 17. Sanchez, C. de Soler-Illia, G. J. Ribot, F. Lalot, T. Mayer, C. R.Cabuil, V. Designed hybrid organic-inorganic nanocomposites from functional nanobuilding blocks. Chemistry of Materials 13, 3061-3083 (2001). 18. Schmidt, H. K., Mennig, M., Nonninger, R., Oliveira, P. W. & Schirra, H. Organic-inorganic hybrid materials processing and applications. Materials Research Society Symposium Proceedings 576, 395-407 (1999). 19. Schmidt, H. K. Geiter, E. Mennig, M. Krug, H. Becker, C. Winkler, R. P. The sol- gel process for nano-technologies: new nanocomposites with interesting optical and mechanical properties. Journal of Sol-Gel Science and Technology 13, 397- 404 (1998). 20. Hajji, P., David, L., Gerard, J. F., Pascault, J. P. & Vigier, G. Synthesis, structure, and morphology of polymer-silica hybrid nanocomposites based on hydroxyethyl methacrylate. Journal of Polymer Science, Part B: Polymer Physics 37, 3172- 3187 (1999). 21. Kaddami, H., Gerard, J. F., Hajji, P. & Pascault, J. P. Silica-filled poly(HEMA) from HEMA/grafted SiO2 nanoparticles: polymerization kinetics and rheological changes. Journal of Applied Polymer Science 73, 2701-2713 (1999). 22. Schaudel, B., Guermeur, C., Sanchez, C., Nakatani, K. & Delaire, J. A. Spirooxazine- and spiropyran-doped hybrid organic-inorganic matrixes with very fast photochromic responses. Journal of Materials Chemistry 7, 61-65 (1997).
251
(a)
(b)
3400 2400 1400 400 wavelength (cm-1)
Figure 7- 1 FTIR spectra of (a) HEMA monomer; (b) dry silica nanoparticles modified with vinyl groups on surface;
252
(a)
(b)
3420 2420 1420 420 wavelength (cm-1)
Figure 7- 2 FTIR spectra of silica nanoparticle sediments after ultracentrifuge (a) with EtOH as solvent; (b) with acetone as solvent.
253
3420 2420 1420 420 wavelength (cm-1)
Figure 7- 3 FTIR spectrum of the new dental composite with vinyl modified silica nanoparticles as inorganic fillers.
254
100 98 96 94 (a) 92 (b) 90
Weight percentage (%) 88 0 200 400 600 800 1000 Temperature (oC)
Figure 7- 4 TGA spectra of silica nanoparticle sediments after ultracentrifuge (a) with EtOH as solvent; (b) with acetone as solvent.
255
Figure 7- 5 TGA spectrum of the new dental composite with vinyl modified silica nanoparticles as inorganic fillers.
256
Figure 7- 6 Picture of the new dental composite with vinyl modified silica nanoparticles as inorganic fillers. For this particular sample, the loading percentage is 51 wt%.
257
Figure 7- 7 AFM picture of nanoparticle sediments after ultracentrifuge.
258 Chapter 8: Summary and Conclusions
All the research works included in this dissertation have been focused on synthesis, processing, evaluation and characterization of nanoporous materials, organic- inorganic hybrid materials and nanocomposites and their applications in enzyme encapsulation, protein folding unfolding, sensor, catalysis and dental materials. These works can be divided into two areas. One is silica nanoporous materials and their applications in bioscience. In this study, by using a novel non-surfactant templated sol- gel method, which was developed in our group, biospecies, such as proteins, polypeptides were encapsulated in nanoporous silica matrix with controlled pore size. By adjusting template molecules and experimental conditions, like pH value, concentration of denaturant and temperature, the folding unfolding behavior of encapsulated protein, and aggregation of encapsulated polypeptide were monitored by various methods. Another aspect of my research is the synthesis, fabrication and characterization of organic- inorganic hybrid nanocomposites. In this area, sol-gel nanoporous silica particles or vinyl modified silica nanoparticles were used as inorganic components; HEMA or mixture of
BisGMA and TEGDMA were used as organic components. Because of the interactions between organic and inorganic phases in those materials, they show great potentials as dental, sensor and catalysis materials.
The main conclusions on these two topics are further summarized in detail in the following sections.
8.1 Nanoporous Materials and Their Application in Bioscience
259 In this area, a novel non-surfactant templated sol-gel method was used to encapsulate biospecies inside nanoporous silica matrix with controlled pore size. The most significant difference of this method form conventional method for the synthesis of mesoporous silica materials is the employment of non-surfactant molecules, such as fructose, glucose and urea was used as template molecules instead surfactants. Several new interesting properties were brought by this difference. First, because the interaction between non-surfactant molecules and silica matrix is not as strong as surfactant molecules, these non-surfactant templates are easy to be removed from silica matrix by simple water extraction at room temperature instead of burning at high temperature which is used for removing surfactant molecules. Second, most of non-surfactant molecules we used in this study are biofriendly. In contrast, many surfactant molecules, especially some ionic surfactants are often toxic. Third, the average pore size of silica matrix via non- surfactant templated pathway can be controlled by changing the concentration of templates.
Owning to these characteristics of non-surfactant templated sol-gel silica, this material can be used for enzyme encapsulation and serves as a unique platform to study protein folding and polypeptide aggregation in confined space. In a typical experimental process, silica precursors, like TMOS or TEOS, were mixed with water and hydrolyzed under acid, HCl. Upon hydrolysis, non-surfactant templates as well as biospecies buffer solution can be added into the mixed sol. As the sol becomes solid gel, biospecies molecular are encapsulated in the silica matrix. After aging and drying, the bio-wet gel becomes glass-like silica monolith. Then, by water extraction, template molecules can be removed from the silica matrix and biospecies molecules are left inside. Since the whole
260 process can be done under room temperature and no bio-toxic reagents are used, biospecies can be encapsulated in their native state. That is very important in bio- encapsulation and bioscience studies.
In this thesis work, several proteins and one polypeptide were encapsulated into the porous silica matrix via this novel method. As the protein on which the most efforts were put in this study, cytochrome c (Cc) was encapsulated in both its folded and unfolded states. In the project on Cc’s refolding behavior, urea was used as both denaturant and template. Thus, Cc was encapsulated in its unfolded state in silica matrix.
Then, urea was removed to give encapsulated Cc an opportunity to refold back to its folded state. Fluorescence spectroscopy and circular dichroism studies of the encapsulated Cc showed that the extent of refolding of Cc increases with the pore size and pore volume of the silica host. The ability of the porous material to mediate the protein refolding process provides an exciting example that mesoporous materials function like a rigid matrix artificial chaperone.
Another project on encapsulated Cc is using resonance Raman (RR) spectroscopy
to study both its unfolding and refolding behaviors. In this project, because biofriendly
non-surfactant fructose was used as the template, Cc was encapsulated in its folded state.
After template removing, the biogel powders were put into different environments, such
as denaturant solutions, acidic media and high temperatures. RR spectroscopy was used
the major analytical method to monitor the encapsulated protein molecules. It was found
that small pores could hinder both folding and unfolding motions of the entrapped
protein more effectively than large pores. The results demonstrate that this new method
of protein encapsulation in silica matrix with controlled pore size has the potential of
261 trapping intermediate states of protein refolding process, thus may be useful in
promoting our understanding of the protein folding and unfolding processes.
One of important polypeptide associated with Alzheimer’s disease (AD), Amyloid
β 1-42 was encapsulated in porous silica by the same method. The aggregation of Aβ1-42 was believed as the main cause of Alzheimer’s disease. To study its aggregation in confined space, Aβ1-42 was encapsulated in its monomeric state in silica matrix with controlled pore size via non-surfactant templated sol-gel method. HFIP, a strong organic solvent was used to dissolve Aβ1-42 and kept the polypeptide in its monomeric state. Then by changing the pH value of the media where the silica biogel powders were immersed in, aggregation behavior of the encapsulated Aβ1-42 was monitored by ThT fluorescence and
CD spectroscopy. It was found that pore size had significant effect on the peptide aggregation behavior. Peptides encapsulated in large pore have more chances to form fibrils than those in small pores. It is postulated that since in large pores peptides have more freedom to move and meet with others to form aggregate. In contrast, in small pores, peptides are more likely fixed in pores and channels in silica matrix and can not move to form aggregates. This is the first study ever reported that the aggregation of Aβ1-42 was studied in confined space to mimic the crowded cellular environments. We believe further investigations based on this material will provide us new insights into amyloid peptides aggregation. Furthermore, the successful encapsulation Aβ1-42 inside the silica porous materials without losing its bioactivity proves that this encapsulation method can be used in various applications, such as biosensor materials, biocatalyst, etc.
8.2 Organic-Inorganic Hybrid Nanocomposites
262 Nanostructure hybrid materials in possession of both advantages of inorganic and organic materials were synthesized and characterized in this thesis work. To achieve homogeneity of the hybrid materials, covalent bonding or physically micro-interlock was utilized to improve interactions between organic ands inorganic phases. In addition, the nano scaled domain size of inorganic phase also contributes to improvement of homogeneity of hybrid materials. Because of versatility and excellent mechanical properties of these hybrid nanocomposites, they show great potentials as dental materials.
Three projects were carried in this research area and two of them were using a colloidal nanoparticles of silica (OG100-31) as raw material. In OG100-31 liquid colloidal, vinyl modified silica nanoparticles were suspended in HEMA monomer.
Because the size of those silica nanoparticles is about 13 nm in diameter and vinyl groups modified on their surfaces enable polymerization reactions happening between silica nanoparticles and HEMA monomers, these silica nanoparticles can be a great candidate as inorganic phase in hybrid materials. In one of the projects, organic-inorganic hybrid nanofibers of poly(2-hydroxyethyl methacrylate) (PHEMA)-silica nanoparticles was prepared by electrospinning. The PHEMA-silica hybrid was prepared by solution radical copolymerization of HEMA with these silica nanoparticles with diameter of 13 nm. After precipitation and re-dissolution, this hybrid polymer-silica solution was electrospun into nanofibers, which were characterized by SEM, TEM, TGA, DSC, etc. The results indicate that silica nanoparticles were embedded in nanofibers with diameter below 200 nm. This might be the first reported electrospun nanofibers with PHEMA as organic component and nanosized inorganic silica particles covalently embedded inside. Because the inorganic domain size in this fibrous material was about to 13 nm and was covalent
263 bonded to organic polymer component, the interactions between organic and inorganic phases were stronger than conventional composite materials. Because of the high surface area of nanofibers and excellent thermal and mechanical properties brought by the hybrid nanocomposite, we believe that with appropriate selection of the organic and inorganic components, nanofibrous material can be used in many applications, such as sensors, catalyst support, and tissue engineering scaffold materials.
The vinyl modified silica nanoparticles were also used in the other project as inorganic fillers for dental materials. These nanoparticles were first separated from the
HEMA colloidal by ultracentrifuging, then mixed with dental resins (e. g. BisGMA:
TEGDMA= 1:1). Upon photo initiated polymerization, hybrid dental materials with vinyl modified silica nanoparticle dense packing as inorganic component were achieved.
Because of the dense packing of silica nanoparticles and covalent bonding between vinyl modified silica nanoparticles, the volume shrinkage arising from polymerization of organic components can be reduced. Our preliminary results showed that at 50 wt % silica loading, the volume shrinkage can be reduced to only about 2 % (v/v). Furthermore, because the size of nanoparticles is 13 nm in diameter, which is far below wavelength of visible light and because the distribution of the silica nanoparticles are even inside the dental resin, the dental materials obtained were transparent. The mechanical properties of this dental material were also enhanced due to the covalent bonds between organic and inorganic phases. We believe that this new hybrid nanocomposite could be an ideal dental material if the problems of poor handling and low silica content loading are solved.
Another attempt to design new dental materials was done by using nanoporous sol-gel silica particles as inorganic fillers. In most of conventional dental composite
264 materials, silica particles are modified with silane coupling reagents as inorganic fillers.
Unfortunately in oral environments, chemical bonds forming between coupling reagents and dental resin may be hydrolyzed to induce separation of organic and inorganic phases.
The purpose of one of this thesis projects to circumvent the problem by interlocking dental resin molecules with nanoporous silica particles physically. In this project, a new nanoporous material with three-dimensional interconnected porous structures inside was synthesized via non-surfactant templated sol-gel method. When this filler is mixed with dental resin monomer before polymerization, the monomer can diffuse inside pores of the filler. After polymerization, dental resin matrix can interlock with nanoporous silica fillers, thus the organic and inorganic phases can tightly interact with each other only via physically interaction. Results showed that nanoporous silica particles filled dental materials were stronger than non-porous silica filled dental materials, even though the non-porous filler had been treated with silane coupling reagents.
265 Appendix A: Supplemental Data of Chapter 2
Table A-1 Data of relative difference, (IU-IT)/IT, in fluorescence intensity between unfolded and refolded Cc for the samples with increasing pore size up to free Cc in solution.
Cyt-C Sample Relative silica Iu Ir per Code differece amount(g) silica(mg) ccu0 23011 18965.6 0.2132791 0.0113 0.008183871 ccu15 24711.2 26277.1 -0.059609 0.010115 0.008511226 ccu30 35264 16518.8 1.1347682 0.01008 0.008183871 ccu40 27727.5 13738.7 1.0181884 0.008183871 0.01008 ccu50 73651.8 13256.3 4.5560261 0.01025 0.008183871 free cc 24528.9 2335.44 6.1488413 tris 2588.77 buffer urea 7833.21
266
25000 Refolded State 20000 Unfolded State 15000
10000
Intensity (cps) 5000
0 300 350 400 450 500 Wavelength (nm)
Figure A- 1 Representative plot of free Cc in its refolded and unfolded state.
267 Appendix B: Supplemental Data of Chapter 4.
90000 80000 pH 2.35 70000 pH 2.97 60000 pH 3.49 50000 pH 4.74 40000 (CPS) 30000 pH 5.57 20000 pH 6.3 10000 pH 7.02 Fluorescence Intensity 0 pH 8.05 465 475 485 495 505 pH 8.9 wavelength (nm)
Figure B- 1 Representative fluorescence spectra of free Aβ 1-42 in buffer with different pH value.
268
4000000
3500000 pH 2.35 3000000 pH 2.97 y (cps t pH 3.49 2500000 ensi
t pH 4.74 n 2000000 pH 5.57 pH 6.3 1500000
scence I pH 7.02 e 1000000 pH 8.05 uor
Fl 500000 pH 8.9
0 460 470 480 490 500 510 Wavelength (nm)
Figure B- 2 Representative fluorescence spectra of Abeta42-0 in buffer with different pH value.
269
3500000 pH 2.35 ps 3000000 c
( pH 2.97 y t i 2500000 pH 3.49 ns e
t pH 4.74
n 2000000
e I pH 5.57 1500000 nc pH 6.3 e c
s 1000000 pH 7.02 e pH 8.05 uor l 500000 F pH 8.9 0 470 475 480 485 490 495 Wavelength (nm)
Figure B- 3 Representative fluorescence spectra of Abeta42-30 in buffer with different pH value.
270
3500000 pH 2.35
ps 3000000 c pH 2.97 y ( t 2500000 pH 3.49 nsi e
t pH 4.74
n 2000000 pH 5.57 1500000 pH 6.3 1000000 pH 7.02 pH 8.05 uorescence I
Fl 500000 pH 8.9 0 460 470 480 490 500 510 Wavelength (nm)
Figure B- 4 Representative fluorescence spectra of Abeta42-50 in buffer with different pH value.
271
Table B- 1 Data of time scale fluorescence study of Abeta42 series samples when the pH value jumping from 2.35 to 7.02 Abeta42- Abeta42- time(s) Abeta42-0 time(s) time(s) 30 50 0 2308430 0 3806149 0 2074780 1 6233658 1 7233982 1 3932003 8 8444321 7 8833055 7 5341856 17 9081818 17 9775828 17 6141823 24 8959713 23 9938836 24 6328529 31 9434841 30 1.02E+07 30 6672265 40 9090146 37 1.12E+07 39 7028617 47 9080535 44 1.16E+07 47 7331136 54 9888511 51 1.15E+07 53 7403910 61 8098850 59 1.09E+07 62 7612547 70 9010930 67 1.08E+07 70 7962658 77 9788136 74 1.17E+07 77 8128025 84 9693687 81 1.11E+07 84 8253591 91 1.04E+07 88 1.20E+07 91 8323072 99 1.00E+07 95 1.26E+07 98 8493093 106 1.02E+07 102 1.20E+07 105 8756384 116 1.05E+07 112 1.15E+07 117 8892312 124 1.06E+07 121 1.18E+07 124 9215611
272 Appendix C: Supplemental Data of Chapter 5.
Figure C- 1 SEM pictures of electrospun hybrid fiber when the ratio of DMF to EtOH in solvent mixture was 20:80.
273
Figure C- 2 SEM pictures of electrospun hybrid fiber when the ratio of DMF to EtOH in solvent mixture was 30:70.
274
Figure C- 3 SEM pictures of electrospun hybrid fiber when the ratio of DMF to EtOH in solvent mixture was 40:60.
275
Figure C- 4 SEM pictures of electrospun hybrid fiber when the ratio of DMF to EtOH in solvent mixture was 60:40.
276
Figure C- 5 SEM pictures of electrospun hybrid fiber when the ratio of DMF to EtOH in solvent mixture was 70:30.
277
Figure C- 6 SEM pictures of electrospun hybrid fiber when the ratio of DMF to EtOH in solvent mixture was 80:20.
278 Appendix D: Fabrication of a New Type Molecularly Imprinted Polymer Membrane
Sensor for Atrazine
(An Independent Research Proposal Defended and Passed On July 30th, 2004 in Partial
Fulfillment of the Requirement for the Ph.D. Candidacy)
ABSTRACT:
Molecularly imprinted polymer (MIP), as an artificial macromolecular receptor, has been widely used in analytical chemistry. This approach includes formation of a complex between functional monomers and guest molecules (templates), and “freezing” of the complex by polymerization. Following template removal by simply washing, polymer binding sites (imprints) are left in the structure, which are specific or complementary to those of the template molecule. Though with many advantages, such as high specificity, great stability, low cost and application of virtually any kind of substance etc., practical applications of MIP are still quite limited due to the long response time and difficulty of fabrication of thin and stable membranes with reproducible properties.
The objective of this study is to fabricate a new type of sensor system for atrazine by spin coating or electrospinning molecularly imprinted post-crosslinked polyurethane on QCM (quartz crystal microbalance). In this study, polyurethane capped with acrylate group is synthesized followed by mixing with template molecule atrazine and photoinitiator. Then the solution is spin coated or electrospun to the surface of mass sensitive sensor (QCM) to form MIP membrane as recognition material. UV curing is used to make linear polyurethane crosslinked. After the atrazine templates are extracted from the MIP nanofibrous membrane, this mass sensitive sensor can be used to detect
279 atrazine either in liquid or gas phase. That is the first time to synthesis nanofibrous MIP membrane sensor material. By this method, the sensitive and reproducibility of imprinted polymer can be enhanced a lot.
INTRODUCTION
Chemical sensors and biosensors are of increasing interest within the field of modern analytical chemistry due to new demands and opportunities appearing particularly in clinical diagnostics, environmental analysis, food analysis and production monitoring, as well as the detection of illicit drugs, genotoxicity, and chemical warfare1,2.
The central part of a chemical or biosensor is the recognition element1,3, which is responsible for specifically recognizing and binding the target analyte often in a complex sample. Therefore to prepare a suitable recognition element is the key part of making sensors.
Polymers have been widely used as recognition or supporting materials in bio or chemical sensors4. Molecular imprinted polymer, as one of the polymer sensor materials, is becoming an important analytical tool5,1,6,7,8. MIP can be considered as the mimic of biomolecular recognition mechanism, which is the underlying principle of many biological processes. Like a key and lock, specialized structures such as antibodies, hormone receptors and enzymes fit perfectly with their natural targets. However, although they are “nature own”, they are far from ideal tools- they are unstable when not in their native environments and often in short supply.
So, people are thinking of building artificial receptors that are capable of recognizing and binding the desired molecules with a high affinity and selectivity. MIP is
280 one of those designed systems. The synthesis of MIP is a process where functional monomers are polymerized in presence of the target analyte (the imprint molecule), which acts as a molecular template. The functional monomers initially form a complex with the imprint molecules, and after polymerization, the functional groups are held in position in the polymer solid. After removing imprint molecules, the binding sites remain in the polymer matrix, which are complementary in size and shape to the analyte. In this way, a molecular memory is introduced into the polymer, which is now capable of rebinding the analyte with a very high selectivity9. If the selectivity factor α is as the ratio of the response of analyte to the other compounds with similar structure, the number of α is always above 10.
There are two distinct approaches to molecular imprinting distinguished by the interactions between monomers and imprinted molecules, which can be either covalent bonds or non-covalent bonds9. Owing to the greater stability of covalent bonds, covalent imprinting protocols yield a more homogenous population of binding sites. On the other hand, the non-covalent imprinting protocols are more flexible and have a greater range of application concerning the theoretical lack of restrictions on size, shape or chemical character of the imprinted molecules. Moreover, it is more similar to natural processes in the way that most biomolecular interactions are non-covalent in nature, which include hydrogen bond, electrostatic interaction, hydrophobic interaction, etc. In this proposed study, a non-covalent bonded MIP sensor is selected. Both of these approaches are depicted in Scheme 1.
281
Noncovalent imprinting
Covalent imprinting
Scheme 1. Two Approaches of Molecular Imprinted Procedures1
The next step, fabrication is a very important in producing sensors1. The use of recognition material can be considered as receiving chemical or bio signals and the use of transducer is to translate the signal and convert it to an easily quantified output signal.
Traditionally, molecular imprinted polymer system can be classified in three groups according to the fabrication processes:
1. The MIP is in the form of powder. In this approach, MIP is polymerized in
presence of imprint molecules in solution to form bulk material followed by
being grinded into powders. After extracting templates by washing, the
particles can be used as recognition elements10,11,12,13,14,15,16. As an alternative,
particles can also be prepared directly in the form of spherical beads of
controlled diameters17,18,19. In this particle form, molecular recognition mostly
occurs at the surface of the particles. And the particle size is usually from 25
282 µm to 50 µm, which leads to slow intraparticle diffusion and consequently
long response time. As noted in previous papers, the sufficient time for target
molecules to be diffused into polymer and bind is around 1-2 hours.
Furthermore, grinding and sieving processes are labor intensive and waste of
useful polymers20.
2. The MIP is prepared as a film on the transducer surface. While working with
films, polymerization can be either achieved by thermo or light
polymerization of methacrylic acid (MMA). Vinylpyridine (Vpy) and
acrylamide derivatives with crosslinker on the surface or directly
electropolymerized conducting polymer, such as polyanniline, polypyrrole
and poly(o-phenylenediamine), on electrode21,22,23,24,25. Another way to
prepare MIP membrane is casting/immersion precipitation phase inversion
method. For MIP membrane, it is difficult to prepare thin and stable
membranes with reproducible properties from highly crosslinked polymers.
Furthermore, mass-transfer in and through MIP membrane is very inefficient
and insufficient for real applications.
3. Molecular imprinting can also be done in self-assembled monolayers
(SAMs)26,27. In a general sense, the formation of rigid nanostructures
organized around the template molecule by SAMs at electrodes surface can be
considered as a form of two-dimensional imprinting. Though molecular
imprinting in SAMs can minimize diffusion barrier due to the thickness in
nanoscale, the main drawback if this form of imprinting is the lack of stability
283 of the film, with possible destruction of recognition sites if the template is
removed.
Though molecularly imprinted polymers have a lot of advantages of being recognition element in chemical or biosensor, such as high selectivity, great stability, low cost and application of virtually any kind of substance etc., practical applications of MIP are still limited due to the long response time and difficulty of integration of these artificial recognition element with transducers. All above-mentioned approaches cannot provide any good solution to these problems.
In this study, we propose to use post-crosslinked polyurethane system as molecularly imprinted polymer combined with spin coating or electrospinning techniques to solve those problems. In this system, the forming of the complex of polyurethane and templates is before crosslinking, thus the complex are still soluble and allow being spin coated or electrospun to get desired membranes.
Spin Coating
Spin coating has been used for several decades for application of thin films. A typical process involves depositing a small puddle of a fluid resin onto the center of a substrate and then spinning the substrate at high speed (typically around 3000 rpm).
Centripetal acceleration will cause the resin to spread to, and eventually off the edge of the substrate leaving a thin film of resin on the surface.
Final film thickness and other properties will depend on the nature the resin
(viscosity, drying rate, percent solids, surface tension, etc.) and the parameters chosen for the spin process, such as rotational speed, acceleration, spin time, etc.
284
Electrospinning
The concept electrospinning, derived from “electrostatic spinning”, can be dated back to more than 60 years ago. From 1934 to 1944, Formalas published a series of patents28,29,30,31, describing an experimental setup for the production of polymer filaments using an electrostatic force. Since 1980s and especially in recent years, the electrospinning process has gained more attention probably due in part to surging interest in nanotechnology. Unlike the other fiber spinning techniques (wet spinning, dry spinning, melt spinning, gel spinning) which can produce polymer fibers in the micrometer range, electrospinning can fabricate polymer fibers in the nanometer range.
The high specific surface area and small pore size of electrospun nanofibers make them interesting candidates for a wide variety of applications including multifunctional membrane, biomedical structural elements (scaffolding used in tissue engineering, wound dressing, drug delivery, artificial organs, vascular grafts), protective shields in special fabrics, filter media for submicron particles in separation industry, composite reinforcement, and structures for nano-electronic machines among others. Besides those applications, electrospun polymer nanofibers also have been used in the fabrication of chemical sensors32.
285
Figure 1. Schematic Diagram to Show Polymer Nanofibers By Electrospinning.
A schematic diagram to describe electrospinning of polymer nanofibers is shown in Figure 1. There are basically three components: a high voltage supplier, a capillary tube with a pipette or a needle of small diameter and a metal collecting screen. The electrospinning process can be easily achieved by applying a high voltage between the capillary filled with the polymer fluid and the collector screen. In this process, a high voltage electric field is subjected to the end of the capillary tube that contains the solution held by its surface tension. This induces a charge on the surface of the liquid. Mutual charge repulsion and the contraction of the surface charges to the counter electrode cause a force directly opposite to the surface tension. With the intensity of the electric field increasing, the hemispherical surface of the fluid at the tip of the capillary tube first
286 elongates to form a conical shape known as the Taylor cone and after a critical value at which the repulsive electrostatic force overcomes the surface tension is attained, the charged jet of fluid is ejected from the tip of Taylor cone. The discharged polymer solution jet undergoes an instable and elongation process, which allows the jet to be very long and thin. At the same time, the solvent evaporates, leaving behind a charged polymer fiber.
It is agreeable that the following three criteria should be applied to ideal nanofibers: (1) the diameters of the fibers should be consistent and controllable, (2) the fiber surface should be defect-free or defect-controllable, (3) continuous single nanofibers should be collectable. To achieve this, the following parameters are needed: (a)
System parameters, like molecular weight, molecular-weight distribution and architecture
(branched, linear etc.) of polymer or viscosity, elasticity, conductivity and surface tension of solution; (b) Process parameters such as electric voltage, flow rate, concentration, distance and angle between the capillary and collection screen.
Generally speaking, one of the most significant parameter influencing the fiber diameter is the viscosity of solution. A high viscosity usually results in a large fiber diameter. Meanwhile, when a solid polymer is dissolved in a solvent, the solution viscosity is proportional to the polymer concentration and molecular weight of the polymers, thus the higher the polymer concentration the larger resulting nanofiber diameters will be. On the other hand, the polymer solution must have a concentration high enough to cause polymer entanglements yet not so high that the viscosity prevents polymer motion induced by the electric field.
287 The other parameter which affects the fiber diameter to a remarkable extent is the distance between capillary jet and collecting screen. In general, a longer spinning distance always results in a smaller fiber diameter.
Another problem encountered in electrospinning is that defects such as beads may occur in polymer nanofibers. Beads can be considered as capillary break-up of the jets by surface tension. Thus, charge density carried by the jet, surface tension and viscoelastic properties of the solution are significant parameters. The higher the solution viscosity is, the fewer beads are formed. And the net charge density influences in the same way.
PROPOSED RESEARCH
The objective of this study is to fabricate a new type of sensor recognition material for atrazine by molecularly imprinted post-crosslinked polyurethane via spin coating or electrospinning. In this study, polyurethane capped with acrylate group is synthesized followed by mixing with template molecule atrazine and photoinitiator. Then the solution is spin coated or electrospun to the surface of mass sensitive sensor to form
MIP membrane as recognition material. And UV curing is used to make linear polyurethane crosslinked. After the atrazine template is extracted from the MIP membrane, this mass sensitive sensor can be used to detect atrazine either in liquid or gas detection. That is the first time to synthesis MIP membrane sensor material, by this method, the sensitive and reproducibility of imprinted polymer can be enhanced a lot.
Theoretical Considerations
288 When designing a sensor system, selectivity and sensitivity are two of the most important criteria that must be considered. Selectivity refers to the extent to which a method can determine a particular analyte in mixture without interferences from other components. MIP sensors usually have inherently high selectivity because of their unique molecular structures, which have cavities left by template molecules thus only the exact same molecules can be again fitted into the space left theoretically. Nevertheless, low sensitivity, long response time and difficulties in fabrication still remain as obstacles which prevent MIP sensors to be widely used. In this proposed study, we are trying to solve those problems in our new MIP system.
Sensors for atrazine. Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5- triazine) is the most widely is used herbicides in the agricultural field to protect crops and corns. Because of its persistence there is a serious risk that soil and drinking water will contain such concentrations that they maybe harmful. Presently, most of the detection is done with established analytical techniques such as high-performance liquid chromatography (HPLC) and gas chromatography with mass spectroscopy (GC/MS).
Normally, pretreatment and preconcentration is necessary, which make these techniques time-consuming, complex and expensive. Therefore a new method is proposed in this study to overcome these problems.
Sensitivity and stability enhancement. Here we propose to use spin coating or electrospinning to fabricate MIP membrane. For spin coating, since the thickness can be easily controlled, the reproducibility of MIP membrane may get improved. The advantage of electrospinning lies in its extremely high surface area to volume ratio.
Theoretically, Electrospun nanofibrous membranes can have approximately 1 to 2 orders
289 of magnitude more surface area than that found in continuous thin films33. Since larger surface area means more recognition sites can be exposed to the environment, it is expected that this large amount of available surface area has the potential to provide desirable high sensitivity and quick response time. As for the case of molecularly imprinted polymers, larger surface area and small fiber diameter usually means more binding sites can be exposed to the surface and faster diffusion rate of analyte molecules into MIPs. Consequently, electrospun MIP nanofiber is supposed to have much higher sensitivity and quicker response than common used MIP sensor.
Mechanism of recognition. To illustrate our proposed study, polyurethane with atrazine as template is chosen as the MIP system. The formation of MIP polymer and recognition process is shown in Figure 2. When mixing atrazine molecules with polyurethane in solution, hydrogen bonds can be simultaneously formed between template molecules and functional groups on polymer backbones, so those functional groups are arranged in an orderly manner complimentary to the template to form complexes. After crosslinking of the polymer molecules by photo initiation, the arrangements of functional groups are fixed even with removal of templates. At this moment, we can say that the structure of the template is memorized in the polymers, providing the target receptors. Then when the sites in MIP encounter target molecules, those targets instead of the other molecules easily form complexes with receptor sites due to the complimentary shape and functional groups arrangement of the sites.
290
Figure 2. Schematic diagram of molecularly imprinting of atrazine using polyurethane as polymer scaffold.
Synthesis of MIP system. Molecularly imprinted polymers have already been used as recognition materials to detect atrazine in aqueous solutions21,34. In previous studies, monomer MAA (methacrylic acid), crosslinker EDMA (ethylene glycol dimethacrylate), initiator AIBN (Azobisisobytyronitrile) and template were mixed in chloroform and polymerized by UV light irradiation.
As noted in previous studies, control of crosslinking level of MIP is very important for the performance of the sensor. Traditional formulas include high crosslinker ratios (5:1 mole of functional monomer), which favor the creation of imprinted sites and obtain the mechanical strength. But it was also found that, with as
291 little as 20% of crosslinker, it was still possible to observe memory effects. Especially when using films as recognition material, some degree of flexibility favors the binding of the templates and improves the adherence to transducer surface. So, there needs to be a compromise between the minimum crosslinker necessary to obtain template memory and flexibility7. In our research, electrospun MIP nanofibrous membrane is used as recognition material instead of single layer MIP film. The same conclusion can also be applied to our material. Therefore, crosslinking is still needed to maintain the specificity of the cavity inside MIP; on the other hand excess crosslinking may cause the material to be too hard and brittle to be integrated with the transducer.
Polyurethane is chosen as the MIP in this project since this polymer has already been proved to be one of good MIP material with corsslinking35,36,37,38. But since highly crosslinked polymer cannot be dissolved in solvent to be electrospun, we must crosslink the polymer fiber in the solid state39,40,41,42 after electrospinning polyurethane solution to nanofibers.
Due to these considerations, we modify the synthesis procedure of MIP nanofiber by post-crosslinking after electrospinning. In our procedure, two monomers, diols and diisocyanates are reacted in particular stoichiometry to form linear polyurethanes with isocyanate as the end groups, then the acrylate groups can be introduced to the polymer chain by reacting with the end isocyanate groups. At this time the polymer molecules are still linear and soluble. After mixed with template atrazine and photoinitiator, the polymer solution can be electrospun to nanofiber followed by UV curing to make the fiber crosslink. Finally, extracting of atrazine templates by washing with MeOH can leave binding cavities inside MIP.
292 The reactions involved in the process can be shown below:
H2 H2 H2 H2 H2 OCN C NCO + HO C C C C OH
H2 H O H2 O H H2 H O H2 O H H2 OCN C N C O C O C N C N C O C O C N C NCO 4 n 4
H2 H O H2 O H H2 H O H2 O H H2 OCN C N C O C O C N C N C O C O C N C NCO 4 n 4
H2C CH H2 + C O C CH2 O OH
H2C CH HC CH2 H2 H2 C O C CH2 H2C C O C O O O O C N N C O H H O OH OH UV C + Ph C Ph
O O
293 H C CH HC CH 2 H2 H2 2 C O C CH2 H2C C O C R + O O O O C N N C O H H O
O
C O H2 H H2 H2 H H2 H H2 R C C C C C C C C C H C O C O C O
O O O
O O O
C O C O C O H2 H2 H H2 H2 H2 C C C C C C C C C C H H H O C O
O
Integration of MIP nanofiber on transducer The performance of sensor device is also determined by the transducer principle and the integration of recognition material and transducer2. In this proposal, mass-sensitive transducers, quartz crystal microbalance
(QCM) are used because QCM sensor is an ultra-sensitive mass sensor which can measure mass change in nanogram range, furthermore it can be miniaturized, mass- fabrication and therefore are inexpensive devices. The heart of QCM is the piezoelectric quartz sandwiched between a pair of electrodes. When the electrodes are connected to an oscillator and an A/C voltage is applied over the electrodes the quartz crystal starts to oscillate at its resonance frequency due to piezoelectric effect. If a sample is deposited on one electrode the resonant frequency will decrease proportionally to the mass of absorbed layer.
294 Figure 3 shows the experimental set up for liquid as well as gaseous phase used for measurements. Whereas the first consists of a flow-cell, where the sensitive area is directly exposed to the samples, in the second case the sensor chamber is separated from the water stream by the means of a thin Teflon membrane with pores.
Figure 3. Liquid and gas cell for determining atrazine43
Integration of MIP nanofibers can be achieved by directly spin coating or electrospinning to the surface of transducer. In the case of electrospinning, it is needed to attach the transducer with the collecting screen. By controlling variables like the distance between capillary tube and collecting screen, spinning time and solution composition etc.,
MIP nanofibrous membrane with desired thickness, surface area can be coated on the surface of transducer.
Experimental
The synthesis of polyurethane has been extensively studied and is not described here in detail. Instead, the modification of polyurethane with an acrylate end group, the
295 entrapment and then extracting of template molecules in polymer, fabrication of electrospun polymer nanofiber on sensor electrospinning of polymer solution and evaluation of recognition efficiency of MIP nanofiber are discussed below.
Synthesis MIP nanofiber process is described below in the flow chart:
Synthesis and modification. A large family of polyurethanes can be obtained by using different monomers. In this proposal, 4,4-dibenzyl diisocyanate can be reacted with
1,4-butanediol in particular stoichiometry with triethylenediamine as catalyst to form polyurethane with both ends as –NCO groups. Then 2-hydroxyethyl acrylate (HEA) is added to react with isocyanate-capped polyurethane. The completion of the reaction can be confirmed by the disappearance of the peak due to N=C=O stretching absorption near
296 2270 cm-1 by IR spectroscopy44. The final product should be diacrylate-capped polyurethane.
Then the template molecule, atrazine and photoinitiator 1-benzyol-1- hydroxylcyclohexane are mixed with this polymer solution and then ready for electrospinning and crosslinking.
Electrospinning and fabrication on sensor. DMF is one of the best solvents for electrospinning of polyurethane while THF serves as a better solvent for polymerization.
So the mixture of THF and DMF may be suitable for both polymerization and electrospinning process. In You-Lo Hsieh’s research45, they found final ratio of
THF/DMF at 8:1 was good. After dissolving, polymer fiber can be either directly electrospun on the sensor surface by attaching the sensor with collector or electrospun on an aluminum film first and then the film can be put on the sensor surface.
Crosslinking. UV curing to crosslink polyurethane by cleavage of C=C in the acrylate group can be achieved by exposing electrospun polymer fiber under UV light.
Photoinitiator 1-benzyol-1-hydroxylcyclohexane is selected because of its high initiation efficiency and its low absorbance in near UV range, which allows a deep-through cure of the sample to be readily crosslinked. In order to avoid the inhibition effect of atmospheric oxygen on the photo initiated crosslinking, samples can be sealed in a reactor equipped with quartz windows, which is evacuated and saturated with nitrogen prior to irradiation.
Extracting template molecules The MIP nanofibrous membrane coated sensor surface can be washed in methanol/acetic acid for several hours to remove the template according to previous studies.
297 Evaluation of imprinting efficiency In the previous steps, MIP nanomembrane has been achieved. The nest essential step is the evaluation of imprinting efficiency (whether or not the imprinted polymer adequately and accurately remembers the templates).
Experimentally, the guest-binding activity of the imprinted polymer is measured by either chromatographic experiments or batchwise guest-binding experiments. Based on our particular material (membrane), we’d better choose the second method to study the binding kinetics. In this method, the guest-binding activity of the imprinted polymer is directly determined in terms of the amount of guest bound by this polymer. First, a predetermined amount of polymer is added to the guest solutions of varied concentrations.
After incubation for a sufficient long period of time until the guest binding reaches the equilibrium, the polymer is removed by centrifugation or filtration, and the concentration of guest in the liquid phase is determined by HPLC, UV, or other analytical methods.
Then we can use Scatchard equation and plot to calculate equilibrium dissociation constant and maximum of binding sites in unit-weight of polymer. The Scatchard equation and plot are shown below:
Bbound/C = (Bmax-Bbound)/KD = -(1/KD) Bbound + Bmax/KD
Where: Bunbound: unbound site
C: concentration of free atrazine
Bbound: bound sites (subtracting C from initial atrazine concentration)
Bmax: maximum of bind sites
KD: equilibrium dissociation constant
298 In the Scatchard plot, Bbound/C is plotted against Bbound. From the slope and the intercept of the straight line obtained, the values of KD and Bmax are determined.
Determination of the binding constant K for the formation of prepolymer-template complex. The formation of the complex prior to crosslinking is crucial., since the structure of the resulting assemblies defined the subsequently formed binding sites, thereby affecting the recognition properties of the materials for the template molecule.
Here, we can use NMR (Nuclear Magnetic Resonance) to determine the binding constant for the formation of prepolymer-template complex. In our system, the most important non-covalent interaction between polyurethane and atrazine is hydrogen bonding. As we know, when a hydrogen bond is formed, the electron density at hydrogen-bonding protons decreases. Accordingly, the NMR signal of this proton shifts toward lower magnetic field by the following equation:
2 2 1/2 ∆δ obs=1/2* ∆δmax{r+1+1/(KC0)-[r +1+1/(KC0) 2r+2r/(KC0)+2/(KC0)] }
Here, r: functional group/template ratio
C0: initial concentration of functional group
∆δmax: maximal change in chemical shift By plotting ∆δobs against r and analyzing the data by the non-linear least square method, the binding constant K can be determined.
Selectivity analysis. The selectivity test can be carried out using a series of herbicides as reference samples, which are selected for their similar structures. The list of those samples is below:
299 H H Cl N NHEt N N N H2N N NH2 Et C(CH3)2CN NN NN NN Cl NHEt
atrazine simazine 1, 3, 5-triazine
H H H H N N N N N N H H C H CH(CH ) Et C(CH3)2CN N N N 2 5 3 2 (H3C)2HC CH(CH3)2 NN NN NN
SCH3 Cl SCH3
cyanazine prometryn ametryn
Instrumentation and characterizations. To further understanding of the structure- property relationship, characterization of MIP nanofibrous material needs to be carried out.
Fourier transform infrared (FT-IR) spectroscopy is to be used to characterize the presence and chemical change of the functional groups in crosslinked polyurethane network. For example, N=C=O stretching absorption is near 2270 cm-1, by monitoring this band in IR spectrum, we can check whether the grafting of acrylate group on polymer chain is completed or not. Another application of FTIR is to evaluate the extent of crosslinking by monitoring the decrease of the peak centered at 1635 cm –1 which
46 corresponding to the stretch of acrylic CH2=CH bond .
UV-VIS spectrometery can be used to measure uptake of atrazine in solution by
MIP nanofibrous membrane. By comparing the concentration of atrazine before and after
300 soaking the membrane in the solution, we can determine the capacity and uptaking time of the membrane.
Gel permeation chromatography (GPC) is to be used to determine the molecular weight and weight distribution. Since crosslinked polymer can not be easily dissolved, this measurement should be done before UV curing.
Nuclear magnetic resonance (NMR) is an effective tool to study the chemical structure of acrylate-grafted polyurethane.
Differential scanning calorimetry (DSC) and thermal gravity analysis (TGA) is to be used to evaluate the thermal properties of the modified or crosslinked polyurethane.
The thermodynamical and mechanical properties of the MIP can be obtained by dynamic mechanical analysis (DMA).
Scanning electronic microscopy (SEM) and transmission electron microscopy
(TEM) are useful tools to observe the shape and morphology of electrospun MIP nanofibers. The diameter of fiber should be important to sensitivity of the sensor.
301 Ph. D. THESIS RESEARCH:
My thesis research is concentrated mainly on encapsulation of biology macromolecules in rigid silica matrix with controlled pore size via non-surfactant template sol-gel chemistry for potential sensor and catalyst applications. Works have been focused on the study of cytochrome c folding unfolding in silica matrix and aggregation and conformation changing of amyloid β in silica matrix, etc. Other work also includes synthesis and characterization of organic-inorganic hybrid nanocomposite material. All the works differ substantially from this proposed research.
302
1 Haupt, K.; Mosbach, K. Molecularly Imprinted Polymers and Their Use in Biomimetic
Sensors Chem. Rev. 2000, 100, 2495-2504
2 Janata, J. Principle of Chemical Sensor Plenum Press, New York, 1989
3 Ballantine, D.S.; White, R.M.; Martin, S.J.; Ricco, A.J.; Zellers, E.T.; Frye, G.C.;
Wohltijen, H. Acoustic Wave sensors Theory, Design, and Physico-chemical applications
Academic Press, 1997
4 Osada, Y.; De Rossi, D. E. Polymer Sensors and Actuators Springer-Verlag, 2000
5 Haupt, K. Molecularly Imprinted Polymers in Analitical Chemistry Analyst, 2001,126,
747
6 Piletsky, S. A.; Turner, A. P. F. Electrochemical Sensors Based on Molecularly
Imprinted Polymers Electroanalysis 2002, 14(5), 317
7 Blanco-Lopez, M. C.; Lobo-Castanon, M. J.; Miranda-Odieres, A. J. Electrochemical
Sensors Based on Molecularly Imprinted Polymers Trends Anal. Chem., 2004, 23(1), 36
8 Merkoci, A.; Alegret, S. New Materials for Electrochemical Sensing IV. Molecular
Imprinted Polymers Trends Anal. Chem., 2002, 21(11), 717
9 Komiyama, M.; Takeuchi, T.; Mukawa, T.; Asanuma, H. Molecular Imprinting: From
Fundamentals to Applications Wiley-Vch Press
10 Kriz, D.; Mosbach, K. Competitive Amperometric Morphine Sensor Based On an
Agarose-immobilized Molecularly Imprinted Polymer Analytica. Chimica. Acta. 1995,
300, 71
303
11 Turkewitsch, P.; Wandelt, B.; Darling, G. D.; Powell, W. S. Fluorescent Functional
Recognition Sites through Molecular Imprinting. A Polymer-Based Fluorescent
Chemosensor for Aqueous Camp Anal. Chem. 1998, 70, 2025
12 Chow, C-F.; Lam, M. H. W.; Leung, M. K. P. Fluorescent sensing of homocysteine by molecular imprinting Analytica. Chimica. Acta. 2002, 466, 17
13 Subrahmanyam, S.; Piletsky, S. A.; Piletska, E. V.; Chen, B.; Karim, K.; Turner, A. P.
F. 'Bite-and-Switch' Approach Using Computationally Designed Molecularly Imprinted
Polymers for Sensing of Creatinine1 Biosensor Bioelectronics 2001, 16, 631
14 Tan, Y.; Zhou, Z.; Wang, P.; Nie, L.; Yao, S. A Study of a Bio-mimetic Recognition
Material for the BAW Sensor by Molecular Imprinting and Its Application for the
Determination of Paracetamol In the Human Serum and Urine Talanta 2001, 55, 337
15 Andrea, P.; Miroslav, S.; Silvia, S.; Stanislav, M. A Solid Binding Matrix/Molecularly
Imprinted Polymer-based Sensor System for the Determination of Clenbuterol in Bovine
Liver Using Differential-Pulse Voltammetry Sens. Actuators, B 2001, 76, 286
16 Tan, Y.; Yin, J.; Liang, C.; Peng, H.; Nie, L.; Yao, S. A Study of a New TSM Bio- mimetic Sensor Using a Molecularly Imprinted Polymer Coating and Its Application for the Determination of Nicotine in Human Serum and Urine Bioelectrochem. 2001, 53, 141
17 Hosoya, K.; Yoshihako, K.; Shirasu, Y.; Kimata, K.; Araki, T.; Tanaka, N.; Haginata,
J., Molecularly Imprinted Uniform-size Polymer-based Stationary Phase for High- performance Liquid Chromatography. Structural Contribution of Cross-linked Polymer
Network On Specific Molecular Recognition J. Chromatogr. 1996, 728, 139
304
18 Vorderbruggen, M. A.; Wu, K.; Breneman, C. M. Use of Cationic Aerosol
Photopolymerization To Form Silicone Microbeads in the Presence of Molecular
Templates Chem. Mater. 1996, 8, 1106
19 Mayers, A. G.; Mosbach, K. Molecularly Imprinted Polymer Beads: Suspension
Polymerization Using a Liquid Perfluorocarbon as the Dispersing Phase Anal. Chem.
1996, 68, 3769
20 Ye, L.; Mosbach, K. Mat. Res. Soc. Symp. Proc. 2002, 723, 51
21 Shoji, R.; Takeuchi, T.; Kubo, I. Atrazine Sensor Based on Molecularly Imprinted
Polymer-modified Gold Electrode Anal. Chem. 2003, 75, 4882
22 Haupt, K.; Noworyta, K.; Kutner, W. Imprinted Polymer-Based Enantioselective
Acoustic Sensor Using a Quartz Crystal Microbalance Anal. Commun. 1999, 36, 391
23 Dickert, F. L.;Tortschanoff, M.; Bulst, W. E.; Fischerauer, G. Molecularly Imprinted
Sensor Layers for the Detection of Polycyclic Aromatic Hydrocarbons in Water Anal.
Chem. 1999, 71, 4559
24 Sergeyeva, T.; Piletsky, S. A.; Piletska, E. V.; Brovko, O. O.; Karabanova, L. V.;
Sergeeva, L. M.; El’skaya, A. V.; Turner, A. P. F. In Situ Formation of Porous
Molecularly Imprinted Polymer Membranes Macromolecules 2003, 36, 7352
25 Percival, C. J.; Stanley, S.; Galle, M.; Braithwaite, A.; Newton, M. I.; McHale, G.;
Hayes, W. Molecular-Imprinted, Polymer-Coated Quartz Crystal Microbalances for the
Detection of Terpenes Anal. Chem. 2001, 73, 4225
26 Mirsky, V.; Hirsch, T.; Piletsky, S. A.; Wolfbeis, O. S. A Spreader-Bar Approach to
Molecular Architecture: Formation of Stable Artificial Chemoreceptors Angew. Chem.
Int. Ed. 1999, 38, 1108
305
27 Lahav, M.; Katz, E.; Willner, I. Photochemical Imprint of Molecular Recognition Sites in Two-Dimensional Monolayers Assembled on Au Electrodes: Effects of the Monolayer
Structures on the Binding Affinities and Association Kinetics to the Imprinted Interfaces
Langmuir 2001, 17, 7387
28 Formhals, A. US patent 1,975,504, 1934
29 Formhals, A. US patent 2,160,962, 1939
30 Formhals, A. US patent 2,187,306, 1940
31 Formhals, A. US patent 2,323,025, 1943
32 Wang, X.; Drew, C.; Lee, S-H.; Senecal, K. J.; Kumar, J.; Samuelson, L. A.
Electrospun Nanofibrous Membranes for Highly Sensitive Optical Sensors Nano. Lett.
2002, 2(11). 1273
33 Gibon, P.; Schreuder-Gibson, H.; Rivin, D. Transport Properties of Porous Membranes
Based on Electrospun Nanofibers Colloids Surf., A 2001, 187
34 Luo, C.; Liu, M.; Mo, Y.; Qu, J.; Feng, Y. Thickness-Shear Mode Acoustic Sensor for
Atrazine Using Molecularly Imprinted Polymer as Recognition Element Anal. Chim. Acta
2001, 428(1), 143
35 Dickert, F. L.; Forth, P.; Lieberzeit, P. A.; Voigt, G. Quality Control of Automotive
Engine Oils with Mass-Sensitive Chemical Sensors- QCMs and Molecularly Imprinted
Polymers Fresenius J. Anal. Chem. 2000, 366, 802
36 Dickert, F. L.; Hayden, O.; Halikias, K. Synthetic Receptors as Sensor Coatings for
Molecules and Living Cells Analyst, 2001, 126, 766
37 Dickert, F. L.; Thierer, S. Molecularly Imprinted Polymers for Optochemical Sensors
Adv. Mater. 1996, 8(12), 987
306
38 Dickert, F. L.; Lieberzeit, P.; Tortschanoff, M. Molecular Imprints as Artificial
Antibodies – a New Generation of Chemical Sensors Sens. Actuators, B 2000, 65, 186
39 Deckert, C.; Zahouily, K.; Keller, L.; Benfarhi, S.; Bendaikha, T. Ultrafast Synthesis of
Bentonite-Acrylate Nanocomposite Materials by UV-Radiation Curing J. Mater. Sci.
2002, 37, 4831
40 Ando, M.; Uryu, T. Synthesis of Polymer Materials by Low Energy Electron Beam. I.
Polyurethane- Acrylate Materials Prepared by the EB and the UV Solid- State
Polymerization J. Appl. Polym. Sci. 1987, 33, 1793
41 Moussa, K.; Decker, C. Semi-Interpenetrating Polymer Networks Synthesis by
Photocrosslinking of Acrylic Monomers in a Polymer Matrix J. Polym. Sci., Part A:
Polym. Chem. 1993, 31, 2633
42 Kaczmarek, H.; Decker, C. Interpenetrating Polymer Networks. I. Photopolymerization of Multiacrylate Systems J. Appl. Polym. Sci. 1994, 54, 2147
43 Dickert, F. L.; Lieberzeit, P. A.; Hayden, O.; Gazda-Miarecka, S.; Halikias, K.; Mann,
K. J.; Palfinger, C. Chemical Sensors- from Molecules, Complex Mixtures to Cells-
Supermolecular Imprinting Strategies Sensors 2003, 3, 381
44 Ando, M.; Uryu, T. Synthesis of Polymer Materials by Low Energy Electron Beam. I.
Polyurethane- Acrylate Materials Prepared by the EB and the UV Solid- State
Polymerization J. Appl. Polym. Sci. 1987, 33, 1793
45 Xie, J.; Hsieh. Y-L. Ultra-High Surface Fibrous Membranes from Electrospinning of
Natural Proteins: Casein and Lipase Enzyme J. Mater. Sci. 2003, 38, 2125
46 Decker, C.; Moussa, K. Photopolymerization of Multifunctional Monomers in
Condensed Phase J. Appl. Polym. Sci. 1987, 34, 1603
307 Vita
Zhengfei Sun was born in Jining, Shan Dong Province, China on March 4th, 1979.
He received B.S. in Chemistry from Fudan University, Shanghai, China, in 2000 and M.S. in Chemistry from Drexel University, Philadelphia, PA in 2002. He joined Drexel
University, Philadelphia, PA since 2000. From 2000 to 2005, he has been a research assistant affiliated with the Center of Advanced Polymers and Materials Chemistry and
Department of Chemistry, Drexel University, under the supervision of Dr. Yen Wei. His research interests include a wide range of fields in polymer and materials chemistry, especially novel sol-gel nanoporous materials, nanocomposites and their applications in bioscience. He was also a teaching assistant in Department of Chemistry, Drexel
University, during 2001 to 2005. His teaching responsibilities include recitation and laboratory sessions in a number of curricular courses within chemistry fields.
His research work has been published and presented in several national and international conferences, including ACS national and regional conferences.
He will join SRD Corp. in the summer of 2005.
Selective Publications and Presentations:
• Y. Wei; ZF. Sun; JY. Zheng; H. Dong; JM. Yuan; GH. Ping, “Rigid Matrix Artificial Chaperone (RMAC)-Mediate Refolding of Heme Protein” Polym. Mater. Sci. Eng. 2002, 87, 252.
• GH. Ping; JM. Yuan; ZF. Sun; Y. Wei, “Studies of Effects of Macromolecular Crowding and Confinement on Protein Folding and Protein Stability” J. Mol. Recog. 2004, 17(5), 433
• ZF. Sun; SX. Li; A. C. Patel; C. Wang; Y. Wei, “Fabrication of Poly(2-hydroxyethyl methacrylate)-Silica Nanoparticle Hybrid Nanofibers via Electrospinning” 228th ACS National Meeting, Philadelphia, PA, United States, August 22-26, 2004 (2004)
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