CHEMICAL DESIGN OF FUNCTIONALIZED
NANOMATERIALS FOR SENSING AND BACTERIAL
TREATMENT APPLICATIONS
by
MONICA NAVARRETO LUGO
Submitted in partial fulfillment of the requirements
for the degree of Doctor of Philosophy
Dissertation Adviser: Prof. Anna Cristina S. Samia
Department of Chemistry
CASE WESTERN RESERVE UNIVERSITY
May, 2019
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
Prof. Carlos E. Crespo-Hernández (Committee Chair, Department of Chemistry, CWRU)
Prof. Robert G. Salomon (Department of Chemistry, CWRU)
Prof. Blanton S. Tolbert (Department of Chemistry, CWRU)
Prof. Anna Cristina S. Samia (Department of Chemistry, CWRU)
Prof. Xiong Yu (Department of Civil Engineering, CWRU)
Date of Defense: March 21, 2019
*We also certify that written approval has been obtained for any proprietary material
contained thein.
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To my loving parents, Madeline and Miguel, my brother, Miguel Jr. my best friend José, and life partner Markell
Acknowledgements
I want to start by expressing my deepest appreciation for my advisor, Prof. Anna
Cristina S. Samia, for her endless guidance and support for my academic and professional pursuits, for her understanding, and for continuously pushing me forward out of my comfort zone. I will always be grateful for her guidance through this process and will always consider her the biggest mentor of my career. Thank you also to my committee members, Prof. Carlos E. Crespo, Prof. Robert G. Salomon, Prof. Blanton S. Tolbert and
Prof. Xiong Yu, who have stimulated thought and created a community supportive of inquiry. Also, want to thank Dr. Ana R. Guadalupe (my first mentor) and Dr. Yanira
Enriquez for believing on me as an undergraduate student, and giving me the necessary tools to succeed later as a graduate student.
I also want to express my sincere gratitude to all of the collaborators who created the dialectic necessary for scientific progress. Moreover, I am grateful for the lab members in Dr. Samia's group: Sameera Wickramasinghe, Minseon (Stella) Ju, and Nathalie
Milbrandt; who are the best coworkers that anyone could ask for and also became close friends through the process. I will also like to thank Shu Situ for her training and help in my transition as a first year student in the group. Also, I want to thank the undergraduate students that worked with me (Jae Hee Lim, Ariel McWhorther and Ryan Wee) for their work, compromise and support; Also, for helping me to develop myself as a mentor.
Moreover, I am especially grateful to have the help and friendship from Charles Kolodziej;
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our passion and enthusiasm for science brought us together, and has created a long lasting friendship. I am thankful for his support, for the long discussions about research that helped to always find solutions, and for always being there. Also, I would like to thank Naishka
E. Caldero, Jesse Davila, Elisa Caloca, Kelsie Ryon and Trevor Ryon, because beyond friends, they became my extended family in Cleveland. Thank you all for always providing the emotional support that pushed me to achieve my academic and professional goals.
Finally, I especially want to dedicate this work to my parents Madeline and Miguel who are the best parents anyone could ask for, and my little brother Miguel Jr., who will always be an example of courage and determination; everything I have done will always be for all of you. José for being my biggest fan, friend and support through many years; and to Markell. Thank you for being there through the process, thanks for enlightening the stressful times and filling them with laughs.
Many thanks to my loving family and close friends for their unconditional love and support. Without their guidance, patience, understanding, encouragement, and laughter, the completion of this work would have never been possible.
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Table of Contents
Acknowledgements ...... i
Table of Contents ...... iii
List of Figures...... ix
List of Schemes ...... xvii
List of Symbols and Abbreviations ...... xix
Abstract ...... xxiv
Chapter 1. Introduction ...... 1
1.1 General Introduction ...... 1
1.2 Nanomaterial Properties ...... 2
1.3 Plasmonic Nanoparticles ...... 3
1.3.1 Electrocatalytic Nanometals ...... 5
1.4 Magnetic Nanoparticles...... 8
1.4.1 Magnetic Properties ...... 8
1.4.2 Single Domain Theory ...... 11
1.4.3 Stoner-Wohlfarth Theory ...... 12
1.4.4 Superparamagnetism ...... 12
1.4.5 Iron Oxide Spinel Ferrites ...... 15
1.5 Nanocomposites ...... 15
1.6 Nanoparticles Synthetic Methods...... 16
1.7 Nanoparticles Characterization Methods ...... 19
iii
1.7.1 Microscopy Methods ...... 20
1.7.1.1 Transmission Electron Microscopy (TEM) ...... 20
1.7.1.2 Scanning Electron Microscopy (SEM) ...... 21
1.7.2 Spectroscopy Methods ...... 22
1.7.2.1 Energy Dispersive X-ray Spectroscopy ...... 22
1.7.2.2 Atomic Absorption Spectroscopy ...... 22
1.7.2.3 Fourier Transform Infrared Spectroscopy ...... 23
1.7.3 Powder X-ray Diffraction ...... 24
1.7.4. Dynamic Light Scattering ...... 25
1.8 Applications of Functionalized Nanomaterials ...... 26
1.8.1 Electrochemical Sensors ...... 26
1.8.2 Magnetic Sensors ...... 29
1.9 Nanoparticles in Photothermal Therapy ...... 30
1.10 References ...... 31
Chapter 2. Designing the Chemistry of Au/Ag Nanostructures for Cortisol Sensing ...... 38
2.1 Introduction ...... 38
2.2 Methods ...... 40
2.2.1 Synthesis of Gold Nanoparticles (Au NPs)...... 40
2.2.2 Synthesis of Au/Ag Nanoboxes (Au/Ag NBs) and Au/Ag Nanocages (Au/Ag
NCs)...... 40
iv
2.2.3 Preparation of Nanocomposites ...... 41
2.2.4 Electrode Modification ...... 41
2.2.5 Electrochemical Analysis – Cyclic Voltammetry (CV) ...... 42
2.3 Results and Discussion ...... 43
2.3.1 Characterization of Au/Ag Nanostructures...... 43
2.3.2 Effect of Nanostructure Morphology and Carbon Matrix Support on
Electrochemical Performance ...... 45
2.3.3 Effect of β-Cyclodextrin (β-CD) Modification on Nanostructure Electrochemical
Performance...... 49
2.3.4 Nanostructure Dispersion Effect on Electrode Surface...... 52
2.3.5 Sensor Application – Detection of the Stress Biomarker, Cortisol ...... 55
2.4 Conclusion ...... 58
2.5 References ...... 59
Chapter 3. Development of a Magnetic Particle Spectroscopic Method for the Detection of Pb2+ ...... 63
3.1 Introduction ...... 63
3.2 Methods ...... 72
3.2.1 Synthesis of Zn Doped Superparamagnetic Iron Oxide Nanoparticles ...... 72
3.2.2 Synthesis of TMS-EDTA-β-CD Ligand ...... 73
3.2.3 Ligand Exchange of Zn/IONP-OA to Zn/IONP-TMS-EDTA ...... 74
3.2.4 Ligand Exchange of Zn/IONP-OA to Zn/IONP-EDTA-β-CD...... 74
v
3.2.5 Magnetic Particle Spectrometry (MPS) Pb2+ Nanosensor ...... 75
3.3 Results and Discussion ...... 75
3.3.1 Characterization ...... 75
3.3.2 Magnetic Particle Spectrometry Volumetric Sensor ...... 80
3.3.3 pH Dependence Study ...... 80
3.3.4 Time Dependence Study ...... 83
3.3.5. Magnetic Particle Spectrometry Pb2+ Sensor ...... 84
3.3.6 MPS as a Tool for Selective Determination of Pb2+ ...... 88
4.4 Conclusion ...... 90
4.5 References ...... 90
Chapter 4. Exploring the Chelation-based Plant Strategy for Iron Oxide Nanoparticle Uptake in Garden Cress (Lepidium sativum) using Magnetic Particle Spectrometry ...... 94
4.1 Introduction ...... 94
4.2 Materials and Methods ...... 98
4.2.1 Materials ...... 98
4.2.2. Synthesis of Iron Oxide Nanoparticles (IONPs) ...... 98
4.2.3 Surface Functionalization of IONP with TMS-EDTA ...... 99
4.2.4 Materials Characterization ...... 99
4.2.5 Seed Germination and Plant Growth ...... 100
4.2.6 Length and Biomass Determination ...... 100
4.2.7 Chlorophyll Measurements...... 101 vi
4.2.8 Magnetic Particle Spectrometry Analysis ...... 101
4.2.9 Elemental Analysis Using Atomic Absorption Spectroscopy (AAS) ...... 102
4.2.10 TEM Sample Fixation ...... 102
4.2.11 Statistical Analysis ...... 103
4.3 Results and Discussion ...... 104
4.3.1 Characterization of Iron Oxide Nanoparticles (IONP) ...... 104
4.3.2 Experimental Growing Cycle for Garden Cress ...... 106
4.3.3 Effect of IONP in Length and Biomass of Garden Cress ...... 108
4.3.4 Effect of IONP in Chlorophyll Concentration ...... 110
4.3.5 Magnetic Particle Spectrometry ...... 115
4.3.6 Magnetic Particle Spectrometry Monitoring of IONP Uptake ...... 118
4.3.7 Magnetic Particle Spectrometry Validation with AAS ...... 120
4.3.8 Magnetic Particle Spectrometry Monitoring of IONP Translocation ...... 121
4.4 Conclusion ...... 124
4.5 References ...... 124
Chapter 5. Chemical Design of Au Nanorods for the Photothermal Treatment of S. aureus Biofilm ...... 129
5.1 Introduction ...... 129
5.2 Methods ...... 131
5.2.1 Synthesis of Au Seeds ...... 131
5.2.2 Synthesis of Au NRs...... 132 vii
5.2.3 Surface Functionalization: Ligand Exchange ...... 132
5.2.4 Atomic Absorption Spectroscopy (AAS): Determination of Au NRs
Concentration...... 133
5.2.5 Photothermal Conversion of Au NRs in Saline ...... 133
5.2.6 Biofilm Formation and PTT Generated Heat Elimination ...... 133
5.3 Results and Discussion ...... 134
5.3.1 Aspect Ratio Effect in Au NRs PTT Heat Production ...... 134
5.3.2 Surface Functionalization Effect in Au NRs PTT Heat Production ...... 138
5.3.3 Excitation Method Effect on Au NRs PTT Heat Production ...... 141
5.3.4 Treatment of Bacteria with Au NRs-PEG-SH and IR Laser ...... 144
5.4 Conclusion ...... 147
5.5 References ...... 147
Chapter 6. Research Outlook ...... 149
6.1. Cortisol Sensing ...... 149
6.2 Pb2+ Sensing ...... 149
6.3 Environmental Monitoring of Magnetic Nanoparticles ...... 150
6.4 Au Nanorods for Photothermal Therapy ...... 151
6.5 References ...... 151
Complete Reference List ...... 152
viii
List of Figures
Figure 1.3.1. Schematic of plasmon oscillation for a sphere. It shows the displacement of
the conduction electron charge cloud relative to the nuclei. ……………….3
Figure 1.3.2. Au/Ag hollow bimetallic structures data, representing the effects of changes
on shape and composition in the optical properties of nanoparticles. (a)
TEM images. (b) UV-Vis characterization. ……………………………….4
Figure 1.3.1.1. MO diagram that represents the interaction of the metal with the adsorbate
…………………………………………………………………………….6
Figure 1.4.1.1. Magnetic moment orientations for various types of magnetic ordering…10
Figure 1.4.1.2. (a) Multi-domain structure of bulk magnetic materials. (b) Single domain
particle showing the absence of domain walls. ……………………..……11
Figure 1.4.4.1. Graphic illustration of change in coercivity as a function of particle's
diameter showing the coercivity values for superparamagnetic (SPM),
single domain (SD) and multi-domain (MD) particles. ……..……………14
Figure 1.6.1. Galvanic replacement reaction. (a) Reaction set-up. (b) Schematic
illustration for the formation of Au/Ag nanocages. …………..…………17
Figure 1.6.2. Graphic illustration of a typical thermal decomposition synthesis setup. ...18
Figure 1.6.3. LaMer diagram showing the nucleation and growth of nanoparticles during
thermal decomposition synthesis. ………………………..………………19
Figure 1.8.1.1. Examples of the most commonly used electrochemical sensors…………28
Figure 1.9.1. Schematic representation for the treatment of bacterial biofilm using Au NRs
in photothermal therapy. …………………………………………………31
ix
Figure 2.3.1.1. TEM images of the (b) Au NP, (c) Au/Ag NB, and (d) Au/Ag NC samples,
respectively; the scale bars indicate 100 nm. …………………………….44
Figure 2.3.1.2. UV-vis absorption spectra of the (a) synthesized Au/Ag nanostructures...45
Figure 2.3.2.1. (a) Cyclic voltammograms (CVs) of 5 mM K3[Fe(CN)6]/ K4[Fe(CN)6] in
PBS (1x, pH 7.4, 0.1 M KCl) obtained using the SPCEs modified with the
Au/Ag nanostructures (i.e. Au NP, Au/Ag NB, and Au/Ag NC,
respectively). (b) The corresponding CVs of the nanocomposites obtained
by mixing the Au/Ag nanostructures with graphene nanoplatelets (Gs) as
carbon matrix support. …………………………………………………..48
Figure 2.3.2.2. Bar graph comparing the anodic peak current response of the nanoparticles
versus the nanocomposites. All CVs were obtained using a scan rate of
200 mVs-1 and normalized by the total metal concentration used in each
measurement. All values are statistically significant with p values less
than 0.05. ………………………………………………………………..49
Figure 2.3.3.1. CVs of 5 mM K3[Fe(CN)6]/ K4[Fe(CN)6] solution obtained using the
nanocomposite-modified SPCEs prepared with and without β-CD
modification: (a) Au NP, (b) Au/Ag NB, and (c) Au/Ag NC,
respectively……………………………………………………………...51
Figure 2.3.3.2. Comparison of the electrochemical responses of the different nanostructure
systems. All values are statistically significant with p values less than 0.05.
……………………………………………………………………….….52
Figure 2.3.4.1. (a) Photograph of a representative SPCE with the areas used in the EDAX
elemental analysis highlighted, and (b) the corresponding scanning electron
x
microscope (SEM) image of two of the spots on the working electron that
were used to assess nanostructure distribution. (c) Summary of the EDAX
analyses. (d) Higher resolution SEM image of a representative SPCE
modified with Au/Ag NC with G matrix support. ……………………….54
Figure 2.3.5.2. CVs of 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] obtained using an SPCE modified
with Au/Ag NC β-CD+G nanocomposite that was used for the indirect
determination of cortisol; the inset shows an amplification of the anodic
peak currents obtained from the CV. (b) Calibration plot with different
cortisol concentrations with n=5. ……………………………………..…57
Figure 3.3.1.1. Transmission electron microscope images of magnetite spherical Zn/IONP.
(a) Zn/IONP-OA and (b) Zn/IONP-EDTA-β-CD. ………………………..77
Figure 3.3.1.2. Dynamic light scattering characterization of Zn/IONP-OA of Zn/IONP-TMS-
EDTA and Zn/IONP-EDTA-β-CD. .……………………………………...77
Figure 3.3.1.3. FT-IR spectra of Zn/IONP-OA, Zn/IONP-TMS-EDTA and Zn/IONP-EDTA-
β-CD. ……………………………………………………………………78
Figure 3.3.1.4. The powder X-ray diffraction patters of the synthesized Zn/IONP-TMS-
EDTA and Zn/IONP-EDTA-β-CD.. ………….……….….……………...78
Figure 3.3.1.5. Field-dependence magnetization curve of Zn/IONP-TMS-EDTA and
Zn/IONP-EDTA-β-CD obtained at T= 300 K.. ….……….……...………..79
Figure 3.3.1.6. Z-potential measurements of Zn/IONP-EDTA-β-CD at various pH. Error bars
correspond to n=3.. …………………………………………………...... 79
Figure 3.3.3.2. pH effect study. (a) Stability of Zn/IONP-EDTA-β-CD. (b) Zn/IONP-
xi
EDTA-β-CD after a 15 min incubation with 15 ppb of Pb2+. (c) ΔMPS
response showing a decrease in the MPS signal due to the formation of
clusters at different pH after incubation with 15 ppb of Pb2+ for 15 min. (d)
DLS measurements. ……………………………..……………………….82
Figure 3.3.4.1. Magnetic particle spectrometry time dependence response for the
Zn/IONP-EDTA-β-CD incubation with 15 ppb of Pb2.………..…………83
Figure 3.3.5.1. MPS response of the incubation of Zn/IONP-EDTA-β-CD with various
concentration of Pb2+ for 5 minutes.. …………………….……………….85
Figure 3.3.5.2. MPS Pb2+sensor using Zn/IONP-EDTA-β-CD. (a) MPS response at various
concentrations of Pb2+. (b) MPS signal intensity showing the capability of this
assay to discern between various concentrations of Pb2+ in the level of concern
for children. Results were obtained with n= 3. …………….………………85
Figure 3.3.5.3. MPS response of the incubation of Zn/IONP-TMS-EDTA with different
concentration of Pb2+ for 5 minutes. Error bars correspond to n= 3……...…..86
Figure 3.3.5.4. MPS Pb2+sensor using Zn/IONP-TMS-EDTA. (a) MPS response at various
concentrations of Pb2+. (b) MPS signal intensity showing the capability of this
assay to discern between various concentrations of Pb2+ in the level of concern
for children. Results were obtained with n= 3.. …………………………….86
Figure 3.3.5.5. Calibration curve for Zn/IONP-EDTA-β-CD. Error bars correspond to
n= 3………………………………………………………………………87
Figure 3.3.5.6. Calibration curve for Zn/IONP-EDTA. Error bars correspond to
n= 3.………………………………………………………………...……87
Figure 3.3.6.1. Zn/IONP-EDTA-β-CD MPS selectivity study (a) response after incubation
xii
with 50 ppb of different cations. (b) MPS signal intensity in response to
different cations.. ……………………………………..………………….89
Figure 3.3.6.2. Zn/IONP-TMS-EDTA MPS selectivity study (a) response after incubation
with 50 ppb of different cations. (b) MPS signal intensity in response to
different cations. .……………….. ……………………………………….89
Figure 4.3.1.1. Characterization of various IONP-EDTAs use for garden Cress treatments. (a)
Scheme of the functionalization of IONPs from oleic acid to TMS-EDTA
capped IONP. (b) High resolution transmission electron microscope (TEM)
images of IONP-EDTA-capped of 10 nm and 20 nm, respectively. Scale bars
are 100 nm; (c) Fourier transform infrared (FTIR) spectra; (d) Vibrating
Sample Magnetometer spectra; (e) powder X-ray diffraction patterns; (f)
Dynamic light scattering results for IONP-EDTA before and after ligand
exchange.. ………...……………………………………………...…….105
Figure 4.3.2.1. Phenotypic observations of the effect of incubation with IONP10-EDTA or
IONP20-EDTA on garden cress harvested after 10 days’ experimental cycle.
(a) Length measuring method. (b) Length measurements. (c) Representation
of the harvested plants exposed to the different treatments. (d) Biomass
measurements. Error bars represent standard errors (n= 50). Each n group was
composed of 10 plants. Asterisk show statistical significance with a p<0.05...
………………………..………………………………………………..107
Figure 4.3.3.1. Phenotypic observations of the effect of incubation with IONP10-EDTA or
IONP20-EDTA on garden cress harvested after 10 days’ experimental cycle.
(a) Length measuring method. (b) Length measurements. (c) Representation
of the harvested plants exposed to the different treatments. (d) Biomass
xiii
measurements. Error bars represent standard errors (n= 50). Each n group was
composed of 10 plants. Asterisk show statistical significance with a p<0.05..
…………………………………………………………………..……..109
Figure 4.3.4.1. Chlorophyll production in garden cress as a result of IONP-EDTA treatments.
(a) Image of representative leaves for each group. Greener leaves were
observed in Garden cress plants exposed to IONP20-EDTA. (b) Spectra of UV-
VIS measurement for the separated Chla and Chlb. (c) The effect of IONP-
EDTA on concentration of chlorophyll a, b and total chlorophyll amount..
…………………………………………………………………………112
Figure 4.3.4.2. TEM images of different parts of a cross sectional areas on a leaf of garden
cress exposed to IONP20-EDTA. Red arrows point to translocated IONP20-
EDTA on plant cells. Red inset show the identification of Fe by elemental
analysis.. …………………………………….…………………………113
Figure 4.3.4.3. Schematic representation for the uptake of Fe and presumed uptake and
translocation of IONP-EDTA. ………………………………………….114
Figure 4.3.5.1. Schematic representation of MPS analysis. (a) MPS instrument picture. (b)
Experimental sample preparation prior to MPS analysis. (c) Application to a
sinusoidal magnetic field to the sample and field free region (FFR) necessary
for signal generation. (d) Representation of signal generation. (e) Point spread
function (PSF) examples showing the MPS response of the different
treatments. (f) Calibration curves obtained from IONP10-EDTA and IONP20-
EDTA. Error bars correspond to with n= 5 and p<0.05..
……………………………………………………...………………….117
Figure 4.3.6.1. Monitoring of the uptake of IONP from hydroponic media. (a) Decrease in
xiv
MPS signal of the IONP treatments in hydroponic media harvest. (c) Δ MPS
showing that most IONP10-EDTA was up taken on the first day of hydroponic
media harvest vs a continuous absorption of IONP20-EDTA through the plant
cycle. Error bars correspond to n= 5 and p<0.05..
…………………………………………………………………..……..119
Figure 4.3.7.1. Comparison and validation of MPS results using atomic absorption
spectroscopy (AAS) (a) IONP10-EDTA treatment. (b) IONP20-EDTA. Error
bars correspond to with n= 5 and p<0.05. …….………………………….120
Figure 4.3.7.2. TEM images of the IONP-EDTA in the hydroponic media used for the
incubation of garden cress (a) IONP10-EDTA and (b) IONP20-EDTA.
…………………………………………………………………………..121
Figure 4.3.8.1. Comparison for the relative response of Fe concentration in the leaves,
stem and roots of Garden cress exposed to the different experimental
conditions. …………………………………………..………………….123
Figure 4.3.8.2. Picture of Garden Cress after incubation with IONP20-EDTA showing the
root hairs surrounded by the IONP. …………………………………….123
Figure 5.3.1.1. Au nanorods with various LSPR. The yellow shaded area of the spectra
represents the biological “water window”, region where aqueous tissue absorbs
relatively little light (700 nm to 1200
nm)……………………………………………...... ……………………135
Figure 5.3.1.2. Au nanorods that absorb in the excitation wavelength of the NIR AC powered
laser (808 nm, represented as a yellow line). (a) UV-VIS spectra of various Au
NRs (CTAB capped Au NRs samples). TEM images, scale bar: 200 nm (b)
AR: 3.5 Au NRs with longitudinal surface plasmon resonance at 750 nm. (c)
xv
AR: 3.7 Au NRs with longitudinal surface plasmon resonance at 810 nm and
(d) AR: 4.4 Au NRs with longitudinal surface plasmon resonance at 925 nm..
………………………………………………………..…………………136
Figure 5.3.1.3. Photothermal heat conversion capacity results for 200 ppm of different Au
NRs-CTAB upon 15 min of NIR irradiation (808 nm). (a) ∆T values from
the different AR Au NRs samples. Graph also shows the ∆T values for the
lost in heat generation immediately after the excitation source is turn off.
(b) Maximum temperature achieved by each Au NRs sample. Experimental
data was collected with n=3. …………………………………………..137
Figure 5.3.2.1. Surface functionalization of Au NRs following a ligand exchange process.
………………………………………………………………………….139
Figure 5.3.2.2. Z-potential results for the Au NRs before and after ligand exchange from
CTAB to m-PEG-SH. …………………………………………………..139
Figure 5.3.2.3. Infrared thermal images showing the photothermal conversion capacity for
200 ppm of Au NRs with different surface chemistries. Data n value equals 3..
……………………………………………………………………….….140
Figure 5.3.3.1. Ti implant used for the Au NRs PPTT conversion studies. (a) Ti implant
dimensions. (b) SEM images showing significant roughness of the Ti
implant surface. ……………………..………………………………….142
Figure 5.3.3.2. PPTT heat conversion capacity of 500 ppm of Au NRs in 1 mL of saline
solution. The gap between the laser and the sample was kept at 4 cm. The
excitation source (808 nm) was kept stationary at quadrant #1, while the
xvi
temperature was recorded on successive quadrants during 11.25 min each.
The heat produced was obtained using a temperature probe. ………….143
Figure 5.3.3.3. Infrared thermal images showing the photothermal conversion for 500 ppm
of Au NRs in saline solution on top of the Ti implant. …………………144
Figure 5.3.4.1. Figure 5.3.4.1. Au NRs-PEG-SH (500 ppm) 45 min PTT in S. Aureus
biofilm on Ti implants. (a) Positive control, (b) Treated S. Aureus, (c)
Negative control………………….……………………………………..145
Figure 5.3.4.2. Confocal micrographs showing live (green) and dead (red) S. aureus. (a)
Positive control, (b) after PTT with 500 ppm of Au NRs-PEG-SH, (c) Data
showing fraction of LIVE/DEAD bacteria. …………………………....146
List of Schemes
Scheme 2.2.2.1. Galvanic replacement reaction scheme for the synthesis of Au/Ag
nanostructures……………………………………………………………41
Scheme 2.2.5.1. Schematic diagram of the electrode surface nanoarchitecture. The Au/Ag
NC sample in conjunction with the G carbon matrix support was used to
modify the working electrode on the SPCE, and was utilized here to
represent the different nanostructures used in the study (Au NP, Au/Ag NB,
and Au/Ag NC). The electrochemical analyses were performed using the
3-/4- Fe(CN)6 redox probe. ……………………………………...... ….……42
Scheme 2.3.5.1. Schematic representation of the nanoarchitecture on the electrochemical
sensing of cortisol. The system presents an indirect determination of cortisol
3-/4- with the use of Fe(CN)6 as a redox probe. ……………………………56
xvii
Scheme 3.1.1. (a) Schematic diagram of the magnetic particle relaxometer. (b) Illustration
of the signal generation process in magnetic particle relaxometer. ………67
Scheme 3.1.2. Schematic representation of MPS instrument and signal generation (a) MPS
instrument picture. (b) Representation of the external magnetic field
application on the sample. (c) Diagram of instrument components.
……………………………………………………………………………68
Scheme 3.1.3. Schematic representation of the relaxation mechanisms that contribute to
the MPS signal (a) Néel and Brownian relaxation. Representation of the
energy barrier that governs the rotation of the magnetic moment of IONPs
(b) IONP relaxation without the interruption of an external magnetic field,
(c) IONP relaxation with external magnetic field, (d) Effect on the
relaxation mechanisms of clusters with and external magnetic dipolar field
phenomenon. …………………………………………………………….70
Scheme 3.1.4. Schematic representation of the Pb2+ MPS sensor. A decrease in MPS
response is observed upon the aggregation of Zn/IONP-EDTA-β-CD in the
presence of Pb2+ ions. The vials show the aggregation of the sample caused
by the addition of 10 ppb of Pb2+. ……………………………………….72
Scheme 3.2.2.1. Synthesis of the EDTA-β-CD ligand. …………………………………74
Scheme 3.2.4.1. Schematic representation of the ligand exchange. …………………….75
Scheme 3.3.3.1. Representation of the complex formation between Pb2+ and β-CD forming
a preferred square planar arrangement. …………………………………..81
xviii
List of Symbols and Abbreviations
% by wt. percent by weight ΔT delta temperature (d-σ) bonding state (d-σ)* antibonding state °C degrees Celsius 3D three dimensional A exchange energy Å angstrom AAS atomic absorption spectroscopy AC alternate current acac acetylacetonate AMF alternating magnetic field aq aqueous Ar argon AR aspect ratio β-CD β-cyclodextrin BET Brunauer–Emmett–Teller
Chla chlorophyll a
Chlb chlorophyll b cm centimeter CTAB cetyltrimethylammonium bromide CV cyclic voltammogram d diameter D diffusion coefficient
Dc critical diameter DI deionized DLS dynamic light scattering DMF dimethylformamide DMSO dimethyl sulfoxide E energy e- electron e.g. example
Ea adsorption energy
EB energy barrier
Eb binding energy ECSA electrochemical active surface area EDAX powder X-ray diffraction EDDHA ethylenediamine-N-N-bis(o-hydroxyphenylacetic)acid
xix
EDTA ethylenediaminetetraacetic acid EG ethylene glycol
Eg band gap emu electromagnetic unit EPA Environmental Protection Agency eq. equation et al. and others EtOH ethanol eV electron volt F Faraday’s constant f frequency FC field cooled Fe iron Fe(acac)3 iron (III) acetylacetonate
Fe(C5H7O2)3 iron (III) acetylacetonate
Fe3O4 magnetite
FeCl2•4H2O iron (II) chloride hexahydrate
FeCl3•6H2O iron (III) chloride hexahydrate FFR field-free region FeO wüstite fT femtotesla FT-IR fourier transform infrared spectroscopy g gram(s) G graphene GC garden cress h hour(s) H magnetic field amplitude HOMO highest occupied molecular orbital i.e. that is IONP(s) iron oxide nanoparticles IR infrared K anisotropy energy K Kelvin kA kiloamp kB Boltzman constant KRA knee replacement arthroplasty kV kilovolts LBK luria broth LOD limit of detection LOQ limit of quantification
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LSPR localized surface plasmon resonance LUMO lowest unoccupied molecular orbital m slope M molar M magnetization m meter MA mugienic acid meV milielectron volt mg miligram min minute(s) mL mililiter mM milimolar mmol milimole MPI magnetic particle imaging MPS magnetic particle spectrometry MRI magnetic resonance imaging
Ms saturation magnetization mV milivolts MW molecular weight MWCNT(s) multi walled carbon nanotube(s) n number of moles of electrons N number of moles NaOL sodium oleate NB(s) nanobox(es) NC(s) nanocage(s)
NH3 ammonia NIR near-infrared nm nanometer nM nanomolar NP(s) nanoparticle(s) NPl(s) nanoplates PBS phosphonated buffer solution ppm parts per million PTT photothermal therapy PSF point spread function PPTT plasmonic photothermal therapy PVP polyvinylpyrrolidone p-xylene para-xylene
QH charge associated with the adsorbed hydrogen monolayer reference charge associated with the adsorbed hydrogen Q ref monolayer
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r radius rpm rotation per minute ROS radical oxygen species s second S. aureus Staphylococcus aureus SEM scanning electron microscopy SHE standard hydrogen electrode SPCE screen printed carbon electrodes T temperature
TB blocking temperature TEM transmission electron microscopy TEOS tetraethylorthosilicate Ti titanium
Tmax maximum temperature TMS-EDTA Trimethoxysilylpropyl ethylenediamine tetracetic acid UV ultraviolet UV-vis ultraviolet-visible spectroscopy V volume V volt
VH hydrodynamic particle volume vs versus YSL yellow stripe-like W watt ZFC zero field cooled β line broadening at half the maximum intensity ΔR change in resistance
ΔRM maximum resistance change η viscosity θ diffaction angle λ wavelength μA microamp μg microgram μL microliter μm micrometer
μo magnetic moment
ρa impurity resistivity
ρo host resistivity
τB Brownian relaxation time
τeff effective relaxation time
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Τmeas measurement time
τN Néel relaxation time
τo attempt time
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Chemical Design of Functionalized Nanomaterials for Sensing and Bacterial Treatment Applications MONICA NAVARRETO LUGO
Abstract
Nanomaterials continue to gain significant attention because of their great potential towards the development of new diagnostic tools and improved biomedical treatments. The interest in nanomaterials is fueled by their unique chemical, physical, and biochemical properties, which can be readily tuned by the chemical design of their size, composition, morphology, and surface chemistry. This thesis work centers on the design of nano-alloys and metal oxides and the optimization of their materials properties and their surface chemistries for the development of electrochemical and magnetic sensors. Three different detection and monitoring methods are explored in this work, proposing novel platform technologies for electrochemical sensors, as well as magnetic sensors for biomedical and environmental applications.
The second chapter presents research work on the systematic engineering of a gold and silver nanocage structure that was modified with β-cyclodextrin for the enhanced indirect electrochemical monitoring of the stress biomarker molecule cortisol. In chapter
3, the surface chemistry of magnetic iron oxide nanoparticles were chemically modified with different organic ligands to evaluate the capabilities of magnetic particle spectrometry
(MPS), as a new technique for the fast and sensitive determination of ions of biomedical relevance. In the succeeding chapter 4, ethylenediaminetetraacetic acid (EDTA) capped iron oxide nanoparticles were evaluated as potential iron fertilizer, and a method was
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proposed to monitor the uptake and translocation mechanisms of magnetic nanomaterials in plants using magnetic particle spectrometry (MPS).
Lastly, in chapter 5, a systematic study of the synthesis of gold nanorods and the optimization of a their surface plasmon absorption properties is presented to enhance their use in the photothermal treatment of pre-formed bacterial biofilms on titanium implants.
This work can contribute to the development of a non-antibiotic treatment for prosthetic joint infections.
All the nanomaterials explored in these studies have been designed to be biocompatible and targeted for their specific applications. This work demonstrates, that tailoring the nanostructure of metals and metal oxides has the potential of significantly enhancing their properties for different kinds of sensing and treatment applications.
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Chapter 1. Introduction
1.1 General Introduction
Nanomaterials (1-100 nm) have become the base of emerging technologies in the medical and environmental industries.1 They are especially attractive due to their unique size-dependent chemical, physical, optical, electronic and magnetic properties.2 Advances in nanomaterials research gave rise to a large variety of functional materials composed of metals, metal oxides, ceramics, insulators, organics, biological materials, ionic compounds, among others.3 Moreover, multiple combinations of these materials have also been reported, and in most cases results have shown that they have synergetic effects in their performance for a wide range of applications.4 The diversity of materials, fabrication methods and possibilities of surface functionalization make nanomaterials one the favorite candidates for the development of sensing devices, diagnosis tools and biomedical treatments.5,6
The progress on the development and understanding of nanomaterials have led to significant advances on sensing platforms based on: optical, colorimetric, fluorescence, electrochemical and magnetic assays.7–10 These specific applications can be targeted by the appropriate selection and combination of materials; and can also be improved by modifications in the surface chemistry or the architecture of the final sensor device. This concept also applies to their use for the development of biomedical treatments. Thus, the design and study of nanostructures is an important area of research with enough space to develop, that can result in the creation of new technologies with promising potential in the development of sensing, diagnosis and treatment tools.
1
This thesis focuses on presenting the chemical design of various nanomaterials and the modification of their surface chemistry in order to optimize their properties for their application in sensing and biomedical treatment technologies. This work shows the synthesis, surface modification and characterization of different nanomaterials and nanocomposites. Moreover, here is presented the use of these nanomaterials in combination with the development of systematic methodologies for their use in electrochemical and magnetic sensing assays. The various proposed nanomaterials were tested on the sensing of different environmental and biologically relevant analytes.
This introductory chapter will provide an overview on the fundamentals of nanomaterials, nanosynthesis and fabrication approaches. Moreover, it presents the state of the art for nano-based sensing technologies and how this work can contribute to them.
1.2 Nanomaterial Properties
Nanomaterials are characterized by their small size that typically ranges from 1 to
100 nm, their high surface area to volume ratios and strong adsorption capacity.11 They have been the protagonists of most of the last decade's research due to their fascinating and relatively easily controllable chemical and physical properties. Through changes in size, composition, shape and crystallinity, specific attributes can be given to these nanostructures.12 Systematic efforts have been made to find and understand the parameters that can have a direct effect on these characteristics. So far, various studies have reported successful tuning of the properties of nanomaterials by changing synthesis precursors, methods and conditions.13,14 These tunable properties, confess them with the capability to increase sensitivity, maximize surface functionalization, minimize technology platform
2
sizes, and facilitates their coupling with a broad range of transducer structures for their study.
1.3 Plasmonic Nanoparticles
One of the most widely studied aspect of metallic nanomaterials are their optical properties. This property has been reported to be dependent on size, shape, and composition; which imparts different colors due to absorption in the visible region.15 For example, 20 nm nanoparticles made from gold (Au), platinum (Pt), silver (Ag) and palladium (Pd) present a characteristic wine red color, yellowish gray, black and dark black colors respectively. These colors also change with particle size on each of the different composition cases.16 One of the main causes for this phenomena is their plasmonic properties. Plasmonic properties emerge from the collective oscillation of the conduction electrons stimulated by incident light and is a common singularity of small size metallic structures (Figure 1.3.1). This is typically observed in silver and gold nanoparticles, even though is not exclusive to them.
Figure 1.3.1. Schematic of plasmon oscillation for a sphere. It shows the displacement of the conduction electron charge cloud relative to the nuclei.
3
The collective oscillation of the conduction electrons creates a strong plasmon band, which is known as localized surface plasmon resonance (LSPR). The oscillating frequency is determined by four main factors: the effective electron mass, the density of electrons, the shape and the size of the charge distribution. This can be observed with the synthesis of different hollow nanostructures, wherein the shape and metallic ratio composition is tuned.
The UV-Vis characterization of these materials show the displacement of the LSPR from the UV to near IR. Figure 1.3.2 shows the transmission electron microscopy images (TEM) of synthesized nanoparticles with different morphology and composition, and the optical properties of the respective samples.
Figure 1.3.2. Au/Ag hollow bimetallic structures data, representing the effects of changes in shape and composition on the optical properties of nanoparticles. (a) TEM images. (b)
UV-Vis characterization.
4
The localized surface plasmon resonance band is dependent on the size, shape dielectric properties, inter-particle spacing and the local environment.17 Mie theory describe the scattering and absorption properties of small spherical particles.18 According to this theory the optical extinction of a sphere of radius r and dimensions smaller than the wavelength of the incident light can be expressed as:
3 2 2 3 2 (1+휒) 8휋 푁푟 휀표푢푡 휀푖(휆) 퐸(휆) = [ 2 2] eq. 1.3.3 3휆푙푛10 (휀푟(휆)+휒휀표푢푡) +휀푖(휆) where, 휀푟 and 휀푖 are the real and imaginary parts of the metal dielectric function, respectively, 휀표푢푡 is the external dielectric constant (surrounding medium), and 휒 is a shape factor whose value is 2 for spheres.
Furthermore, the absorption intensity and wavelength maximum of these nanoparticles are sensitive to the dielectric constant of the environment, which makes them potentially useful as sensors.19
1.3.1 Electrocatalytic Nanometals
Among the broad variety of properties that nanomaterials exhibit, their electrocatalytical properties are one of the most promising characteristics in areas such as renewable energy production devices and sensing applications. By taking into consideration that the specific chemical design of nanomaterials has a direct effect on its properties, small nanoparticle composites can be created in order to develop materials with enhanced electrocatalytic capabilities. The design involves taking into consideration factors like decreasing the size of the nanomaterials and altering their morphology in order to increase the surface fraction of atoms per unit of mass. Altering this property, can have a direct effect on the contact area between the catalyst and the reactants, maximizing the
5
capability of the catalyst.20 Furthermore, some theories have established that small particle
sizes make the atoms on the surface of the nanoparticles under-coordinated and thus more
active for catalytic reactions in terms of the d-band center.21 The principle that supports
this theory is described by the chemisorption theory that says that the binding energy of an
adsorbate to a metal surface, is directly dependent on the electronic structure of the
catalyst’s surface.22 Moreover, it has been established that the dominant contributions to
molecular adsorption, are through electron-donation and back-donation interactions of the
highest occupied molecular orbital–lowest unoccupied molecular orbital (HOMO-LUMO)
levels of the adsorbate with the valence sp and d-bands of the metal (Figure 1.3.1.1).
Metal d-band (d-σ)*
Adsorbate σ orbital
(d-σ)
Figure 1.3.1.1. MO diagram that represents the interaction of the metal with the adsorbate.
6
Electrocatalyst metal's interactions with an analyte are dependent on the filling of their molecular orbitals. On the electrocatalyst, the extent of filling of the (d-σ)* state depends on the electronic structure of the metal at the surface. An increased filling of the antibonding (d-σ)* state corresponds to a destabilization of the metal-adsorbate interaction, and thus weaker binding. The extent of filling of the (d-σ)* MO is well correlated to the location of the d-band center,23 which is size dependent, and it has been shown to shift towards the Fermi level with a decrease in cluster size.24
One of the most effective electrocatalysts is platinum (Pt). However, due to its high cost, its use for large scale applications is limited. This motivates the study of alternative metallic electrocatalysts. Among others, gold (Au) and silver (Ag) have been broadly studied as substitutes for Pt in sensor and biosensor applications. Their catalytic activity is not as high as Pt, but the ability to tune the properties of nanomaterials by altering their shape, size and composition promotes the development of new structures with potential synergistic effects. For this type of materials, the specific surface area plays a crucial role, as it determines the overall adsorption and electron transport properties, which directly impact the electrocatalytic performance. For instance, different shapes of Au nanostructures can provide different surface controlled structures, such as, cubic nanocubes enclosed by (100) facets; truncated octahedral Au nanocubes enclosed by
(100) and (110) facets; nano-octahedra (111) facets, and multitwined quasi-spherical Au
NPs (111) and (100) facets.25 The different facets have distinguishable chemical activities, which is a characteristic that can be use in order to develop materials for specific applications.26 Moreover, the chemical composition of nanomaterials can combine two or
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more elements, facilitating the prevention of surface poisoning due to the high active metal surface area.
1.4 Magnetic Nanoparticles
Magnetic nanoparticles (MNPs) have been vastly investigated for their great potential in environmental, biomedical, and clinical applications owing to their many unique properties.27 They are particularly interesting due to their unique physicochemical properties such as size, shape, surface area and magnetic properties that make them suitable for in vivo and in vitro applications. Some of these applications are drug delivery, hyperthermia treatments and magnetic relaxation sensors, among others.28–30 Many of the applications for magnetic nanoparticles rely on the use of external magnetic fields in order to manipulate their properties. This makes the final application of these materials dependent on the particle magnetic moment and the field gradient.31 This makes relevant the understanding of the synthetic parameters that will give rise to specific magnetic properties, and how to tailor the surface chemistry of these materials in order to make them suitable for specific applications.
1.4.1 Magnetic Properties
The properties exhibited by different magnetic materials are dependent on the arrangement of their magnetic moments. These differences divide magnetic materials into five main groups: paramagnetic, diamagnetic, antiferromagnetic, ferromagnetic, and ferrimagnetic. Paramagnetic materials have a small susceptibility to magnetic fields. They have randomly aligned magnetic moments, which makes their overall structure have a zero net magnetization. These materials are characterized by the presence of unpaired electrons and in the presence of an external magnetic field, the moments align to produce a small net
8
crystal magnetization. Some examples of paramagnetic materials are gadolinium, magnesium, lithium, and tantalum.32 Moreover, diamagnetic materials (e.g., bismuth, boron, copper, phosphorous, N2, sulfur, silver, gold and most organic compounds) have no unpaired electrons, thus no moment. These materials show a very weak response to an applied external magnetic field and do not retain a magnetic moment when the external field is removed.33
On the other hand, ferromagnetic materials have magnetic interactions that favor the parallel alignment of spins, which results in a net magnetization even in the absence of an external magnetic field. Some materials that exhibit ferromagnetic ordering are nickel, cobalt and iron. Antiferromagnetic materials spontaneously align their spins at relatively low temperatures into opposite or antiparallel arrangements throughout the material. This arrangement exhibits almost no gross external magnetism due to the cancelation of the magnetic moments. Transition metal compounds such as CoO, CuCl2, and MnO are examples of antiferromagnetic materials. Like antiferromagnets, ferrimagnetic materials have their magnetic dipole moments aligned antiparallel, but the magnitude or number of moments in one direction is different from the magnitude or number pointing in the opposite direction. This results in a net magnetization in the absence of an external field.
Some examples of ferrimagnetic materials are spinel ferrites. Figure 1.4.1.1 illustrates the magnetic ordering of the different types of magnetic materials.
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Figure 1.4.1.1. Magnetic moment orientations for various types of magnetic ordering.
Magnetic nanomaterials research has brought special attention to their size dependent properties, which are different from their bulk counterparts.34 Bulk magnetic materials exhibit a multi-domain structure, where regions of uniform magnetization
(groups of spins all pointing in the same direction) are separated by domain walls (Figure
1.4.1.2a). These walls have a characteristic width and are formed in order to minimize magneto-static energy.35 However, when the size of magnetic materials is tuned to nano 10
scales, the formation of domain walls is not thermodynamically favorable, thus favoring a single magnetic domain structure (Figure 1.4.1.2b).36 This phenomena observed in nanomaterials brings to the table a new set of magnetization reversal or relaxation dynamics.
Figure 1.4.1.2. (a) Multi-domain structure of bulk magnetic materials. (b) Single domain particle showing the absence of domain walls.
1.4.2 Single Domain Theory
During the mid twentith century, the first estimate of the critical size for the
37 existence of a single domain particle was predicted. The critical diameter (Dc) for most magnetic materials ranges under 100 nm and is dependent on the: saturation magnetization
(Ms), the anisotropy energy (K), the magnetic moment (μo), and the exchange energy (A) of a material as described in in equation 1.4.2.1.
11
1 36 (퐾퐴) ⁄2 퐷푐 = 2 eq. 1.4.2.1 휇표푀푠
The Dc value sets a maximum size for the synthesis of a material uniformly magnetized
(with the magnetic moments in each particle pointing in the same preferred direction, or the easy axis).
1.4.3 Stoner-Wohlfarth Theory
Stoner and Wohlfarth theory was developed in 1948, and describes the magentization reversal through the coherent rotation of the magnetization vector for an assembly of single domain nanoparticles with uniaxial anisotropy.38 Equation 1.4.3.1 describes the energy barrier (EB) for coherent magnetization reversal for single domain nanoparticles.
2 퐸퐵 = 퐾푉 푠푖푛 휃 eq. 1.4.3.1
Here, K is the magnetic anisotropy, V is the volume of the nanoparticle, and θ is the angle between the magnetic moment and the easy axis of the nanoparticle.
1.4.4 Superparamagnetism
The concept of superparamagnetism was first proposed by Frenkel and Doefman in
1930.39 This phenomenon appears when the crystal size of a magnetic material is small enough that the thermal energy kT (being k the Boltzmann’s constant and T the absolute temperature) may be sufficient to cause fluctuations of the magnetization direction.40 The fluctuations in direction happen between two stable orientations that are antiparallel to each other. Moreover, instead of a single magnetic moment per atom (paramagnetic material),
12
each nanoparticle contains many magnetically coupled spins. This results in a nanomaterial that behaves like a paramagnetic material with a giant magnetic moment, and that after eliminating the magnetic field the particle will no longer show magnetic interaction.
Moreover, superparamagnetic nanomaterials can also be described by the relaxation time (τ) of magnetization reversal. This relaxation time basically describes the average time for a magnetic spin reorientation and is defined as the Néel relaxation time
(τN). The τN is heavily dependent on the absolute temperature (T), nanoparticle volume (V) and the magnetic anisotropy constant (K).41
휏푁 = 휏표푒푥푝 (퐾푉⁄푘퐵푇) Eq. 1.4.4.1
where, τo is the attempt time (which depends on a number of factors including saturation magnetization, temperature, nanoparticle volume, gyromagnetic ratio, applied field, and damping constant, but is typically considered a constant). From Néel’s calculations, the τo theoretical value is 10−9, but reported experimental values range from 10-9 to 10-13.42
At a specific temperature, the magnetic moment could be undergoing constant relaxation by thermal fluctuation when the measurement time is longer than the relaxation time. This represents the behavior of a superparamagnetic state. On the other hand, if the measurement time is shorter than the relaxation time, then magnetic moment relaxation will be blocked. This is an indication that the thermal energy is not sufficient to reorient the magnetic moment. When the temperature at the magnetic moment time equals the measurement time, it is known as the blocking temperature. Since the Néel relaxation time heavily depends on the nanoparticle’s volume and the intrinsic magnetic properties
(magnetic anisotropy, K), the Néel relaxation time can be controlled by tuning the size and
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chemical composition of the nanoparticles. Unlike other single domain nanoparticles,
superparamagnetic nanoparticles have zero coercivity at room temperature (Figure
1.4.4.1).
Figure 1.4.4.1. Graphic illustration of change in coercivity as a function of particle's diameter showing the coercivity values for superparamagnetic (SPM), single domain (SD) and multi-domain (MD) particles.
14
Superparamagnetic nanoparticles are of great interest in environmental and biomedical research because of their sensitive response to external magnetic fields, which make them excellent candidates for the development of different kind of magnetic sensors and adsorbents for pollutant removal.
1.4.5 Iron Oxide Spinel Ferrites
The crystal structure of many magnetic oxide nanoparticles is based on a spinel structure with general chemical formula AB2O4. The unit cell of a spinel structure contains cubic close packing (ccp) of oxygen anions, as well as divalent and trivalent metal cations occupying tetrahedral (A sites) and octahedral (B sites) holes. There is a diversity of metal cations than can be integrated in this structure creating an extensive library of hybrid spinel
2+ 2+ structures. This work presents various spinel ferrites (MFe2O4 with M being Fe or Zn ) in order to exploit the magnetic properties of these particles for their use in diverse applications.
1.5 Nanocomposites
Nanocomposites are solid materials that are comprised by multiple components, of which at least one has a nanoscale structure.43 These materials are characterized by their synergistic properties that are dependent on the morphology and interfacial characteristics of each component.44 The development of nanocomposites is a growing area of research that forecast the development of promising materials that can overcome serious common issues that independent materials have not been able to address in biomedical and industrial applications.
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1.6 Nanoparticles Synthetic Methods
There are two major approaches for the synthesis of nanomaterials: the “top-down” or “bottom-up” methods.45 The top-down approach refers to the slicing or cutting of a bulk material to get a nano structure, while the bottom-up starts with atoms or molecules that get together to form small clusters that turn into nanoparticles. From each approach, multiple synthesis methods have been developed in order to synthesize nanoparticles with controlled shape, size and crystal structure. Among these methods can be found: ball milling, vapor condensation, coprecipitaion, sol-gel processing, hydrothermal synthesis, and microemulsion methods.46 However, even though these methods can provide large quantities of magnetic nanoparticles during a one-pot synthesis, the resulting nanoparticles usually are not uniform and have poor size distribution. Lack of size distribution can negatively affect their performance for some applications. This is an important issue for the development of nanoparticles since size and shape are important parameters for the control of specific properties of nanostructures. This is the main argument that justifies the use of a bottom-up approach for the synthesis of the electrocatalytic and magnetic nanomaterials present in this thesis. For these specific applications, it is essential to control the shape and narrow the size distribution.
For Au/Ag electrocatalysts, the polyol method was used for the synthesis of an Ag nanocube template.47 This sacrificial template was later used to synthesize Au/Ag nanocages using a galvanic replacement method.48 The galvanic replacement reaction is
− based on the differences in potential between AuCl 4/Au (0.99 V vs. SHE)
> AgCl/Ag (0.22 V vs. SHE) which promotes the reduction of three Ag atoms per each
16
atom of Au. This process promotes the deposition of Au and the formation of a hollow structure that we named nanocage (Figure 1.6.1).
Figure 1.6.1. Galvanic replacement reaction. (a) Reaction set-up. (b) Schematic illustration for the formation of Au/Ag nanocages.
On the other hand, a thermal decomposition method was used for the synthesis of the magnetic nanomaterials with highly uniform shape and size. This method starts with the decomposition of a metal precursor in the presence of a surfactant ligand and a high boiling point solvent Figure 1.6.2. This method allows fine tuning the size and shape of the
17
particles by controlling the synthesis conditions such as heating rate, reflux temperature and the concentration of metal precursors.
Figure 1.6.2. Graphic illustration of a typical thermal decomposition synthesis setup.
The thermal decomposition synthesis relies on the crystallization of the nanoparticles from a solution. In general, nanoparticle formation is divided in two main stages: nucleation and particle growth.49 The first stage starts with the decomposition of a metal precursor at a high temperature to rapidly increase the concentration of active metal precursor over the supersaturation threshold.50 Once the metal precursor concentration is above the supersaturation threshold, nucleation occurs and a burst of nanoparticle formation is observed (Figure 1.6.3). Immediately after the nanoparticle nucleation stage, the concentration of metal precursor is depleted, and the second stage of the particles formation starts. During the growth stage, any additional precursor can only accumulate on the nuclei from the nucleation stage, with the smaller nanoparticles growing faster than the ones with larger size due to the higher free energy in the smaller nanoparticles. This promotes a narrower size distribution of the particles collected at the end. On the other
18
hand, if the nanoparticles continue to grow beyond the size focusing stage, the metal precursor in the synthesis is completely depleted and the smaller nanoparticles start to dissolve, resulting in a large size distribution. This effect is known as “Ostwald ripening”.51
Taking this into consideration, in order to carefully achieve precise control over size, size distribution and shape it is necessary to regulate the metal precursor’s concentration.
Figure 1.6.3. LaMer diagram showing the nucleation and growth of nanoparticles during thermal decomposition synthesis.
1.7 Nanoparticles Characterization Methods
The characterization of nanomaterials is key for understanding their properties and optimizing them for specific applications. However, due to their small size, advance
19
analytical instrumentation is needed in order understand their structural and chemical properties. Among the most commonly used characterization techniques for nanomaterials are microscopy, spectroscopy and vibration sample magnetometery (VSM). Microscopy techniques like TEM and SEM are fundamental to perform structural analysis of nanomaterials. Spectroscopy techniques such as energy dispersive X-ray spectroscopy
(EDX), fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy
(UV-vis), and atomic absorption spectroscopy (AAS) have been adapted to obtain chemical information on nanomaterials; like the crystal structure, information of the surface chemistry, shape, composition and concentration of components. Powder X-ray diffraction
(PXRD) and vibrating-sample magnetometer (VSM) are common instrumentation to determine the crystal phase and magnetic properties of the nanomaterials. The detailed descriptions of common characterization techniques utilized throughout this thesis can be found in the following sections.
1.7.1 Microscopy Methods
1.7.1.1 Transmission Electron Microscopy (TEM)
Transmission electron microscopy (TEM) is a powerful and unique tool for the characterization of micro and nanostructures. This technique has been widely applied for the study of the size, morphology and crystalline structure of a variety of nanomaterials.
TEM produces a gray scale image by transmitting a high voltage (80-200 keV) electron beam through an ultra-thin sample holder. The electrons interact with the nanoparticles, which have higher density than the sample holder, and are diffracted.52 This process forms a shadow-like representation of the nanoparticle morphology onto a fluorescent screen or photographic plate. Subsequently, the image is detected by a charge-coupled device (CCD)
20
camera. Due to the small de Broglie wavelength of electrons, TEM can capture images of materials that are thousands of times smaller than the smallest objects seen in optical microscopes. TEM is used to evaluate the morphology and size of all nanoparticles presented in this thesis. The samples were prepared on a 400 mesh Formvar-coated copper grid by dropping 5 μL of a previously diluted nanoparticle suspension. After sample deposition on the grid, the solvent is left to dry (1 hour for toluene samples and 2-4 hours for samples dispersed in water). TEM images were obtained with a FEI Tecnai G2 Spirit
BioTWIN transmission electron microscope operated at 120 kV. The mean particle size and size distribution were evaluated by measuring up to 200 nanoparticles per sample.
1.7.1.2 Scanning Electron Microscopy (SEM)
Scanning electron microscopy (SEM) uses a focused beam of high-energy electrons in order to generate signals that derive from electron-sample interactions. These signals reveal important information about the sample like: morphology, chemical composition and crystalline structure. Similar to TEM, scanning electron microscopy (SEM) also utilizes a high energy electron to study the structural properties of nanomaterials. However, unlike TEM, SEM scans the electron beam over the surface of the sample. As the electron beam strikes on the surface, it interacts with the specific atoms present on the surface and generates secondary electrons, backscattered electrons, and characteristic X-rays.53 This mechanism for the creation of the signal, allows SEM to provide information about the topography of the sample. In order to acquire useful images from SEM, the samples need to be electrically conductive at the surface. Cases in which the sample is nonconductive, usually a thin layer of electrically conductive material (usually gold) can be sputter coated on to the surface. Samples in liquid form can be dropped onto a cleaned silicon wafer chip
21
or strips of solid sample can be taped onto carbon tape for SEM imaging. For this thesis
SEM images were obtained using a PhenomProX desktop scanning electron microscope.
High resolution SEM images were obtained using the Hitachi S-2600N scanning electron microscope (1-30 kV).
1.7.2 Spectroscopy Methods
1.7.2.1 Energy Dispersive X-ray Spectroscopy
Energy dispersive X-ray spectroscopy (EDX) is an advanced chemical microanalysis technique that is usually coupled with SEM or TEM. The high-energy electron beam used in SEM and TEM can be utilized to simulate X-ray emission from the sample, thus providing chemical information on the material during imaging. This information provides insights about both quantitative and spatial elemental analysis of nanomaterials. The fundamental principle of EDX is based on the unique atomic structure of individual elements that gives rise to a characteristic X-ray emission spectrum after the sample interacts with the source X-ray excitation.54 When SEM is coupled with EDX, a fine electron probe is used to scan over the sample and generate an elemental map instead of a single point measurement, thus enabling a quantitative and qualitative spatial analysis of a sample. SEM-EDX was employed in this work to determine the composition of bimetallic structures and study the distribution of nanomaterials on an electrode surface.
1.7.2.2 Atomic Absorption Spectroscopy
Atomic absorption spectroscopy (AAS) was first observed in 1802 and later explained in 1859 by Kirchoff and Busen, but it was not until 1953 when Alan Walsh fabricated the first analytical atomic absorption spectrometer.55 AAS is another spectroscopic technique that allows the analysis of metallic elements. This method is
22
accepted as a standard method, because of its high element recognition capabilities that provides analytical sensitivities at the parts-per-million level and less. It is an essential tool for quantitative analysis for chemical elements through measuring the absorption of optical radiation by free atoms in the gaseous state. For the experimental determination of the concentration of a typical iron oxide nanoparticle sample, it is first dissolved in low pH solutions (usually HCl) overnight. Then the sample is diluted and atomized in a high temperature flame. The electrons of the metal ions present in the solution can absorb the thermal energy from the flame that promotes it to excited energy state.56 This gives off a radiation with a specific wavelength that is unique to a specific electron transition in a particular element. The difference between background and sample radiations detected is presented as the absorbance. A calibration of standards with known concentrations is used to establish the relationship between the measured absorbance and analyte concentrations.
This information is used to make a calibration curve that can be used to calculate the unknown analyte concentration through the Beer-Lambert Law.57 All of the AAS elemental analysis work presented in this thesis was performed using a fast sequential atomic absorption spectrophotometer Varian 220FS AA.
1.7.2.3 Fourier Transform Infrared Spectroscopy
Fourier transform infrared (FT-IR) is a common spectroscopy technique use to determine the chemical structure of materials and surface functionalities. FT-IR spectroscopy probes molecular vibrations, allowing the identification of functional groups that can be associated with characteristic infrared absorption bands. A normal mode of vibration is infrared active if there is a change in the dipole moment of the molecule during the course of the vibration, thus symmetric vibrations are usually not detected.58
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In FT-IR measurement, radiation can be absorbed by the sample or transmitted through, giving rise to spectrum that is characteristic for specific molecular absorption or transmission. The chemical identity of the specific material is revealed by obtaining the absorption or transmittance spectrum that is unique for the specific combination of atoms.
This work presents FT-IR analysis to study the surface functionalization of different types of nanostructures.
1.7.3 Powder X-ray Diffraction
Powder X-ray diffraction (PXRD) is an advanced instrument that allows the identification of the crystalline structure of nanomaterials, which provides qualitative and quantitative information on phase composition and particle size. Diffraction occurs when light is scattered by a periodic array that can produce constructive interference at specific angles. Since atoms in a crystal are periodically arranged, a diffraction of light is produced.
The wavelength of X-rays are similar to the distance between atoms.59 The basic principle of XRD relies on the principle of constructive interference of a monochromatic x-ray and the sample material. The correlation between the wavelengths of electromagnetic radiation, the diffraction angle and the lattice spacing of a crystalline sample is described by Bragg’s
Law (nλ = 2d sin θ).60
During a typical measurement, the characteristic pattern based on the diffracted X- ray from different lattice directions is obtained from scanning the sample through a range of 2θ angles. The diffraction pattern obtained is unique to an individual crystal lattice and can be used to estimate d-spacings (for phase identity), purity of the crystaline phase, and the size of the crystalline particle.
24
The crystallographic phase of the sample can be identified by matching the peak positions and intensities from the diffraction patterns to standard diffraction patterns found in a database. Common database sources include JCPDS (Joint Committee on Powder
Diffraction Standards), ICDD (International Crystallographic Diffraction Database) and
COD (Crystallography Open Database).61
The Scherrer equation uses PXRD data to calculate the nanoparticle size from the peak broadening on nanoparticles with a size smaller than 100 nm.62
푑 = 퐾휆/(훽푐표푠휃) eq. 1.5.4
Equation 1.5.4 is the Scherrer equation in which d is the average crystalline dimension perpendicular to the reflecting phases, K is a constant, λ is the wavelength of the X-ray, β is the size broadening provided by the full width at half-maximum (FWHM) and θ is the
Bragg angle.
This thesis presents the PXRD analysis of iron oxide nanoparticles in order to obtain structural information of the iron oxide nanoparticles.
1.7.4. Dynamic Light Scattering
Dynamic light scattering (DLS) also known as photon correlation spectroscopy is a powerful technique that allows the determination of the hydrodynamic size and shape of nanomaterials.63 DLS reveals the interparticle interaction between nanoparticles in solution and the colloidal stability of a nanoparticle dispersion.64 During the DLS measurement, the sample is exposed to a monochromatic light, which encounters the sample that scatters the
25
incident light in various directions; The scattered light angle and intensity is recorded by a detector. The diffraction of the light is dependent on the hydrodynamic volume and shape of the nanoparticles in solution.
The resulting intensity fluctuations are caused by the constructive and destructive inference from the diffracted light, which are used to calculate the particle size (r) using the Stokes-Einstein equation:
r= kBT/6πηD eq. 1.5.5
where, T is the temperature, kB is the Boltzmann’s constant, η the viscosity of the solvent, and D the diffusion coefficient. The DLS measurements for this work were performed using a Brookhaven ZetaPALS particle size analyzer with a TurboCorr correlator.
1.8 Applications of Functionalized Nanomaterials
Nanomaterials are promising materials due to their diverse properties that make them suitable for broad range of applications. Their performance for specific uses can be improved by tuning properties like size, shape and composition. Moreover, further work on the surface chemistry of these materials can improve their performance. Specific functionalization of the nanomaterials surface can be used for drug delivery, selective sensors, and absorbents for heavy metals treatment and for the development of hydrophobic surfaces.
1.8.1 Electrochemical Sensors
Sensors are one of the most important tools for the progress of biomedical research, monitoring of disease related biomarkers, pharmaceutical analysis, food safety and environmental monitoring.65–67 Due to their extensive use for such a broad range of
26
applications, it is important to understand their fundamental components and how to tune the properties of the materials used on them in order to optimize them for specific applications. Key factors for the development of sensors are: durability, costs, simplicity, and potential for real time monitoring. Considering these requirements, electrochemical approaches result in the most promising candidate technologies due to their simplicity, high sensitivity and specificity, rapid response times, and portability.68 Some of the most commonly used electrochemical sensors are the glucometer, the alco-sensor and carbon monoxide sensors (Figure 1.8.1.1). The chemical design of nanomaterials with specific composition, size, shape and surface chemistry have the potential to significantly improve the sensitivity and selectivity of sensing technologies. These kinds of advances can promote the development of novel early-stage detection and diagnosis of disease related biomarkers. Nanomaterials can improve sensor technologies through functions like effective catalytic activity, fast electrode kinetics and increased active surface area.69
27
Figure 1.8.1.1. Examples of the most commonly used electrochemical sensors.
Over the past decades, a number of electrochemical techniques have been employed for the development of nanomaterials-based electrochemical sensors. Some of these techniques are ampero-metric/potentiometric sensors, electrochemical impedance sensors, electrochemical luminescence sensors and photoelectrochemical sensors.70–73 However, regardless of the type of electrochemical sensors, nanomaterials usually participate on the interface component where they play an important role on signal amplification via catalytic activity and/or conductivity. In some instances, they also participate in the chemical and biological recognition of relevant analytes. However, beside all the advantages of nanomaterial-based electrochemical sensors, there are numerous characteristic draw-backs like mass transport and electron transfer (due to the dispersion of the material, interparticle
28
interactions and or surface poisoning). This thesis presents the chemical design of different nanomaterials in where their size, shape, composition and surface chemistry were fine tune in order to address these drawbacks and improve their performance for specific applications.
1.8.2 Magnetic Sensors
During the last decades, considerable efforts have been made to develop and explore magnetic nanoparticles (MNPs) due to their advantages such as their size, physicochemical properties and low cost production. These particles are characterized by their supermagnetism that is exhibited in size ranges from 10 nm to 25 nm.74 This small sizes promote large surfaces area and consequently high mass transference which are characteristics that are especially important for sensing applications. So far, chemically design MNPs with tailored surface properties have been prepared for the development of waste water treatments, disease therapy, disease diagnosis (through magnetic resonance imaging), cell labelling and imaging, tissue engineering and sensors.75,76 Moreover, MNPs have been use to enhance sensitivity and stability of sensors for clinical analytes, food and other environmental applications.
To date, one of the biggest issues associated with the use of magnetic nanomaterials for sensing applications is the chemical design of an appropriate surface chemistry that promote the desired chemical recognition of a specific analyte and also maintains a balance with the adequate stability of the particle in order to avoid aggregation. This thesis presents the chemical design of superparamagnetic iron oxide nanoparticles with specific compositions, size and surface chemistry moieties and the test of these designs for sensing
29
applications from environmental monitoring of nanomaterials in plants systems to the selective determination of heavy metals.
1.9 Nanoparticles in Photothermal Therapy
Photothermal therapy (PTT) is a relatively new therapeutic strategy that employs near-infrared (NIR) laser photoabsorbers to generate concentrated heat that can be used to treat cancer cells or bacterial infections.77 Compared with traditional therapeutic modalities, PTT exhibits unique advantages like high specificity, minimal invasiveness and precise spatial-temporal selectivity.78 Recent studies have explored the properties of nanomaterials for this application. Some nanostructures have the particularity of having electrons in the conduction band that can undergo synchronized oscillations when they are irradiated by laser light. These results in either the absorption or scattering of the applied light.79 These nanomaterials can be made out of various materials including organic compounds or metal nanostructures, however, gold nanoparticles (Au NPs) have proved to offer several major benefits.80 However, it is important to take into consideration that the engineering of NPs (specific shape, size and surface chemistry) needs to be designed to enhance their properties for PTT. 81
Au NRs have been used in various disease treatments, due to their unique optical properties, namely, their localized surface plasmon resonance (LSPR) as well as their inherently low toxicities. This thesis presents the synthesis of different types of Au NRs as well as their surface functionalization in order to improve their properties for their use in
PTT. Moreover, it presents a systematic study that aims to contribute to the treatment of bacterial biofilm infections caused by S. Aureus (Figure 1.9.1).
30
Figure 1.9.1. Schematic representation for the treatment of bacterial biofilm using Au NRs in photothermal therapy.
1.10 References
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Chapter 2. Designing the Chemistry of Au/Ag
Nanostructures for Cortisol Sensing
Adopted from the published article:
Navarreto-Lugo, M.; Lim, J.; Samia, A.C.S*. “Engineering of Au/Ag Nanostructures for
Enhanced Electrochemical Performance” Journal of Electrochemical Society. (2018),
165, 3, B83-B88. DOI:10.1149/2.0361803jes.
37
Chapter 2. Designing the Chemistry of Au/Ag Nanostructures for Cortisol Sensing
2.1 Introduction
Nanoparticle based electrochemical sensors have received considerable attention in recent years due to their exceptional attributes including high sensitivity, good reliability, and stability.1-4 Moreover, engineered electrocatalytic nanomaterials provide a means to develop simple-to-contruct5 electrochemical sensors that can be used for routine point-of- care health monitoring and diagnostics.6,7 However, for most biomedical applications a next generation sensor platform technology will require significant improvements in the nanomaterials’ properties to facilitate lower detection limits and higher sensitivities under lower costs for mass production.8,9
An effective electrochemical sensor relies heavily on the surface architecture of the working electrode in order to allow the recognition process to occur under short response time, achieve good signal-to-noise (S/N) ratio, and detect the analyte of interest at low limits of detection (LOD) with high selectively.9-11 For these purposes, Au nanoparticles have been heavily explored for their use as modifiers of the electrochemical sensor’s interface due to their unique chemical attributes such as high conductivity,12 stability,13 ease of enabling surface chemical modifications,13,14 and their large surface-to-volume ratios.15,16 Moreover, Au nanoparticles are less susceptible to suffer from surface poisoning compared to other commonly used electrocatalytic nanometals.17 Due to the many promising attributes of Au-based nanostructures, this nanomaterial has become a good scaffold for different electrochemical sensing applications. The properties of Au-based nanostructures can be readily tuned by changing particle size, shape, composition or by preparing hybrid composites with carbon based nanomaterials.18,19
38
While some studies have indicated that Au is not the best electrocatalytic material due to its filled d-band, which leads to the weak chemisorption of electroactive analyte species.20,21 It has also been demonstrated that by tuning its morphology, the electrocatalytic activity of Au nanostructures can be improved.22 For example, recent studies have demonstrated how shape tuning with the incorporation of corners and edges on synthesized Au-based nanoboxes and nanocages can provide more electroactive sites for improved performance in electrocatalysis.23 Other studies support the fact that smaller nanoparticles show higher catalytic activities due to their higher surface-to-volume ratios, however, just smaller Au NPs might still not be the optimum candidates for the electrochemical monitoring of different types of redox reactions.24 A rational behind this argument is that as the Au NPs become smaller in size, the electron transfer process becomes more dependent on good electrical connections between particles, specifically in situations where the reduction and oxidation sites occur on different particles.24 In addition, smaller Au NPs can be more prone to particle aggregation, which can directly influence the available electroactive surface area. This is an issue that can be addressed by changing the capping agent of the particles or by the incorporation of a conducting carbon matrix support to help disperse the NPs.25
In this work, we synthesized a series of Au-based nanostructures: solid spherical
Au NPs, hollow Au/Ag nanoboxes, and porous-hollow Au/Ag nanocages, in order to systematically investigate the effect of nanoengineering on the electrochemical performance of Au/Ag nanostructures for electrochemical sensing. In addition, we explored the effects of nanostructuring with G as carbon matrix support and the
39
modification of the surface chemistry with β-cyclodextrin on the electrochemical performance of the Au/Ag nanomaterials.
2.2 Methods
2.2.1 Synthesis of Gold Nanoparticles (Au NPs).
Solid, spherical Au NPs with an average diameter of 15 nm were prepared following a citrate-based synthesis approach.26 In a typical Au NP synthesis, a solution of
10 mL of 7 mM HAuCl4 in deionized water is reduced with the addition of trisodium citrate
(1 mL of 3.8x10-2 M citrate solution). The reaction mixture is heated and then kept at 100
°C under constant stirring (300 rpm) until a wine red color was observed, which indicates the formation of Au NPs (~3-4 minutes).
2.2.2 Synthesis of Au/Ag Nanoboxes (Au/Ag NBs) and Au/Ag
Nanocages (Au/Ag NCs).
Silver nanocubes were first synthesized by adopting a procedure developed by Xia et al., and were used as sacrificial nanoparticle templates in the galvanic replacement reaction for the formation of Au/Ag NBs and NCs.27 For the NBs’ and NCs’ preparation,
50 µL of Ag nanocubes were dispersed in an aqueous solution of 9 mM poly(vinylpyrrolidone) (PVP) and the solution was titrated with different amounts of a 0.1 mM HAuCl4 aqueous solution until the desired wavelength of surface plasmon resonance
(SPR) peaks were obtained. Specifically, two sets of Au/Ag nanostructures were synthesized with different Au compositions and different morphologies: hollow Au/Ag
NBs with 20% Au, and porous-hollow Au/Ag NCs with 40% Au content. For the β-
40
cyclodextrin (β-CD) modified particles, a similar procedure was followed but the PVP was replaced with a 10 mM β-CD aqueous solution.
Scheme 2.2.2.1. Galvanic replacement reaction scheme for the synthesis of Au/Ag nanostructures.
2.2.3 Preparation of Nanocomposites
Graphene nanoplatelets (G) were selected as the conducting carbon matrix support for the synthesized Au/Ag nanostructures. For the preparation of the nanocomposites, 2 mg of the different nanomaterial samples dispersed in 1 mL of deionized water were separately vortex mixed with 1.2 mg of G for 5 minutes. The nanocomposite mixtures were sonicated for an additional 30 minutes. The nanocomposite mixtures were then subjected to a washing process and re-dispersed in a mixture of 4 mL deionized water, 1 mL isopropanol and 0.025 mL of 5% Nafion. AAS and EDAX data were used to determine the metal concentration on the samples used for the electrode modifications, and those values were used to normalize the electrochemical data.
2.2.4 Electrode Modification
Prior to analysis, the SPCEs were modified using the different nanostructure and nanocomposite samples. For the surface modification, four successive depositions of 10
41
µL (each nanostructure/nanocomposite sample) were deposited on the carbon working electrode. After each deposition step, the modified SPCE was placed in an oven set at 80
°C to allow the solvent to evaporate prior to the next round of deposition.
2.2.5 Electrochemical Analysis – Cyclic Voltammetry (CV)
To evaluate the electrocatalytic efficiencies of the different nanostructure/nanocomposite samples, the modified SPCEs were washed with DI water
3-/4- and subjected to CV analysis. For the CV experiments, 5 mM Fe(CN)6 solutions were prepared in PBS (1x, pH 7.4) and 0.1 M KCl (as supporting electrolyte) and scanned between -1.0 V to 1.0 V at a scan rate of 200 mV/s at room temperature.
Scheme 2.2.5.1. Schematic diagram of the electrode surface nanoarchitecture. The Au/Ag
NC sample in conjunction with the G carbon matrix support was used to modify the working electrode on the SPCE, and was utilized here to represent the different nanostructures used in the study (Au NP, Au/Ag NB, and Au/Ag NC). The electrochemical
3-/4- analyses were performed using the Fe(CN)6 redox probe.
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2.3 Results and Discussion
2.3.1 Characterization of Au/Ag Nanostructures.
Hollow Au/Ag NBs and porous-hollow Au/Ag NCs were synthesized using the galvanic replacement synthetic approach and their properties were evaluated and compared against the conventional solid spherical Au NP analog. The nanostructure morphology of the synthesized nanomaterials was investigated by transmission electron microscopy
(TEM) and their optical properties were evaluated by UV-vis absorption spectroscopy.
Shown in Figure 2.3.1.2 is the evolution of the localized surface plasmon resonance
(LSPR) peak of the synthesized Au/Ag nanostructures. As the morphology (i.e. particle shape and porosity) of the nanostructure evolves from solid spheres to hollow cubic nanobox and nanocage architectures, the LSPR peak shifts from 520 nm to 500 nm for the
Au/Ag NBs, and to a longer wavelength at 920 nm for the Au/Ag NCs, respectively. The change in LSPR band peak can be attributed to the changes in particle structure and composition, which is reflected in the obtained TEM images from the synthesized nanostructures Figure 2.3.1.1. The TEM image of the synthesized Au NPs that exhibits a spherical shape with an average diameter of 15 nm. In contrast, both the Au/Ag NB and
Au/Ag NC samples exhibit cubic morphology with an average side length of 50 nm (Fig.
1c-d). Moreover, the presence of pores and contrast change observed for the NBs and NCs reflects their open and porous structure. Elemental AAS and EDAX analyses of the samples reveal 100%, 20% and 40% Au-content for the NP, NB, and NC samples, respectively.
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Figure 2.3.1.1. TEM images of the (b) Au NP, (c) Au/Ag NB, and (d) Au/Ag NC samples, respectively; the scale bars indicate 100 nm.
44
Figure 2.3.1.2. UV-vis absorption spectra of the (a) synthesized Au/Ag nanostructures.
2.3.2 Effect of Nanostructure Morphology and Carbon Matrix Support
on Electrochemical Performance
Au nanomaterials have been investigated in electrochemical sensing applications
with great interest largely in part due to their stability and the ease of modifying its surface
chemistry.28-30 Moreover, synergistic effects on the electrocatalytic activity of Au-based
nanostructures as a result of alloying with other metals have been shown to bring
improvements in performance for specific sensing applications.31,32 As such, in our study
we have prepared a set of alloyed Au/Ag nanostructures with different morphologies in
order to develop and characterize different nanomaterial systems for the improved
detection of electroactive analytes.
45
In this study, we evaluated the electrochemical performance of different Au/Ag
3-/4- nanostructures toward the direct determination of the Fe(CN)6 redox probe using CV.
Shown in Scheme 2.2.5.1. is an illustration of the nanostructure architecture and
3-/4- electrochemical system used in our study. The Fe(CN)6 redox probe has been employed in different studies as a valuable tool and model system to study and monitor the electrochemical characteristics of surface-modified electrodes.33 The redox reversibility of this electroactive couple makes it versatile and readily adaptable to test many electrochemical sensor models.
Comparison of the measured CV curves in Figure 2.3.2.1. showcases a much
3-/4- higher anodic current density from the electroactive Fe(CN)6 probe when it was exposed to the SPCEs modified with hollow cubic Au/Ag NBs and NCs in comparison to the SPCE modified with the solid spherical Au NPs. This result can be attributed to the increased surface area in the open porous architecture of the hollow NBs and NCs morphologies and the good conductivity brought by the synergistic effect provided by the alloyed Au/Ag bimetallic structure. These characteristics facilitate the transfer of electrons in the system by increasing the contact points of the electroactive probe with the electrocatalyst.
Furthermore, the open cavity on the NB and NC architectures provide a confinement effect known as the nanoreactor cage effect that facilitates more metal exposure (capping agent free), which then provides more active sites for the diffusion of the electroactive probe, therefore facilitating more direct contact with the electrocatalysts.34 In addition, ligands or capping agents play an important role on the synthesis of nanostructures as they have a crucial role on controlling particle growth and particle dispersity during and after the synthesis process. Depending on the nanostructure shape, size, and the type of ligand by
46
which the particle is coated, different types of intermolecular interactions can lead to aggregation, which can considerably affect the electrocatalytic performance of the nanomaterial.35,36 Moreover, the electron transport process is directly influenced by the level of how well the electrocatalysts is protected and consequently how accessible the catalytic sites are during the electrochemical measurements.34,37 As such, the degree of ligand passivation on the nanostructure surface provides another rational for the proportional increase in measured peak current from using the Au NPs to transitioning to the Au/Ag NB and NC morphologies. The opening of the cavity in the hollow cubic architecture leads to more exposed metal sites with less passivation by the capping agent.
This in turn provides metal spots with available surface atoms for the electroactive redox probe to diffuse and interact, leading to an increase in anodic peak current as observed for the Au/Ag NB and NC modified SPCEs. The highest signal was observed for the Au/Ag
NC-modified SPCE, where the effect of the porous-hollow cubic structure is more pronounced and there’s a significant increase in exposed corners and edges, which are characterized by being enriched with chemically unsaturated surface atoms that can act as catalytically active sites.24,38,39
47
In this study, we also explored the effect of the introduction of G, as carbon matrix support, on the measured anodic peak current. As evident in Figure 2.3.2.1. and Figure
2.3.2.2 there is a significant increase in peak current with the incorporation of G to all the nanostructure samples. Graphene nanoplatelets are unique nanostructures that have the ability to increase the surface area, improve conductivity, and help enhance the electrocatalytic activity of metal nanomaterials by providing more interconnectivity between the particles.40,41 These attributes lead to an overall increase in the load of electroactive probe and the amplification of the electrochemical response.
Figure 2.3.2.1. (a) Cyclic voltammograms (CVs) of 5 mM K3[Fe(CN)6]/ K4[Fe(CN)6] in PBS (1x, pH 7.4, 0.1 M KCl) obtained using the SPCEs modified with the Au/Ag nanostructures (i.e. Au NP, Au/Ag NB, and Au/Ag NC, respectively). (b) The corresponding CVs of the nanocomposites obtained by mixing the Au/Ag nanostructures with graphene nanoplatelets (Gs) as carbon matrix support.
48
Figure 2.3.2.2. Bar graph comparing the anodic peak current response of the nanostructures versus the nanocomposites. All CVs were obtained using a scan rate of 200 mVs-1 and normalized by the total metal concentration used in each measurement. All values are statistically significant with p values less than 0.05.
2.3.3 Effect of β-Cyclodextrin (β-CD) Modification on Nanostructure
Electrochemical Performance.
To study the effect that the capping agent has on the electrocatalytic performance of the synthesized nanostructures the same particle types were synthesized and prepared with and without β-CD. This organic polysaccharide has been widely used in the modification of different nanomaterials because of its ability to form inclusion complexes
49
with hydrophobic molecules.42,43 In addition, the open cavity of the β-CD structure facilitates the transfer of electrons directly between the metal and the electroactive redox
3-/4- probe. Figure 2.3.3.1 shows the electrochemical response of the 5 mM Fe(CN)6 solution when it is exposed to the modified SPCE with the different nanocomposites: Au NP+G,
Au/Ag NB+G and Au/Ag NC+G and their counter versions prepared with β-CD modified nanostructures. The comparison between the different systems show a significant increase in the anodic peak current obtained from Au/Ag NC β-CD+G in comparison with the other nanosystems investigated. This result can be attributed to the interactions between the
3-/4- electroactive Fe(CN)6 redox probe with the β-CD capping agent that facilitates the electron transfer to the metal nanostructure surface. Overall, the modification with β-CD aides in improving the proximity of the electroactive probe to the metal nanostructure surface, thereby, influencing the stability, enhancing the current response, and improving reproducibility of the electrochemical measurements.
50
Figure 2.3.3.1. CVs of 5 mM K3[Fe(CN)6]/ K4[Fe(CN)6] solution obtained using the nanocomposite-modified SPCEs prepared with and without β-CD modification: (a) Au NP, (b) Au/Ag NB, and (c) Au/Ag NC, respectively.
51
Figure 2.3.3.2. Comparison of the electrochemical responses of the different nanostructure systems. All values are statistically significant with p values less than 0.05.
2.3.4 Nanostructure Dispersion Effect on Electrode Surface.
The different factors previously discussed act in a synergistic way to affect the electrocatalytic performance of the investigated nanomaterials. However, another important factor that has not been thoroughly discussed in previous reported studies is the influence of the nanomaterial interactions on the working electrode surface and how this affects the surface coverage and electroactive surface area on the SPCE. Figure 2.3.4.1. presents the SEM-EDAX analyses of representative Au NP+G and Au/Ag NC β-CD+G
52
(the worst and best performing nanostructures, respectively) modified SPCEs. The images show the distribution of the nanostructures on the SPCEs. The results present the uneven distribution of the Au NPs on the SPCE working electrode surface, possibly due to desorption of the capping ligand during the electrode preparation. The Au/Ag NC β-CD+G sample, on the other hand, is well spread throughout the entire working electrode surface.
This result is reflected in the calculated electroactive surface areas (using the Randles-
Sevcik equation): 0.03 cm2 and 0.073 cm2 for the Au NP+G and the Au/Ag NC β-CD+G modified electrodes, respectively. The improved electrocatalyts dispersity on the electrode surface can be attributed to the unique cubic architecture of the synthesized Au/Ag NCs, which has been similarly observed when comparing Pt-Ag NCs with Pt NPs,44 and the steric effects brought about by the G and β-CD modifications.
53
Figure 2.3.4.1. (a) Photograph of a representative SPCE with the areas used in the EDAX elemental analysis highlighted, and (b) the corresponding scanning electron microscope (SEM) image of two of the spots on the working electron that were used to assess nanostructure distribution. (c) Summary of the EDAX analyses. (d) Higher resolution SEM image of a representative SPCE modified with Au/Ag NC with G matrix support.
54
2.3.5 Sensor Application – Detection of the Stress Biomarker, Cortisol
The best performing nanocomposite, Au/Ag NC β-CD+G, was used to test an electrochemical sensor model for cortisol. Scheme 2.3.5.1. shows the working electrode architecture and the electrode’s surface chemistry. The sensor was built following an indirect determination method, where β-CD shows preference for the formation of inclusion complexes with cortisol, creating an insulating layer on the surface of the SPCE and leading to a decreased current response with the increase in cortisol concentration.
Figure 2.3.5.2. shows the CV response curves obtained with the Au/Ag NC β-CD+G modified SPCE. The CV curve shows the presence of two anodic peaks. The 0.5 V peak
3-/4- corresponds to the Fe(CN)6 redox pair and the 0.1 V peak indicates the residual of Ag remained in Au/Ag NCs. The results show the decrease in anodic peak current with the increase in cortisol concentration. The nanostructure electrochemical sensor platform demonstrated a linear response in the 1 pM-100 nM cortisol concentration range with a regression coefficient of 0.995, and a limit of detection of 0.9 pM.
55
Scheme 2.3.5.1. Schematic representation of the nanoarchitecture used in the electrochemical sensing of cortisol. The system presents an indirect determination of 3-/4- cortisol with the use of Fe(CN)6 as a redox probe.
56
Figure 2.3.5.2. CVs of 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] obtained using an SPCE modified with Au/Ag NC β-CD+G nanocomposite that was used for the indirect determination of cortisol; the inset shows an amplification of the anodic peak currents obtained from the CV. (b) Calibration plot with different cortisol concentrations with n=5.
57
2.4 Conclusion
In summary, a systematic comparative study of the effect of nanostructure revealed that the porous-hollow cubic Au/Ag NCs modified with β-CD and G exhibited the best electrochemical performance among the different nanostructures investigated in the direct
3-/4- detection of the Fe(CN)6 redox probe. This result can be attributed to the increase in active surface area, the nanoreactor cage effect, and the edge effect, which lead to an increase in available catalytic sites for electron transfer. Furthermore, the channel effect caused by modification with β-CD also provides direct contact/exchange of electrons between the electroactive redox probe and the electrocatalytic nanometal. Moreover, the unique cubic morphology and the particle interactions with the β-CD and G produce better dispersion of the particles throughout the working electrode, thereby improving the overall electrochemical activity of the nanocomposite system.
The nanocomposite with the optimal electrochemical performance was evaluated for the indirect determination of cortisol and displayed a linear detection range between 1 pM-100 nM with a regression coefficient of 0.995 and a limit of detection of 0.9 pM. This result showcases a novel sensor platform system for the direct or indirect quantification of an analyte for electrochemical sensor applications.
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Chapter 3. Development of a Magnetic
Particle Spectroscopic Method for the
Detection of Pb2+
Manuscript in preparation for submission:
Author List: Navarreto-Lugo, M.; Ju, M.; Milbrant, N.; Wickramasinghe, S.; Samia,
A.C.S*.
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Chapter 3. Development of a Magnetic Particle Spectroscopic Method for the
Detection of Pb2+
3.1 Introduction
Heavy metals pollution has become a pervasive problem worldwide mainly due to the anthropogenic exploitation through industry development.1 Their accumulation has proven to increase and affect especially the regions close to mining industries, foundries, smelters and coal burning power plants.2 This group is composed mainly by arsenic, cadmium, chromium, mercury and lead.3 The extensive use of lead (Pb2+) for agricultural, domestic and industrial applications promotes more routes of contamination for exposure of the most vulnerable groups: pregnant women and children.4 To date, several studies have reported that even small concentrations of Pb2+ can cause serious organ failure and health problems especially for children younger than 6 years. Even though no concentration of
Pb2+ has been reported to be safe, the Centers for Disease Control and Prevention USA
(CDC), has estimated that 535,000 US children between 1-5 years have BLL ≥ 5 µg/dL
(50 ppb), and determined > 10 µg/dL (100 ppb) as the level of concern for children.5 The toxicity in children is of greater concern, since their systems that are still under development have higher gastrointestinal uptake, and more so, their permeable blood-brain barrier.6 Pb2+ poisoning of children has been directly associated with significantly negative effects on their neurological development, consequently contributing to behavioral problems and learning deficits.7 Currently the main tool for the detection of Pb2+ is through blood samples. This test gives an account of the Pb2+ concentration in circulating blood, however, its analysis is time consuming due to the need of a multistep sample preparation process.8 Moreover, the main treatments for Pb2+ poisoning consist of dimercaprol and
63
succimer.9 These are both chelation based treatments that can be given orally (succimer and penicillamine) or parenterally (dimercaprol and edentate); The biggest problem with these therapies is their lack of selectivity. This issue brings further health problems like anemia or Ca2+ deficiency.10 Pb2+ poisoning is a serious medical issue that is in need of the development of more effective detection techniques and improved selective chelating agents for its treatment.
During the last decades, the development of magnetic relaxation based assays or sensors have gain special attention due to their sensitivity, low detection limits, and mostly, due to their high signal-to-noise ratio (SNR). Magnetic relaxation sensors response signal depends exclusively on the behavior of the magnetic material on a specific environment;
The negligible magnetic susceptibility of practical samples allows magnetic relaxation sensors to generate background-free detection in complicated matricies.11 However, despite the great potential of these assays, and the fact that this type of sensing technique addresses the most important requirement for sensor technologies; the most commonly used magnetic relaxation assays still have several unaddressed limitations. To the moment, the biggest challenges for the development of these types of assays are measurement costs, time consuming sample preparation, slow analyte response rates and the sample incubation times necessary to resolve substantial changes in analyte interaction-dependent response signal.12 This work explores the advantages of magnetic particle spectrometry (MPS) for magnetic-based sensing applications. Here is presented the study of the ability of MPS to discern between the chelation ability of two chemically design Zn doped superparamagnetic iron oxide nanoparticle (Zn/IONP) towards Pb2+. The results show promising potential of MPS for the development of fast response sensor technologies that
64
can be used for environmental and biological systems, as well as for the study of new chelation agents for chelation therapy.
Iron oxide nanoparticles (IONP) are biocompatible materials that provide the opportunity of easily fine tuning their properties to make them suitable for sensing and biomedical applications.13-15 This work presents the surface modification of 18 nm Zn- doped superparamagnetic IONP with β-cyclodextrin-EDTA-TMS (Zn/IONP-EDTA-β-
CD) and TMS-EDTA. Previous studies have shown improved selectivity of β-cyclodextrin towards Pb2+ among other biologically relevant cations, and we were interested in exploring this results with MPS.16 Moreover, Zn-doped magnetite spherical nanoparticles have shown increase saturation magnetization (Ms) due to the incorporation of 13% Zn- dopant.17 When Zn2+ ion are incorporated in the unit cell of a spinel structure (x<0.4), they tend to occupy the tetrahedral sites. This effect induces the partial removal of antiferromagnetic coupling interaction between Fe3+ ions in the tetrahedral and octahedral
18,19 sites, significantly enhancing the Ms value. The Ms enhancement of nanomaterials with this bimetallic combination, make this structure an ideal candidate for the development of magnetic assays that produce magnetic relaxation changes due to analyte recognition.
This work proposes the use of magnetic particle spectrometry (MPS) to test the ability of various ligands to detect different concentrations of Pb2+ using a target-mediated clusters formation of surface modified Zn/IONP, that produce an effect on the relaxation magnetization dynamics of the nanoparticles. The aggregation and cluster formation of the nanoparticles is directly correlated to the concentration of the analyte of interest, facilitating the development of a sensing device that can distinguish between different Pb2+ concentrations in a quick, easy to use and sensitive manner.
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Magnetic particle spectrometry was developed for the assessment of the magnetic suspension suitability for magnetic particle imaging (MPI).20 However, recent studies have explored its advantages (sensitivity, rapid measurements and ability to provide quantitative determination) for different applications.21 MPS, also known as magnetic particle relaxometry, works by applying a strong magnetic field gradient that magnetically saturate the IONPs within the sample. The selective saturation of nanoparticles that participate on the signal generation within the sample, is generated at the field-free region (FFR). This region is a location in space at which the magnetic field is zero. At that point the nanoparticles are able to respond to a superimposed AC driving magnetic field that periodically drives IONP magnetization in and out of saturation.22 This process induces a signal in the receive coils that is transformed to the Fourier domain to observe a harmonic response (scheme 3.1.1).23
The MPS instrument allows the study of the relaxation dynamics of magnetic nanoparticles through a combination of multiple key components: a high power, low- distortion excitation transmit chain, a low-noise receive chain that rejects excitation field feedthrough and amplifies the harmonic signal from the IONP, and an electromagnetic shield to reject ambient interference (Scheme 3.1.2).
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Scheme 3.1.1. (a) Schematic diagram of the magnetic particle relaxometer. (b) Illustration of the signal generation process in the magnetic particle relaxometer.
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Scheme 3.1.2. Schematic representation of MPS instrument and signal generation (a) MPS instrument picture. (b) Representation of the external magnetic field application on the sample. (c) Diagram of instrument components.
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The signal response from the IONP comes from a combination of Brownian and
Néel relaxation mechanisms (Scheme 3.1.3a).24,25 The shape of the magnetization reversal is the response of IONP and is represented by a one-dimensional point-spread function
(PSF).26 The PSF response shape, height, position and half-width directly correspond to the relaxation magnetization dynamics of the nanoparticles. IONP are characterize by their ability to align with an easy axis that is the energetically favorable direction of spontaneous magnetization. The opposite direction along the easy axis are separated by an energy barrier (∆퐸) that the IONP must overcome in order for the magnetization reversal process to take place. As the IONP become smaller in size, the ∆퐸 for magnetization reversal decreases reaching superparamagnetism. For these particles, there is a possibility that the magnetic moment of a single IONP hops from one energetically favorable direction to another (Scheme 3.1.3b). The time between two consecutive flips is known as Néel relaxation time (휏푁). Néel relaxation occurs when the thermal energy overcomes the anisotropy energy barrier, leading to a rotation of the internal magnetic moment. Moreover, when this particle is exposed to an external magnetic field H, the 휏푁 decreases by the
Zeeman energy (Ez) as shown in Scheme 3.1.3c. Furthermore, if the sample aggregates, promoting the formation of clusters, the local dipolar magnetic fields produced by the surrounding IONP begin to alter the effective magnetic field on each particle. This phenomenon, reduces the Zeeman energy to Ez’, affected by the overall dipolar field generated by neighboring IONPs (Scheme 3.1.3d).
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Scheme 3.1.3. Schematic representation of the relaxation mechanisms that contribute to the MPS signal (a) Néel and Brownian relaxation. Representation of the energy barrier that governs the rotation of the magnetic moment of IONPs (b) IONP relaxation without the interruption of an external magnetic field, (c) IONP relaxation with external magnetic field,
(d) Effect on the relaxation mechanisms of clusters with and external magnetic dipolar field phenomenon.
70
On the other hand, for bigger nanoparticles the Brownian relaxation process dominates. Brownian relaxation or rotation diffusion refers to the physical rotation of the particle with respect to the surrounding fluid.27 For these particles the effective relaxation time is dominated by Brownian relaxation mechanism making (휏 ≈ 휏퐵) . Either way, MPS tracers (magnetic nanoparticles) achieve magnetic reversals with an effective relaxation time (휏) that comes from the contribution of both processes (Brownian and Néel relaxation). Scheme 3.1.3 shows IONP magnetization reversal mechanisms that contribute to the generation of the MPS. Under the application of a fixed applied frequency and time, this signal will be dependent on the particle size, interactions, surface functionalization and surrounding environment.20 On this assay, IONPs are specifically functionalized with β- cyclodextrin to selectively recognize and complex Pb2+.28 This complexation occurs by the multivalent binding of Pb2+ with ligands between different particles, promoting the aggregation and formation of clusters. The formation of clusters restricts the nanoparticles movements, consequently, suppressing the Brownian relaxation contribution to the MPS response signal (Scheme 3.1.4). The ability of Zn/IONP-EDTA-β-CD to complex Pb2+ and sensitively discern between different concentrations in the ppb range was successfully explored and compared with Zn/IONP-TMS-EDTA. Moreover, the ability of the technique and the chemically designed Zn/IONP to discern between other biologically relevant ions like Ca2+, Zn2+ and Fe2+ was also explored. The ability of MPS to provide reproducible changes in the relaxation dynamics of the dispersed and aggregated particles in small periods of time, make this assay ideal for the development of magnetic sensors. These results present the potential of MPS as a sensing technique for the determination of Pb2+,
71
and for the study of promising chemically design IONPs that can be used as new chelating agents to treat heavy metals poisoning.
Scheme 3.1.4. Schematic representation of the Pb2+ MPS sensor. A decrease in MPS response is observed upon the aggregation of Zn/IONP-EDTA-β-CD in the presence of Pb2+ ions. The vials show the aggregation of the sample caused by the addition of 10 ppb of Pb2+.
3.2 Methods
3.2.1 Synthesis of Zn Doped Superparamagnetic Iron Oxide
Nanoparticles
To synthesize iron oxide nanoparticles with spherical shape, Fe(acac)3 (2.2 mmol),
ZnCl2 (2.1mmol), oleic acid (12.7 mmol), oleylamine (24.3 mmol), trioctylamine (2.3
72
mmol) and dioctyl ether (6.65 mmol) were mixed in a 50 mL round bottom flask at 70 °C for 1 h under Ar atmosphere. The reaction was then heated slowly to 300 °C (3 °C/min) and maintained under reflux for 1 h. The resulting nano-spheres were precipitated with a
1:1 ethanol: toluene mixture and centrifuged for 20 min at 7000 rpm for the isolation of the
Fe3O4 nano-spheres with an average size of 18 nm diameter.
3.2.2 Synthesis of TMS-EDTA-β-CD Ligand
To synthesize the TMS-EDTA-β-CD ligand, first a β-CD/chitosan composite was fabricated. 3.0 g of β-CD and 0.5 g of chitosan were dissolved in 60 mL of a 1 M HCl solution on a three-neck round bottom flask. The mixture was left to react for 30 min at 85
˚C under constant stirring (1200 rpm). Then, 1.5 mL of 50% glutaraldehyde was added and left to react for 1.5 h. After 1.5 h, the pH was adjusted to 8 using 1 M NaOH and left to react 30 more min. The products obtained were cooled and washed with ethanol and distilled water. Finally, 70% of the composite was obtained after drying overnight in the vacuum oven.
The composite was used to allow the attachment of the β-CD to TMS-EDTA. 0.5 g of the composite was dispersed in 10 mL of 10% (v/v) acetic acid, and 3.0 g TMS-EDTA was suspended in 50 mL of methanol with under stirring (1200 rpm). The two solutions were mixed and stirred at 1200 rpm for 20 h at room temperature. Finally, the resulting products were washed with distilled water and ethanol, and dried overnight in a vacuum oven. A yield of 83% was obtained. Scheme 3.2.2.1 show the TMS-EDTA-β-CD ligand synthesized.
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Scheme 3.2.2.1. Synthesis of the TMS-EDTA-β-CD ligand.
3.2.3 Ligand Exchange of Zn/IONP-OA to Zn/IONP-TMS-EDTA
Oleic acid capped nanoparticles (4 mM, 4 mL) were mixed with a solution of ammonium hydroxide (NH4OH) in 1-butanol (1 M, 4 mL), triethylamine (1.4 mL), water
(0.5 mL), and N-[(3-Trimethoxysilyl)propyl] ethylenediamine triacetic acid trisodium salt
(TMS-EDTA) (100 μL) on a 20 mL glass vial. The mixture was left to react for 1 h in a homogenizer (7000 rpm). The product was centrifuged at 7000 rpm for 20 min. The supernatant was removed and the particles were re-dispersed in water.
3.2.4 Ligand Exchange of Zn/IONP-OA to Zn/IONP-EDTA-β-CD
The oleic acid capped nanoparticles (4mM, 4mL) were mixed with a solution of ammonium hydroxide (NH4OH) in 1-butanol (1 M, 4 mL), triethylamine (1.4 mL),
Millipore water (0.5 mL), and the TMS-EDTA-β-CD ligand (4mM, 100 μL) in a 20 mL glass vial. The mixture was left to react for 1 h in a homogenizer (7000 rpm). The product was centrifuged at 7000 rpm for 20 min. The supernatant was removed and the particles were re-dispersed in water (Scheme 3.2.4.1).
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Scheme 3.2.4.1. Schematic representation of the ligand exchange.
3.2.5 Magnetic Particle Spectrometry (MPS) Pb2+ Nanosensor
The magnetic particle spectrometry measurements were performed by preparing five solutions of Zn/IONP-TMS-EDTA and Zn/IONP-EDTA-β-CD (0.5 mg/0.450 µL) respectively. Each solution was incubated with Pb2+ in order to generate solutions in 0 ppb,
5 ppb, 15 ppb, 50 ppb, 100 ppb and 500 ppb. These solutions were vortex mixed for 5 s and then placed on a custom made x-space magnetic particle relaxometer.23 Measurements were collected at 30 s for 5 minutes.
3.3 Results and Discussion
3.3.1 Characterization
Transmission electron microscopy (TEM) analysis confirmed a spherical morphology and mono-dispersion of Zn/IONP-EDTA-β-CD with a median core size of
17.8 ± 0.7 nm (Figure 3.3.1.1), which agrees with the results obtained from dynamic light scattering (DLS). DLS shows a total hydrodynamic diameter of 18.2 nm for Zn/IONP-OA and 19.8 nm 20.1 nm after its surface modification with TMS-EDTA and TMS-EDTA-β-
CD respectively (Figure 3.3.1.2).
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The infrared spectroscopy spectra (FTIR) confirmed the surface functionalization with TMS-EDTA-β-CD, showing the appearance of a broad adsorption band located in at
3298 cm-1 corresponding to the stretching vibration of OH and adsorption bands at 2920 cm-1 and 2860 cm-1 correspond to the stretching of the C-H. The peaks at 1150 cm-1 and
1027 cm-1 correspond to the antisymmetric (O-C-O) vibrations from the composite, and the peak at 693 cm-1 to the skeleton vibrations of the β-CD. Moreover, the peak at 1610 cm-1 corresponds to the C=O stretching vibration of –NHCO- that demonstrated the presence of EDTA in the composite. On the other hand, the disappearance of the adsorption bands at 2913 cm-1 and 3031 cm-1 that correspond to the stretching vibrations of C-H bonds in the oleic acid ligand confirm the modification of the surface capping ligand. (Figure
3.3.1.3).
The crystallographic structure of Zn/IONP-EDTA-β-CD was evaluated using X- ray diffraction (XRD) (Figure 3.3.1.4). The field-dependent magnetic behavior was measured at 300 K, and the Ms value was determined to be 123 emu/f Fe. (Figure 3.3.1.5).
The surface charge of the particles in aqueous solution at pH 9 was determined to be negative (Figure 3.3.1.6).
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Figure 3.3.1.1. Transmission electron microscope images of magnetite spherical Zn/IONP. (a) Zn/IONP-OA and (b) Zn/IONP-EDTA-β-CD.
Figure 3.3.1.2. Dynamic light scattering characterization of Zn/IONP-OA of Zn/IONP-TMS- EDTA and Zn/IONP-EDTA-β-CD.
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Figure 3.3.1.3. FT-IR spectra of Zn/IONP-OA, Zn/IONP-TMS-EDTA and Zn/IONP-EDTA-β-CD.
Figure 3.3.1.4. The powder X-ray diffraction patters of the synthesized
Zn/IONP-TMS-EDTA and Zn/IONP-EDTA-β-CD.
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Figure 3.3.1.5. Field-dependence magnetization curve of Zn/IONP-TMS-EDTA and
Zn/IONP-EDTA-β-CD obtained at T= 300 K.
Figure 3.3.1.6. Z-potential measurements of Zn/IONP-EDTA-β-CD at various pH. Error bars correspond to n=3.
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3.3.2 Magnetic Particle Spectrometry Volumetric Sensor
MPS volumetric sensors are based on the change in Brownian relaxation in IONP due to the formation of clusters. When IONP cross-link upon the recognition and binding to a specific analyte, these clustered particles experience suppression of their capabilities to undergo through Brownian relaxation mechanisms. This work presents an assay that is based on the formation of clusters of Zn/IONP-EDTA-β-CD caused by the recognition of
Pb2+ resulting in capping ligand-analyte interactions, which results in a larger phase lag φ detected by the harmonics (longer effective relaxation time (휏)), and consequently a decrease in the MPS response signal. Within the assumption that Néel and Brownian relaxation processes are completely independent, the effective relaxation time is dictated by:
휏 휏 휏 = 푁 퐵 eq. 3.3.2.1 휏푁+휏퐵
Thus affecting the Brownian relaxation of the IONP will result in a direct effect on the effective relaxation of the sample.29
3.3.3 pH Dependence Study
The immobilization of this chemically design capping ligand, facilitates the aggregation of nanoparticles and formation of clusters upon chelation of Pb2+ by two β-CD attached in different Zn/IONPs. Upon the encounter of Pb2+ ions with β-CD, a coordination number of four, with the ligands arranged in a square-planar arrangement is produced
(Scheme 4.3.1).30 Previous studies showed that Pb2+ forms complexes with β-CD only in solution with medium alkalinity, and that complexation is less favorable on a media with
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pH<9.31 For the determination of the optimum pH for performing the analysis, Zn/IONP-
EDTA-β-CD showed stability on a pH range from 7-10. MPS and DLS were used to test the formation of clusters at different pH values. Results showed no significant changes in
MPS response of Zn/IONP-EDTA-β-CD from pH 7-10. However, significant changes in delta MPS (ΔMPS) were observed upon the addition of 15 ppb of Pb2+ in the same pH range. This suggested a dependence between the pH of the media and the capability of the
Zn/IONP-EDTA-β-CD to complex Pb2. At pH values larger than the zero-point charge, the secondary hydroxyl groups of the peripheral side of β-CD get deprotonated, producing a negative charge on the surface of the particle, that have strong coordinative affinity towards positively charged Pb2+ (Figure 3.3.3.1). The electrostatic forces of attraction allow the deprotonated hydroxyl groups to capture the Pb2+ through surface complexation, forming chelate complexes.32 Also, DLS measurements confirmed an increase in hydrodynamic size that match with the formation of clusters of two to three nanoparticles. These results support the performance of the analysis at pH=9. Higher pH was not preferred, since pH>10 promote the formation of Pb(OH)2 complexes, which will decrease the efficiency of the analysis.
Scheme 3.3.3.1. Representation of the complex formation between Pb2+ and β-
CD forming a preferred square planar arrangement.
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Figure 3.3.3.2. pH effect study. (a) Stability of Zn/IONP-EDTA-β-CD. (b) Zn/IONP- EDTA-β-CD after 15 min incubation with 15 ppb of Pb2+. (c) ΔMPS response showing a decrease in the MPS signal due to the formation of clusters at various pHs after incubation with 15 ppb of Pb2+ for 15 min. (d) DLS measurements.
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3.3.4 Time Dependence Study
To determine the maximum time needed to resolve substantial changes on the MPS signal due to capping ligand-analyte interactions, 15 ppb of Pb2+ was incubated with a
([Fe]= 0.5 mg/450µL) Zn/IONP-EDTA-β-CD solution. The samples were vortex mix
(1200 rpm) for 3 s, followed by the immediate placement on the MPS instrument. Time dependence complexation study for 15 ppb of Pb2+ was performed every 20 seconds for 20 minutes. Results showed no further decrease of the MPS signal after five minutes of incubation with Pb2+. Five minutes was selected for the MPS sensing analysis.
Figure 3.3.4.1. Magnetic particle spectrometry time dependence response for the Zn/IONP-EDTA-β-CD incubation with 15 ppb of Pb2.
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3.3.5. Magnetic Particle Spectrometry Pb2+ Sensor
To study the sensing capabilities of MPS, samples of Zn/IONP-EDTA-β-CD [Fe]=
0.5 mg/450µL were incubated with various concentrations of Pb2+ (0 ppb, 5 ppb, 50 ppb,
100 ppb, and 250 ppb). The samples were vortex (1200 rpm) for 3 s, followed by the immediate performance of the MPS measurements. Measurements were collected each 20 s for 5 min. All measurements showed reproducible results with n= 3.
Decrease in MPS signal corresponds to the formation of nanoparticle clusters in the presence of Pb2+. The formation of clusters restricts the movement of the nanoparticles, consequently, suppressing the Brownian relaxation contribution to the magnetic relaxation response of the Zn/IONP-EDTA-β-CD. All the analyses were also performed using
Zn/IONP-TMS-EDTA. The comparison with another ligand allows evaluation of the capabilities of the instrument to discern between the chelation capabilities of different ligands. EDTA is a commonly use chelation ligand for Pb2+.
Results show the stability of the MPS signal at T= 300 s (5 min) for Zn/IONP-
EDTA-β-CD in the presence of all the various concentrations of Pb2+ (Figure 3.3.5.1).
Moreover, the MPS results show a decrease of the signal with the increase in analyte concentration, and the ability to discern between the different concentrations of Pb2+ in the level of concern for children (Figure 3.3.5.2). Similar results were observed with Zn/IONP-
TMS-EDTA (3.3.5.3 and Figure 3.3.5.4). These results confirm the capability of Zn/IONP-
EDTA-β-CD to chelate Pb2+, and the ability of this MPS assay to distinguish between the chelation abilities of different ligands. Figures 3.3.5.5 and Figure 3.3.5.6 show the calibration curves for Zn/IONP-EDTA-β-CD and Zn/IONP-TMS-EDTA respectively.
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Figure 3.3.5.1. MPS response of the incubation of Zn/IONP-EDTA-β-CD with various concentration of Pb2+ for 5 minutes.
Figure 3.3.5.2. MPS Pb2+sensor using Zn/IONP-EDTA-β-CD. (a) MPS response
at various concentrations of Pb2+. (b) MPS signal intensity showing the capability
of this assay to discern between various concentrations of Pb2+ in the level of
concern for children. Results were obtained with n= 3
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Figure 3.3.5.3. MPS response of the incubation of Zn/IONP-TMS-EDTA with different concentration of Pb2+ for 5 minutes. Error bars correspond to n= 3.
Figure 3.3.5.4. MPS Pb2+sensor using Zn/IONP-TMS-EDTA. (a) MPS response at various concentrations of Pb2+. (b) MPS signal intensity showing the capability of this assay to discern between various concentrations of Pb2+ in the level of concern for children. Results were obtained with n= 3.
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Figure 3.3.5.5. Calibration curve for Zn/IONP-EDTA-β-CD. Error bars correspond to n= 3.
Figure 3.3.5.6. Calibration curve for Zn/IONP-TMS-EDTA. Error bars correspond to n= 3.
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3.3.6 MPS as a Tool for Selective Determination of Pb2+
The absorption of Fe2+, Ca2+ and Zn2+ by chelation agents used in heavy metals poisoning, are the primary causes of the secondary effects of the treatment: anemia, bone marrow depression and teratogenic effects.34 For this reason, the development of selective chelation agents for treating heavy metal poisoning is of vital importance. Here is evaluated the selectivity of Zn/IONP-EDTA-β-CD and Zn/IONP-TMS-EDTA towards 50 ppb of
Pb2+, versus Fe2+, Ca2+ and Zn2+. Magnetic relaxation studies on Zn/IONP-EDTA-β-CD, showed a reproducible 10% decrease of the MPS signal in the presence of 50 ppb of Pb2+ in comparison with 48%, 52% and 44% for Fe2+, Ca2+ and Zn2+ respectively (Figure
3.3.6.1). The results suggest the ability of Zn/IONP-EDTA-β-CD to chelate the different metal ions with different affinities. These results differ with Zn/IONP-TMS-EDTA, which present similar ∆푀푃푆 percentages 54%, 54% and 55% and 43% for 50 ppb of Fe2+, Ca2+,
Zn2+ and Pb2+ respectively. Figure 3.3.6.2 shows the MPS data for Zn/IONP-TMS-EDTA.
These results suggest the ability of MPS to discern between the chelation ability of different ligands toward specific analytes. Moreover, support the ability of MPS to selectively determine small concentrations of analytes by using the volumetric sensor assay presented in this work.
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Figure 3.3.6.1. Zn/IONP-EDTA-β-CD MPS selectivity study (a) response after incubation with 50 ppb of different cations. (b) MPS signal intensity in response to different cations.
Figure 3.3.6.2. Zn/IONP-TMS-EDTA MPS selectivity study (a) response after incubation with 50 ppb of different cations. (b) MPS signal intensity in response to different cations.
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4.4 Conclusion
In conclusion, it was successfully shown that Zn/IONP-EDTA-β-CD and Zn/IONP-
TMS-EDTA have the ability to chelate Pb2+ and form clusters that can be monitored through MPS measurements. Moreover, this work showed a successful MPS assay that can be used to detect different concentrations of Pb2+ in the relevant range for point-of-care applications in children. Selectivity studies showed the capability of MPS to identify the ability of different ligands to chelate and identify small concentrations metal ions like Pb2+,
Fe2+, Ca2+, Zn2+. Overall, this work presents the advantages of MPS based assays, and showcases the technique as an attractive tool for the fast, easy to use and sensitive detection of different analytes in the presence of specifically design IONP. This work has the potential to accelerate the development of new IONPs for a safer heavy metal removal treatment.
4.5 References
(1) Nagajyoti, P. C.; Lee, K. D.; Sreekanth, T. V. M. Environ. Chem. Lett. 2010, 8 (3),
199–216.
(2) Imperato, M.; Adamo, P.; Naimo, D.; Arienzo, M.; Stanzione, D.; Violante, P.
Environ. Pollut. 2003, 124 (2), 247–256.
(3) Srivastava, N. K.; Majumder, C. B. J. Hazard. Mater. 2008, 151 (1), 1–8.
(4) Villalobos, M.; Merino-Sánchez, C.; Hall, C.; Grieshop, J.; Gutiérrez-Ruiz, M. E.;
Handley, M. A. Sci. Total Environ. 2009, 407 (8), 2836–2844.
(5) Wheeler, W.; Brown, M. J. Morb. Mortal. Wkly. Rep. 2013, 62 (13), 237–244.
(6) Järup, L. Br. Med. Bull. 2003, 68, 167–182.
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(7) Lanphear, B. P.; Hornung, R.; Khoury, J.; Yolton, K.; Baghurst, P.; Bellinger, D.
C.; Canfield, R. L.; Dietrich, K. N.; Bornschein, R.; Greene, T.; et al. Environ. Health
Perspect. 2005, 113 (7), 894–899.
(8) Wani, A. L.; Ara, A.; Usmani, J. A. Interdiscip. Toxicol. 2015, 8 (2), 55–64.
(9) Coull, B.; Nie, H.; Hu, H.; Schwartz, J. Environ. Heal. 2009, 19 (1), 111–120.
(10) Flora, S. J. S.; Pachauri, V. Int. J. Environ. Res. 2010, 7 (7), 2745–2788.
(11) Zhang, Y.; Yang, H.; Zhou, Z.; Huang, K.; Yang, S.; Han, G. Bioconjugate Chem.
2017, 28 (4), 869–879.
(12) Chanpimol, S.; Seamon, B.; Hernandez, H.; Harris-love, M.; Blackman, M. R.
Chem. Commun. (Camb). 2014, 50 (27), 3595-8.
(13) Alcantara, D.; Lopez, S.; García-Martin, M. L.; Pozo, D. Nanomed. Nanotech.,
Biol. Med. 2016, 12 (5), 1253–1262.
(14) Laurent, S.; Forge, D.; Port, M.; Roch, A.; Robic, C.; Vander Elst, L.; Muller, R.
N. Chem. Rev. 2008, 108 (6), 2064–2110.
(15) Vallabani, N. V. S.; Singh, S. 3 Biotech. 2018, 8 (6), 1–23.
(16) Aswathy, B.; Avadhani, G.; Suji, S.; Sony, G. Front. Mat. Sci. 2012, 6 (2), 168-
175.
(17) Bauer, L.; Situ, S.; Griswold, M. A.; Samia, A. C. S. Nanoscale 2016, 8 (24),
12162-9.
(18) Szczerba, W.; Jan, Z.; Safonova, O.; Shmeliov, A.; Nicolosi, V.; Schneider, M.;
Granath, T.; Oppmann, M.; Mandel, K. Phys. Chem. Chem. Phys. 2016, 18, 25221.
(19) Nanoparticles, D. M.; Jang, J.; Nah, H.; Lee, J.; Moon, S. H.; Kim, M. G.; Cheon,
J. Angew. Chem. Int. Ed. 2009, 48, 1234 –1238.
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(20) Garraud, N.; Dhavalikar, R.; Maldonado-Camargo, L.; Arnold, D. P.; Rinaldi, C.
AIP Advances 2017, 7, 056730.
(21) Gandhi, S.; Arami, H.; Krishnan, K. M. Nano Lett. 2016, 16 (6), 3668–3674.
(22) Tay, Z. W.; Hensley, D. W.; Vreeland, E. C.; Zheng, B.; Conolly, S. M. Biomed
Phys Eng Express. 2017, 3 (3), 1–21.
(24) Bauer, L. M.; Hensley, D. W.; Zheng, B.; Tay, Z. W.; Goodwill, P. W.; Griswold,
M. A.; Conolly, S. M. Rev. Sci. Instr. 2016, 87, 055109.
(25) Ferguson, R. M.; Minard, K. R. Med. Phys. 2011, 38 (3), 1619–1626.
(26) Perreard, I. M.; Reeves, D. B.; Zhang, X.; Kuehlert, E.; Forauer, E. R. Weaver, j.
B. Phys Med Biol. 2014, 59(5), 1109–1119.
(27) Arami, H.; Krishnan, K. M. IEEE Tran.s Magn. 2013, 49(7), 3500–3503.
(28) Arami, H.; Ferguson, R. M.; Khandhar, A. P.; Krishnan, K. M. Med. Phys. 2013,
40 (7), 071904.
(29) Aswathy, B.; Avadhani, G. S.; Suji, S.; Sony, G. Front. Mater. Sci. 2012, 6 (2),
168–175.
(30) Wu, K.; Su, D.; Saha, R.; Wong, D.; Wang, J. Journal of Physics D Applied Physics
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Chapter 4. Magnetic Particle Spectrometry as
a Tool for the Monitoring of the Uptake and
Translocation of Different Size Iron Oxide
Nanoparticles in Plant Systems
Manuscript in preparation for submission:
Author List: Navarreto-Lugo, M.†, Ju, M. † Wickramasinghe, S. McWhorter, A.; Samia,
A.C.S*. †: Equal author contribution
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Chapter 4. Exploring the Chelation-based Plant Strategy for Iron Oxide Nanoparticle
Uptake in Garden Cress (Lepidium sativum) using Magnetic Particle Spectrometry
4.1 Introduction
After decades of struggle, the remediation of iron deficiency in crops remains one of the strongest agronomic challenges.1,2 Even though iron is the fourth most abundant element on earth and in soil, under aerobic conditions and neutral to high pH (of the rhizosphere), the Fe3+ oxidation state is favored.3 This form of iron commonly promotes the formation of insoluble ferric oxide-hydroxides making it inaccessible for plant uptake.4
It is estimated that about 30% of the world’s cropland is too alkaline for optimal plant growth, and some staple crops, like rice, are particularly susceptible to iron deficiency, thereby requiring the need for continued research in developing iron based fertilizers.5
Crops like rice can be especially benefited by the development of iron based fertilizers.
Rice feeds more than half people in the world, is a front line crop against world hunger and one of the most popular gluten-free grains for people with celiac disease.6 Moreover, it is one of the preferred crops for the development of iron fortified goods for the prevention and treatment of iron deficiency anemia in regions with a rice based diet. This extensive list of reasons for the potential of rice, made relevant the development of iron additives that can enhance its production. Untreated iron deficiency promotes the development of lime induced-chlorosis, which is visibly manifested in the yellowing of plant leaves, which if left for a prolonged time can result in plant death.7 Evolution in plants has promoted the development of two principal mechanisms for solving this issue: the reduction based strategy and the chelation based strategy.8,9 These mechanisms have facilitated the absorption/uptake of iron. However it has also promoted the overload of iron uptake adding
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more possible hazardous expositions to the plants.10 The reduction based strategy or
Strategy I, IRT1 gene (specifically expressed in the epidermis of roots) encodes the ferric reductase and iron transporter involved in iron uptake.11 The problem comes from the lack of selectivity of the IRT1 gene which can also take up Zn, Mn, Co, Ni and Cd present in the growing media. This decreases the absorption of iron in a competitive assay.12–14 On the other hand, the chelation based strategy or Strategy II, is based on the release of chelators of the mugineic acid (MA) class named phytosiderophores. This chelator’s functionalities allow the absorption of Fe3+ through the formation of an Fe-MA complex, which favors the uptake of iron aided by the YSL (yellow stripe-like) family of transporters in the roots surface.15 However, even though these mechanisms have improved the iron uptake process in plants, a significant amount of cultivated soils still suffer from low iron availability, which force agronomists to use fertilizers as the standard approach to treat lime induced-chlorosis.16 This brings new challenges due to incorrect or over application of fertilizers that under aerobic conditions, promote the formation of reactive oxygen-based radicals as byproducts of Fenton reactions, which damage vital cellular constituents.17
Currently, iron in most of the correctly applied fertilizers still remain unavailable to plants due to several factors such as leaching and degradation by hydrolysis, insolubility and decomposition.18 For this reason, the development of chelated iron forms that help maintain iron availability and promote its uptake and translocation is of strong interest. Chelated iron fertilizers like iron ethylene diaminetetraacetic acid (Fe-EDTA) and iron ethylenediamine-N-N’-bis(o-hydroxyphenylacetic) acid (Fe-EDDHA) were showento be the most suitable carriers of iron, by improving its availability to plants, when compared with the non-chelated analogs.19,20 Most likely this is due to the similarities of chemical
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structure and functional groups with MA. However, even though these chelating ligands improve the mobilization of iron ions, the stability constant of this complex (ferric ion-
EDTA is 25.0)19 promotes the lability of this ligand inside plant conditions. The release of these organic molecules, as the metal ions are absorbed and translocated within the plant, have shown toxicity effects on cellular division, chlorophyll synthesis and plant development.21,22
Alternatively, recent studies demonstrated the potential of using iron oxide nanoparticles (IONPs) as fertilizers to address iron deficiency in plants and humans.22 The potential of this material has been explored in the treatment of iron deficiency anemia in adult patients since its FDA approval under the name of Feraheme in 2009.23 This is because, IONPs represent a potential carrier for the smart storage and translocation of adequate concentrations of iron ions, and also a promising nutritional tool for biological organisms. However, while some studies performed in plants have demonstrated significant enhancement in phenotypic traits like growth, biomass or chlorophyll production upon treatment with IONPs, other investigations have shown conflicting results.23–26 This phenomenon can be largely attributed to the wide variations in experimental parameters pertaining to IONP size, shape, surface coating, plant type and growth conditions, and variations in plant organisms between species.
We believe that the potential of this material can be optimized by the development of the correct recipe for each plant wherein the ingredients will consist of (1) small, monodispersed and stable IONPs at (2) a specific concentration, (3) at a specific start day for incubation time in (4) specific growing media. To address these requirements, we developed a synthetic approach to produce highly stable IONPs that mimic the chelating
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ligands found in commercial chelated iron fertilizers, promoting their uptake in plants and the slow release of nutritional iron through the different plant tissues.
However, in order to determine these specific parameters and provide the risk assessment of new fertilizer products, a systematic study of the effect of nanoparticles in plants needed to be performed. Currently, IONPs plant uptake studies have utilized atomic absorption spectroscopic (AAS) methods, which involve laborious strong acid digestion sample processing, and it still is unable to distinguish between plant uptake of intact IONPs versus leached iron ions. Here we effectively demonstrated that the plant uptake process and translocation of IONPs can be efficiently monitored using magnetic particle spectrometry (MPS). This is an important contribution to the ability to monitor the biotransformation and the fate of magnetic nanomaterials inside of biological systems, which is an area that still lacks methodologies that can detect, quantify and characterize.27
On this report, we present the use of MPS to monitor the absorption and translocation of various sized (10 nm and 20 nm) EDTA-coated iron oxide nanoparticles (IONP10-EDTA and IONP20-EDTA) in garden cress (Lepidium sativum) plants. Garden cress (GC) was picked for its short growth cycle and its nutritional value. We evaluated the IONP-EDTA effect on plant biomass, growth, and chlorophyll production, in comparison to effects of plant exposure with a commercial Fe-EDTA fertilizer. Our MPS studies were validated by
AAS, and we demonstrated that this method is reliable, sensitive, and an effective analytical tool for the development and study of IONP based fertilizers.
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4.2 Materials and Methods
4.2.1 Materials
Iron (III) chloride hexahydrate (98%), anhydrous iron (II) chloride (98%), oleic acid (90%), 1-octadecene (90%), trimethylamine (99%), toluene (HPLC grade, 99%), ethanol (HPLC grade, 90%), were purchased from SigmaAldrich (Milwaukee, WI, USA).
Ammonium hydroxide (14.8 M), hydrochloric acid (37%), and the iron reference standard solution (1000 ppm) for the atomic absorption spectroscopic analyses were purchased from
Fischer Scientific (Pittsburg, PA, USA). N-(trimethoxysilylpropyl) ethylenediaminetriacetate, trisodium salt, 35% in water was purchased from Gelest.
EDTA-chelated iron (13.20%) was purchased from Greenway, Biotech inc. All of the chemicals and reagents were used as received. The LED lamp (18 W) dual head plant growth light with LED colors: red (660 nm, 24 pcs) and blue (460 nm,12 pcs) was bought from LEDMEI (Pembroke, NC, USA). The garden cress seeds were bought from The
Sprout House (Lake Katrine, NY, USA).
4.2.2. Synthesis of Iron Oxide Nanoparticles (IONPs)
Spherical monodispersed oleic acid (OA) capped IONPs were synthesized by a thermal decomposition method. This synthesis involves a two-step process: the thermal decomposition of iron oleate to wüstite phase (FeO) nanoparticles and a later mild oxidation step that gives rise to the magnetite (Fe3O4) phase. For the formation of the wüstite nanoparticles, the synthesis parameters were controlled in order to produce samples with 10 nm and 20 nm hydrodynamic ratios. For the synthesis of 10 nm nanoparticles, iron oleate (5.8 mmol), oleic acid (3.2 mmol) and 18 mL of 1-octadecene were mixed in a 25 mL round bottom flask. The reaction mixture was heated following a rate of 3 ºC/m till it
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reached 320 ºC. Then was left to reflux for 1 h at this temperature. The final product was left to cool at room temperature and collected by centrifugation (7000 rpm, 20 min). For
20 nm nanoparticles, 12.7 mmol of oleic acid and 10 mL of 1-octadecene were used.
For the second step, the wüstite nanoparticles (1.67 mmol) were further slowly oxidized by mixing oleic acid (1.52 mmol), 20 mL 1-octadecene and 10 mg of trimethylamine N-oxide (oxidizing agent). The mixture was left to react under constant stirring for 2 h at 130 ºC, followed by reflux at 260 ºC for 1 h. The resultant IONP10-OA and IONP20-OA were isolated by the addition of 1:1 ethanol: toluene solvent mixture (30 mL) and subsequent centrifugation at 7000 rpm for 20 min.
4.2.3 Surface Functionalization of IONP with TMS-EDTA
The IONPs (4 mM, 4 mL) were mixed with solution of ammonium hydroxide
(NH4OH) in 1-butanol (1 M, 4 mL), triethylamine (1.4 mL), milli-Q water (0.5 mL), and
N-[3-(trimethoxysilyl)propy] ethylenediaminetriacetic acid trisodium salt (TMS-EDTA)
(100 μL) in a 20 mL glass vial. The mixture was left to react for 1 h in a homogenizer (7000 rpm). The resulting product was centrifuged at 7000 rpm for 20 min, the supernant removed and the particles are re-dispersed in milli-Q water.
4.2.4 Materials Characterization
A JEOL 1200CX transmission electron microscope (TEM) operated at 80kV was used to evaluate size and morphology. The software ImageJ was used to process the TEM data and measure the particle size using an average of 200 nanoparticles. High resolution
TEM (200 kV microscope, Tecnai G2 F20) was used to obtain images of the IONP inside of the leaf. TEM samples were prepared by adding a drop of the IONP diluted sample on a 50 mesh copper grid and allowed to dry. The particle size distribution was analyzed by
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dynamic light scattering (DLS) on a ZetaPALS particle size analyzer (Brookhaven, Upton,
NY, USA) at a scattering angle of 90°. Elemental analysis was accomplished using atomic absorption spectroscopy (AAS) Varian 220FS AA. The surface functionalization of the
IONP as evaluated using attenuated total reflectance ̶ Fourier transform infrared spectroscopy (ATR-FTIR) in a range of 500−4000 cm−1 using a Thermo Scientific Nexus
870 ATR-FTIR spectrometer. The powder X-ray diffractometer (PXRD) patterns of the samples were collected using a Rigaku MiniFlex X-ray powder diffractometer with Cu Kα radiation (γ = 0.154 nm).
4.2.5 Seed Germination and Plant Growth
Garden cress (Lepidium sativum) seeds were placed into a plastic container with 4 by 6 pots (2x2 cm). Each pot contained a total of ten seeds in 3 mL of hydroponic media
(volume that was kept through the entire growing cycle. The sample was kept at room temperature and placed under a red (660 nm, 24 pcs) and blue (460 nm, 12 pcs) LED light lamp (18 W) for 12 h. Five days after seedling emergence, samples were injected with 500 ppm of the various sized IONP-EDTA, and left to grow for five more days (total growing cycle of ten days). Garden cress plants exposed to tap water and EDTA chelated iron (Fe-
EDTA, commercial fertilizer) were used as control groups and grew simultaneously on every trial.
4.2.6 Length and Biomass Determination
At the end of the growing cycle, the plants were harvested and washed with milli-
Q water until all visible traces of IONP sample was detached from the external surface.
Then the plants were stretched, taped and measured. Followed by separation of the plant components (leaves, stems and roots). Each pot samples’ separated components (10x) were
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placed in small Eppendorf tubes and dried with a vacuum pump for 4 h. The dried samples were weighed to obtain the biomass. After this step, samples were used to perform the MPS analysis.
4.2.7 Chlorophyll Measurements
For chlorophyll measurements, an equivalent of 60 leaves (6 leaves per each plant in a 10 plants pot) were taken from sacrificial samples of: control, Fe-EDTA, IONP10-
EDTA and IONP20-EDTA. Each separate sample was placed in 5 mL glass vials and weighted by difference. Milli-Q water was added to each vial, and the samples were boiled for 10 min. After the 10 min, the water was removed. Ethanol (95%, 3 mL) was added and the samples were placed in a 50 °C water bath for approximately 1 h. Complete bleach of the leaves was observed. Chla and Chlb were separated by using a alumina column chromatography with 100% hexane, 90:10 hexane: acetone and a 80:20 hexane: acetone as stated in the Owen M. McDougal previously reported method.49 After the chlorophyll extraction UV-VIS measurements were performed.
4.2.8 Magnetic Particle Spectrometry Analysis
To study the absorption of IONPs in garden cress, an MPS analysis of sacrificial hydroponic media was performed for IONP10-EDTA and IONP20-EDTA. The hydroponic media solution was sacrificed since the first day of incubation with the treatments, till the end of the cycle. Each collected sample was vortex mix (1200 rpm) for 1 min and then aliquots of 450 µL were collected and placed into small eppendorf tubes to perform the
MPS analysis. The concentrations were calculated by using a calibration curve generated with each particle type (IONP10-EDTA and IONP20-EDTA) adjusting to the total initial volume. All analyses were performed in triplicate.
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For the study of the translocation of IONPs through the plant components, once the samples were cleaned, separated into leaves, stem and roots, dried and weighed, MPS measurements were performed. A total of 5 pots were evaluated per sample (control, Fe-
EDTA, IONP10-EDTA and IONP20-EDTA). This equals 50 sets of 6 leaves, 50 stems and
50 root samples per condition. The concentrations were calculated by using a calibration curve generated with each particle (IONP10-EDTA and IONP20-EDTA).
4.2.9 Elemental Analysis Using Atomic Absorption Spectroscopy (AAS)
Samples used for the MPS analysis, were finely 5.2.1ground in a tissue grinder to facilitate the digestion of the sample. For total leaf, stem and root Fe concentration, the samples were acid-digested at 150 °C for 12 h in a Teflon line vessel with 3 mL of 70% nitric acid. Finally, the samples were diluted with milli-Q water to 20 mL and analyzed for
Fe using flame AAS.
4.2.10 TEM Sample Fixation
For TEM analysis of the leaves, the plant sample was first rehydrated in 70%, 50% then 30% ethanol for 5 min. Then the sample was fixed with fixative (2.5% glutaraldehyde/
4% paraformaldehyde in 0.2 M cacodylate buffer) overnight at 4ºC. Fixation stage was done by washing the sample three times with sodium cacodylate buffer (0.2 M, pH7.3), for
5 min. Followed by removal of buffer solution, post-fixation was processed by adding 1% osmium tetroxide in water and was left for 60 min at 4 ºC. The sample was then washed two times with sodium cacodylate buffer solution (5 min each), rinsed with maleate buffer solution (pH 5.1) once, and stained with 1% uranyl acetate in maleate buffer for 60 min.
Uranyl acetate was then removed by washing the sample with maleate buffer three times.
Dehydration was then performed in 30%, 50%, 75%, and 95% ethanol solution for 20 min
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each at 4 ºC, followed by dehydration three times for 15 min dehydration in 100% ethanol at room temperature. Dehydration was settled with washing the sample three times with propylene oxide for 15 min each. Propylene oxide was removed by addition of 1:1 and 1:2 mixtures of propylene oxide/eponate12 medium at room temperature overnight foe each.
The final solution was then changed to pure eponate12 medium for overnight infiltration at room temperature. On the following day, the sample was polymerized for 24 h for the embedding process.
To be observed in TEM, the embedded sample was cut into sections following two different processes; i) Semi-thin sections of 1 µm were cut with a diamond knife, stained with Toluidin Blue, and then observed with a Leica DM5500 light microscope, and ii)
Ultra-thin sections of 85 nm were cut with diamond knife, stained with uranyl acetate and lead citrate, and then observed with a Tecnai G2 SpiritBT, electron microscope operated at 60 kV.
4.2.11 Statistical Analysis
All of the experimental results were evaluated using an analysis of variance
(ANOVA). The data was examined using Microsoft Excel and Origin. Significance was tested as compared between all different treatments with a p < 0.05.
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4.3 Results and Discussion
4.3.1 Characterization of Iron Oxide Nanoparticles (IONP)
Iron oxide nanoparticles capped with EDTA (IONP-EDTA) were fabricated by a thermal decomposition method followed by a ligand exchange (Figure 4.3.1.1a). IONP-
EDTA transmission electron microscopy (TEM) analysis confirmed a spherical morphology and mono-dispersion with a median core size of 9.9 ± 0.7 nm and 19.5 ± 1.1 nm (Figure 4.3.1.1b). The infrared spectra (FTIR) confirmed the surface functionalization showing the appearance of a broad adsorption band located in at 3298 cm-1 corresponding to the stretching vibration of OH and the 1651 cm-1 of the new carbonyl on carboxylic acid groups in EDTA. Moreover, the disappearance of the adsorption bands at 2913 cm-1 and
2842 cm-1 that correspond to the stretching vibrations of C-H bonds in the oleic acid ligand confirm the successful surface modification (Figure 4.3.1.1c). The superparamagnetic magnetite character was confirmed through analysis of its crystal structure using vibrating sample magnetometer (VSM) measurements (Figure 4.3.1.1d) and X-ray diffraction
(XRD) (Figure 4.3.1.1e). Finally, dynamic light scattering (DLS) analysis confirmed a hydrodynamic diameter of 14.4 nm and 23.8 nm after the surface modification with EDTA
(Figure 4.3.1.1f).
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Figure 4.3.1.1. Characterization of various IONP-EDTAs use for garden Cress treatments. (a)
Scheme of the functionalization of IONPs from oleic acid to TMS-EDTA capped IONP. (b) High resolution transmission electron microscope (TEM) images of IONP-EDTA-capped of 10 nm and 20 nm, respectively. Scale bars are 100 nm; (c) Fourier transform infrared (FTIR) spectra; (d)
Vibrating Sample Magnetometer spectra; (e) powder X-ray diffraction patterns; (f) Dynamic light scattering results for IONP-EDTA before and after ligand exchange.
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4.3.2 Experimental Growing Cycle for Garden Cress
To evaluate the effect of IONP-EDTA on the phenotypical traits of garden cress, this plant was exposed to an experimental cycle of 10 days in hydroponic media with a nanoparticles incubation time of 5 days. Similar to a typical green-leaf plant, the growing cycle of garden cress plant begins with a flat, brown colored seed. After one day, the seed will sprout and start producing a tiny, immature plant called rooting/sprouting. The further growth of the plant transforms it into a mature plant by day 4, which is the stage in which the appearance of leaves is evident. At this stage it is suitable for the plants to be incubated with the IONP-EDTA. At early maturing stages, plants require additional nutrients intake
(especially if the growing media is lacking them) in order to facilitate growing and photosynthetic processes. This justifies the addition of IONP-EDTA on the 5th experimental day (Figure 4.3.2.1).
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Figure 4.3.2.1. Experimental life cycle of garden cress. From seedling (Day 1) to translocation of IONPs in mature plant (Day 10). Garden cress was incubated in hydroponic media, with 500 ppm of IONP10-EDTA or IONP20-EDTA for the last five days.
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4.3.3 Effect of IONP in Length and Biomass of Garden Cress
IONP10-EDTA and IONP20-EDTA treatments were evaluated after 10 days of seedling
(5 days of incubation time with 500 ppm of IONP-EDTA). Figure 4.3.3.1a shows the method used to measure the length of the samples, and Figure 4.3.3.1b the length measurement values obtained from the shoot and root of the samples after 5 days of exposure with the different treatments. The shoot length showed no significant difference
(P<0.05, n= 50) among the control, Fe-EDTA and IONP10-EDTA, even though, IONP20-
EDTA showed slightly higher length value. However, even though, no significant changes were observed for the length measurements, the phenotypic images showed an increase in plant thickness for those plants exposed to the IONP treatments (Figure 4.3.3.1c). This characteristic was reflected on the biomass measurements, in which a more evident difference was observed, reflecting an 8x fold increase in biomass for those plants treated with IONP20-EDTA versus the control or Fe-EDTA (Figure 4.3.3.1d). Comparable length measurements between garden cress experimental and control groups reflect no signs of toxicity for IONP10-EDTA or IONP20-EDTA treatments. Biomass increase in plants exposed with IONP was also observed by Sheykhbaglou et al and Dhoke et al.28,29 This group, observed the increase in biomass of soybean plants and mung after treatment with
IONP using a foliar application method. The significantly higher biomass results observed in our work could be justified by the application of the treatments in hydroponic growing media, which provides more moving channels for the nanomaterials to reach all plant components.
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Figure 4.3.3.1. Phenotypic observations of the effect of incubation with IONP10-EDTA or
IONP20-EDTA on garden cress harvested after 10 days’ experimental cycle. (a) Length measuring method. (b) Length measurements. (c) Representation of the harvested plants exposed to the different treatments. (d) Biomass measurements. Error bars represent standard errors (n= 50). Each n group was composed of 10 plants. Asterisk show statistical significance with a p<0.05.
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4.3.4 Effect of IONP in Chlorophyll Concentration
Besides an increase in biomass, there was also an increase in chlorophyll production. Under normal circumstances (like those that apply to the control group), most green plants can convert only 2% to 4% of the available energy in radiation into new plant growth via photosynthesis.30 More specifically, chlorophyll uses sun light to convert carbon dioxide and water into oxygen and glucose. Oxygen and glucose are further used to supplement plant growth. However, in order for chlorophyll to be produced, there is needed a lot more than sunlight and water. Plants need various minerals, with special importance to iron (Fe) due to its multiple participation through the process. In plant cells, at least 80% of Fe can be found in plant leaves, and it is known to participate in all electron transfer complexes such as photosystem I (PSI), photosystem II (PSII), cytochrome complex, and ferredoxin molecule which all are photosynthetic apparatus.31 Both chlorophyll a and b play important roles in photosynthesis by absorbing light in the antenna complex via PSI and PSII, with consequent electron transport that is enabled mostly by Fe-S proteins.32
These Fe-S proteins are composed of Fe-S clusters that are known as ferredoxins which are characterized by their very high negative redox potential. These proteins are also used as carrier proteins that work as sensors and serve to avoid the toxicity associated with free iron at the same time that it allows the transport of Fe at lower intracellular concentrations.
Fe-S electron transfer and its role in chlorophyll production are the initial steps in the process of production of plant energy, growth and development.33 Previous literature reported that a 30% enhancement in photosynthesis can only produce an increase of relative
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growth of 10%. This results were rationalized by the limitations of plants systems to process big amounts of carbohydrate availability in short periods of time.30
Our studies demonstrated an increased production of total chlorophyll in the plants exposed to IONP20-EDTA, which supports the phenotypical observations. Moreover, a higher concentration of Chla relative to Chlb was obtained (Figure 4.3.4.1). These suggests an efficient production of Chla which we attribute to the possibility of very slow leaching of Fe from IONP20-EDTA that was able to translocate to the leaves as demonstrated in the
TEM images (Figure 4.3.4.2). This slow leaching of Fe, provides a slow but continuous supply of Fe to the Fe-S proteins that facilitates electron transfer needed in the production of Chla, which will consequently boost the production of energy for the plant (Figure
4.3.4.3). These results suggest that IONP20-EDTA can promote a significant increase in plant thickness and chlorophyll production by providing very small but continuous amounts of suitable concentrations of Fe. Moreover, even though TEM images proved the translocation of IONP through aerial components, it does not provide quantitative information, which important information for understanding the effect of these materials in biological systems. Nanoparticle absorption in plants has been a controversial topic in agricultural sciences due to the contradictory set of information about their absorption and translocation in different plant species and under different growth conditions.34–37
However, the exploration of IONPs as a potential fertilizer agent for some plant species, still is a promising area of study, an idea that is in fact supported by the phenotypic response results obtained after incubation of garden cress plants with IONP20-EDTA, and observed by other groups in various plant species.38–40
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Figure 4.3.4.1. Chlorophyll production in garden cress as a result of IONP-EDTA treatments.
(a) Image of representative leaves for each group. Greener leaves were observed in Garden cress plants exposed to IONP20-EDTA. (b) Spectra of UV-VIS measurement for the separated
Chla and Chlb. (c) The effect of IONP-EDTA on concentration of chlorophyll a, b and total chlorophyll amount.
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Figure 4.3.4.2. TEM images of different parts of a cross sectional areas on a leaf of garden cress exposed to IONP -EDTA. Red arrows point to translocated IONP -EDTA on plant cells. Red 20 20 inset show the identification of Fe by elemental analysis.
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Figure 4.3.4.3. Schematic representation for the uptake of Fe and presumed uptake and translocation of IONP-EDTA.
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4.3.5 Magnetic Particle Spectrometry
Here magnetic particle spectrometry is presented as a tool for the study of IONP in plant systems. In this study, MPS was used for the monitoring of the absorption of IONPs in sacrificial samples of hydroponic media, and their translocation inside of the various plant components. The magnetic particle spectrometer (also known as magnetic particle relaxometer) used for the analysis was a custom x-space magnetic particle relaxometer at
Case Western Reserve University (Figure 4.3.5.1a). This relaxometer is a sensitive tabletop system that facilitates the monitoring of superparamagnetic iron oxide nanoparticles in plant systems by requiring minimal sample preparation. This statement applies to samples in solution and inside of biological systems. This work shows the diversity of the capabilities of MPS analysis by measuring the concentration of IONP in solution (for the study of the absorption of IONP from hydroponic media) and also inside of the plant parts
(for translocation studies) for which the sample was simply separated in different eppendorf tubes and later placed into the sample holder (Figure 4.3.5.1b). Once the sample is in the sample holder it can be placed in the instrument where the data acquisition and reconstruction is controlled using a 16-bit DAQ and MATLAB with a total experimental time of 30 seconds per measurement. MPS is a zero-dimensional MPI scanner that applies a sinusoidal magnetic field (with sufficiently large amplitude) to superparamagnetic iron oxide nanoparticles within the sample, this periodically drives the IONP magnetization into and out of saturation and collects information from the field-free region (FFR) where the magnetic field is zero (Figure 4.3.5.1c).41 Since the particles have a nonlinear magnetization curve, the magnetization reorientation that takes place in the FFR induces a
42 signal in the receive coils at the drive frequency f0 and higher harmonics. MPS allows
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the study of the relaxation dynamics of magnetic particles through a combination of multiple key components: a high power, low-distortion excitation transmit chain, a low- noise receive chain that rejects excitation field feedthrough and amplifies the harmonic signal from the IONP, and an electromagnetic shield to reject ambient interference (Figure
4.3.5.1d).43 After their exposure to a sinusoidal magnetic field, in order to generate the signal, IONP must undergo a magnetization reversal by using a combination of Brownian and Néel relaxations.44,45 The shape of the magnetization reversal is the response of IONP and is represented by a one-dimensional point-spread function (PSF) as shown in the MPS
46,47 characterization of IONP10-EDTA or IONP20-EDTA (Figure 4.3.5.1e). The MPS response, depends on the size, capping ligand, surrounding environment and
48 concentration. IONP20-EDTA showed a higher MPS signal due to and increased surface magnetization (Ms) and a reduced disordered layer radius in comparison with IONP10-
EDTA. Particle calibration curves showed a detection limit as low as 0.02 ppm (Figure
4.3.5.1f).
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Figure 4.3.5.1. Schematic representation of MPS analysis. (a) MPS instrument picture. (b) Experimental sample preparation prior to MPS analysis. (c) Application to a sinusoidal magnetic field to the sample and field free region (FFR) necessary for signal generation. (d) Representation of signal generation. (e) Point spread function (PSF) examples showing the
MPS response of the different treatments. (f) Calibration curves obtained from IONP10-EDTA and IONP20-EDTA. Error bars correspond to with n= 5 and p<0.05.
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4.3.6 Magnetic Particle Spectrometry Monitoring of IONP Uptake
For quantification of the total Fe absorbed by the plant, the decrease in concentration change was monitored in the hydroponic growing medium using MPS
(Figure 4.3.6.1). The results showed that IONP10-EDTA was taken up faster in the first 24 h after incubation. On the other hand, IONP20-EDTA showed a continuous uptake through the course of the 5 days. These results suggest a correlation of the particle size and the uptake of IONP. Comparing the IONP20-EDTA MPS response of the hydroponic media with the phenotypic observations of enhanced biomass and chlorophyll production several suggestions can be made. Previous studies showed that Fe ions present in the rhizosphere are recognized by FRD3 which is a protein expressed in the roots of the vasculature. This protein is in charge of complexing Fe with citrate in order to form a Fe-Citrate complex that helps avoid the precipitation of Fe3+ species and the formation of oxidizing forms on its path through the xylem of the stem (Figure 4.3.4.3).17 Non-chelated forms of Fe produce toxic effects due to its Fenton reaction by products, which indicates that this chelation strategy is the mechanism that the plant has developed for the uptake and translocation of the maximum amount of iron from the rhizosphere while protecting themselves from the toxic effects of iron overload. However, this chelation strategy is not enough to protect the plant in several instances, due to the lability of the Fe-ligand bond, overload of iron in the plant often cannot be controled by the presence of chelating ligands. In our study, we engineered an IONP capped with TMS-EDTA because of the reported favorability of Fe-
EDTA uptake in plants, and because silane terminated ligands provide three anchor groups for the capping ligand on the IONP. These three groups favor a stronger bond and enhanced protection of the particle. This promotes a slower release of Fe ions through the
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translocation process that keeps providing the plant with iron without the necessity of
multiple fertilizing cycles, and also, helps to considerably reduce the release of free toxic
-3 EDTA ligands. We hypothesize, that EDTA functionalities mimic citrate (C6H5O7 ) acting
as a surrogate for nanoparticles that allows them to be absorbed and translocated in aerial
plants. This idea supports our results of a 98% uptake of IONP10-EDTA and 87% uptake
of IONP20-EDTA. The slower uptake of IONP20-EDTA could represent an alternative for
a more efficient uptake of iron that avoids iron deficiency and/or overload that leads to
increase in free electron careers in the plant and the formation of toxic ROS species.
Figure 4.3.6.1. Monitoring of the uptake of IONP from hydroponic media. (a) Decrease in MPS signal of the IONP treatments in hydroponic media harvest. (c) Δ MPS showing that most
IONP10-EDTA was up taken on the first day of hydroponic media harvest vs a continuous absorption of IONP20-EDTA through the plant cycle. Error bars correspond to n= 5 and p<0.05.
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4.3.7 Magnetic Particle Spectrometry Validation with AAS
The MPS calculated concentration of Fe in the hydroponic media measurements were validated by atomic absorption spectroscopy (AAS) and compared in order to determine the sensitivity of the proposed technique. MPS and AAS results showed no significant difference after performing the F test and the student T test with a 95% confidence level (Figure 4.3.7.1). TEM studies were performed on the hydroponic media harvested in the final day. This result shows no signs of change in shape or size of the
IONP (Figure 4.3.7.2), which allows us to keep constant the variables that affect the saturation magnetization of the particles in this media.
Figure 4.3.7.1. Comparison and validation of MPS results using atomic absorption
spectroscopy (AAS) (a) IONP10-EDTA treatment. (b) IONP20-EDTA. Error bars correspond to
with n= 5 and p<0.05.
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Figure 4.3.7.2. TEM images of the IONP-EDTA in the hydroponic media used for the incubation of garden cress (a) IONP10-EDTA and (b) IONP20-EDTA
4.3.8 Magnetic Particle Spectrometry Monitoring of IONP
Translocation
For further exploration of the MPS sensing capabilities, the sensitivity of the method was tested by measuring the magnetization response of the particles in the leaves, stem and roots. The signal obtained from the relaxation magnetization of the particles inside each component of the plant was used in conjunction with the calibration curve to obtain the concentration of Fe in each component of the plant. These results were compared with the total iron concentration results obtained by AAS analysis. Both techniques, show higher concentration of Fe in the roots, followed by the leaves and less in the stems (Figure
4.3.8.1). These data support the results obtained from the hydroponic media MPS analysis that predicted absorption of the nanoparticles in the plants. Higher concentration on the roots is justified by the role of the root hairs as the first source for the uptake of nutrients from the media (Figure 4.3.8.2). All sizes used for this study should theoretically be able
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to be absorbed on the roots of garden cress. Nanoparticles may enter the plant through their pores (680 nm wide), driven by the recognition of the capping ligand EDTA, osmotic pressure and capillary forces. The results showed similar trends of translocation of IONP in the leaves, stem and roots indicating translocation of both kinds of particle treatments to aerial parts of the plant in which pore sizes decrease to a range closer to 20 nm. Higher concentration of iron was found in the leaves for the sample treated with IONP20-EDTA, however the highest amount of number of particles was found for IONP10-EDTA. These results could be explained by the decrease in the size of nutrient transportation channels on the leaves in comparison with the stem and roots, that allows smaller particles to translocate faster to upper levels in larger amounts, but since IONP20-EDTA are double the size they contain higher concentration of Fe.
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Figure 4.3.8.1. Comparison for the relative response of Fe concentration in the leaves, stem
and roots of garden cress exposed to the various experimental conditions.
Figure 4.3.8.2. Picture of garden cress after incubation with IONP20-EDTA showing the root hairs surrounded by the IONP.
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4.4 Conclusion
In summary, this work reports the effects of IONP-EDTA on the phenotypical characteristics of garden cress, showing enhancement on biomass and chlorophyll production. The results show IONP20-EDTA as a potential fertilizer material to treat chlorosis and fortify plants with nutritional value. Moreover, we proved that MPS can be used for the determination of magnetic materials in plant systems decreasing the sample preparation time and facilitating the performance of the measurements.
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Chapter 5. Chemical Design of Au Nanorods
for the Photothermal Treatment of S. aureus
Biofilm
Manuscript preparation in progress:
Author List: Wickramasinge, S., Ju, M., Navarreto-Lugo, M.; Milbrant, N. Samia,
A.C.S*.
Contribution: Design, synthesis, and characterization of the Au nanorods.
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Chapter 5. Chemical Design of Au Nanorods for the Photothermal Treatment of S. aureus Biofilm
5.1 Introduction
The acquisition of nosocomial infections is one of highest risks associated with surgeries.1 This is an alarming issue especially for immunocompromised patients, children and geriatric patients. Several reports have shown that the most common hospital-acquired infections are multi-drug-resistant nosocomial organisms that include methicillin-resistant
Staphylococcus aureus, and vancomycin-resistant enterococci Pseudomonas aeruginosa and Klebsiella pneumonia.2 Currently, periprosthetic infections are one of the main causes of failure in total knee replacement arthroplasty (KRA) which is one of the most commonly perform surgical procedures in the US. However, even though multiple control and sterilization methods are employed (the application of antibiotic therapies and control of surgical techniques) the incidence of infection still around 1-2% in primary arthroplasties and 3.5-5% in revision surgeries.3 Currently, the treatment of KRA infections involves the surgical removal of the focus infection and multiple doses of antibiotic treatment against the biofilm producing bacteria.4 This means, that in order to treat the infection, the patients get exposed to additional post-operative complications. Multiple combinations of antibiotic treatment, have been explored in order to approach this problem in a non-invasive way.
However, considering that pathogenic bacteria constantly adapt and evolve to be resistant to an increasing number of antibiotics, this problem is still a challenge, and the development of biologically safe methods for the inactivation of bacterial viability and proliferation is of significant importance.5
129
Various studies have suggested photothermal therapy (PTT) as a promising tool for the treatment of pathogenic bacteria due to its unique advantages when compared with other traditional therapeutic modalities. PTT is characterized by high specificity, minimal invasiveness and precise spatial-temporal selectivity.6 But more specifically, what makes
PTT such an attractive technique for the treatment of post-operative infections, is the ability to penetrate 10 cm of soft tissue, which provides the hope for the development of non- invasive therapies.7 PTT is based on the generation of heat from photo-absorbers in response to an exogenous applied near-infrared (NIR) laser.8,9 Gold-based nanoparticles
(Au NPs) are the most attractive photo-absorbers for this application since they offer: biocompatibility, small diameters, the ability of simple surface modification, ability to tune their properties and efficient light to heat conversion.9 Moreover, the most relevant characteristic of Au NPs for this application, is the ability to be tuned in order to have the capability to absorb NIR light, which penetrates tissue deeper than other wavelengths.
However, there is an extensive variety of gold nanomaterials available for them to be suitable for PTT applications they must have plasmon resonance tunability, high photothermal conversion efficiency and specific surface functionalization.10,11 These properties can be controlled by tuning their size, shape and aspect ratio. Previous studies promoted Au nanorods (Au NRs) for PTT due to the ability of tuning their absorption range by changing the aspect ratio (AR, length/diameter). For the application of PTT in vivo, it is important that the particle can absorb light within the first (650-850 nm) NIR window because these wavelengths of light can safely and deeply penetrate healthy tissue to reach
Au NRs on subcutaneous implants, for the treatment of bacterial infections. However, despite the potential of this nanomaterial for PTT, to our knowledge, very few reports have
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shown systematic studies on how to optimize these materials in order to enhance their PTT heat conversion ability under real biomedical conditions relevant to this application. Most of the existent research used planktonic, free-swimming bacteria to study the ability of Au
NRs to eliminate bacteria. These results cannot be correlated to the treatment of bacteria in biofilms (bacteria attached to a surface and encapsulated in a protective layer of extracellular polymers). Biofilm formation is common on the surface of implant-associated infections.
This work shows the synthesis and optimization of Au NRs for a systematic study to tune their properties for PTT. Here the aspect ratio and surface chemistry of the material was explored in order to target biocompatibility and photothermal conversion capability.
Moreover, experimental conditions, e.g. Au NRs concentration and laser application logistics effects on the heat conversion process were also evaluated. The potential utility of the synthesized Au NRs in PTT was explored against Staphylococcus aureus (S. Aureus) biofilms. The results were used to develop a systematic method for the disruption of biofilm and killing of bacteria in Titanium (Ti) implants for biomedical applications. This work is part of a bigger project that aims to develop a composites for the complete eradication of bacteria in prosthetic joint infections.
5.2 Methods
5.2.1 Synthesis of Au Seeds
For the synthesis of Au NRs, Au seeds (3-4 nm) were first synthesized by the chemical reduction of chloroauric acid (HAuCl4) with a strong reducing agent sodium borohydride (NaBH4) in the presence of a capping agent, cetyltrimethylammonium bromide (CTAB). The first solution was prepared by mixing a HAuCl4 (0.5 mM, 5 mL)
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solution with a CTAB solution (0.2 M, 5 mL) in a 20 mL scintillation vial. Simultaneously, a solution of NaBH4 (0.01 M, 0.6 mL) was prepared. The NaBH4 solution was injected into the Au (III)-CTAB solution under stirring (1200 rpm) for 2 min. Finally, the solution was aged at room temperature for 30 min before use.
5.2.2 Synthesis of Au NRs
For the synthesis of Au NRs, 0.18 g of CTAB and 0.0269 g of sodium oleate
(NaOL) were dissolved in 5 mL of warm water (50 ˚C) in a 20 mL scintillation vial. The solution was allowed to cool down to 30 ˚C before the addition of a silver nitrate solution
(4 mM, 360 µL). The solution was left undisturbed for 15 min. Then a solution of HAuCl4
(1 mM, 5 mL) was added with stirring (700 rpm) for 1.5 h. Then, the pH of the solution was adjusted to 3-5 (depending of on the desired aspect ratio of the NRs), under constant stirring (400 rpm) for 15 min. Then a solution of ascorbic acid (0.064 M, 25 µL) was added with fast stirring (1200 rpm) for 30 s. Finally, the seed solution (4 µL) was added at 1200 rpm for 3 µs. The seeds solution served as nucleation sites for the anisotropic growth of
Au NRs. The solution was left to react undisturbed for 12 h and the final product was centrifuged (7,000 rpm, 30 min). The supernatant was removed, and the particles were re- dispersed in water.
5.2.3 Surface Functionalization: Ligand Exchange
A solution of Au NRs (3.5 mM, 2 mL) was mixed with a solution of m-PEG-Thiol
(1 M, 2 mL) for 1 h under constant stirring (1200 rpm). The final product was centrifuged, re-dispersed in saline solution and stored in the dark at 4 ˚C until use.
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5.2.4 Atomic Absorption Spectroscopy (AAS): Determination of Au
NRs Concentration
For total Au concentration, 10 µL of Au NRs samples were digested in 1 mL of
HCl (12 M) for 24 h and dilute in milli-Q water to 10 mL. The solutions were vortex mix and analyzed for Au using flame AAS using a standard calibration curve.
5.2.5 Photothermal Conversion of Au NRs in Saline
Various concentrations of Au NRs (CTAB and m-PEG-thiol capped) were studied in open and closed environments. For the closed environment, Au NRs samples were diluted in 1 mL of saline solution in an eppendorf tube. For the open environment experiments, Au NRs samples were diluted in 2 mL of saline and deposited on top of biofilms on Ti implants. These samples were exposed to a near-infrared (NIR) AC powered laser (808 nm) at 2 W/cm2 for various radiation times at an application distance of 4 cm.
The temperature increase of the solutions was measured by placing an optical probe directly into the samples and by a FLIR EX series thermal camera.
5.2.6 Biofilm Formation and PTT Generated Heat Elimination
S. aureus were cultured overnight in kanamycin sulfate (200 ppm) containing luria broth (LBK) with agitation (200 rpm) at 37 °C. Biofilm formation was evaluated under static conditions using a Ti implant in a polystyrene petri dish (Fisherbrand). Bacterial cultures made overnight were further diluted in LBK broth for the desired cell amount at
OD600 and each dish was filled with 3 mL of the diluted bacterial solution and incubated at 37 °C for 72 h. The samples were then incubated with the Au NRs solutions and then treated with the excitation NIR laser (808 nm) placed at 4 cm from the samples. After treatment, the plates were gently washed with phosphate buffered saline (PBS, 1×)
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swapped and re-platted in order to confirm the presence of viable bacteria cells. For the confocal microscopy assay the biofilm treated with the Au NRs were stained with BacLight
LIVE/DEAD (Thermo Fisher Scientific) according to the manufacturer's method. The area covered by dead (red) or live (green) bacteria were evaluated using ImageJ.
5.3 Results and Discussion
5.3.1 Aspect Ratio Effect in Au NRs PTT Heat Production
Au NRs are promising materials for PTT owing to their unique optical properties, resulting from their surface plasmon band resonance (LSPR), as well as their low toxicity.12
The Au NRs plasmonic properties can be used for photothermal therapy by photo exciting their conduction electrons to induce surface plasmon band oscillations, that can result in non- radiative relaxation through electron-phonon and phonon-phonon coupling.13 This relaxation mechanism results in the generation of localized heat that can be used for biofilm disruption and bacteria eradication. Most work performed using plasmonic photothermal therapy (PPTT) has been done for the selective photothermal ablation of epithelial carcinoma cells in vitro.14 However, several groups consider that similar mechanisms can be used as an alternative or complement to antibiotic treatment for nosocomial infections.
To produce Au NRs (Au NRs capped with CTAB) with different photothermal conversion abilities, the size, aspect ratio and surface functionality needs to be fine tune.
Au NRs samples were synthesized with various longitudinal surface plasmon resonances, in order to generate a library to study the PPTT conversion capacity of different NRs that absorb on the NIR range (Figure 5.3.1.1).
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From this library, Au NRs that absorb in the excitation wavelength (808 nm) of the NIR
AC powered laser: 750 nm, 810 nm and 925 nm (Figure 5.3.1.2), were used to evaluate the effect of aspect ratio (AR) on the heat conversion capacity (Figure 5.3.1.3).
Figure 5.3.1.1. Au nanorods with various LSPR. The yellow shaded area of the spectra represents the biological “water window”, region where aqueous tissue absorbs relatively little light (700 nm to 1200 nm).
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Figure 5.3.1.2. Au nanorods that absorb in the excitation wavelength of the NIR AC powered laser (808 nm, represented as a yellow line). (a) UV-VIS spectra of various Au NRs (CTAB capped Au NRs samples). TEM images, scale bar: 200 nm (b) AR: 3.5 Au
NRs with longitudinal surface plasmon resonance at 750 nm. (c) AR: 3.7 Au NRs with longitudinal surface plasmon resonance at 810 nm and (d) AR: 4.4 Au NRs with longitudinal surface plasmon resonance at 925 nm.
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Figure 5.3.1.3. Photothermal heat conversion capacity results for 200 ppm of different Au NRs-CTAB upon 15 min of NIR irradiation (808 nm). (a) ∆T values from the different AR Au NRs samples. Graph also shows the ∆T values for the loss of heat generation immediately after the excitation source is turn off. (b) Maximum temperature achieved by each Au NRs sample. Experimental data was collected with n= 3.
The photothermal conversion capacity experiments were performed by placing solutions of 200 ppm of the various Au NRs (samples with AR of 3.5, 3.7 and 4.4) on a custom made plate (later used to perform the experiments on the implant surface). A temperature probe was used to monitor the temperature changes while the solutions were exposed to a continuous excitation with NIR radiation at 2 W/cm2 for 15 min. Results showed ∆T values of 4 ˚C, 16 ˚C and 3 ˚C for Au NRs with respective resonant wavelengths of 750 nm, 810 nm and 925 nm. The Au NRs 810 nm with AR of 3.7, showed significantly higher ∆T values under these experimental conditions. The main factors influencing these
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results are the laser wavelength used to perform the experiments (808 nm) and the distance at which the field decays. Since samples with LSPR wavelength at 750 nm and 925 nm were not excited with the correspondent wavelengths, their extinction value of the Au NRs sample is expected to decrease. Moreover, the distance at which the field decays (which is the distance that the heat is dissipated away from the particle surface) determines the overall heating transferred to the surrounding environment. Because the field decays exponentially as it separates from the surface of the particles, both the maximum field enhancement value and the particle size play a role in how far the enhanced field extends away from the particle.7 Work done by El-Sayed et al. showed that particles with smaller
AR generate stronger fields, however it extends just a few nanometers away from the particle surface. Consequently, the results of this work indicate that both, AR and excitation wavelength, play a role in the ability of Au NRs to produce heat. Also, that the Au NRs sample with LSPR wavelength of 810 nm and AR 3.7, is the optimum for the optimization experiments using our experimental set-up and laser.
5.3.2 Surface Functionalization Effect in Au NRs PTT Heat Production
The surface functionalization of Au NRs is of vital importance for biomedical applications. As synthesized Au NRs are capped with CTAB, which is an essential ligand for control of the shape, size and stability of Au NRs during their synthesis. However,
CTAB alone is quite toxic to cells (even at submicromolar dose), due to its highly positive charge surface.15 Free CTAB molecules in gold solution samples can be generated due to desorption or incorrect purification. This risks can be eliminated by changing the capping ligand through ligand exchange. However, the capping ligand plays an important role on the dispersion of the nanoparticles, which can affect the heat conversion capacity. Here
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CTAB capping ligand was exchanged with m-PEG-SH through a ligand exchange process
(Figure 5.3.2.1). Z-potential measurements confirmed the significant decrease in surface
charge, to almost neutral values (Figure 5.3.2.2).
Figure 5.3.2.1. Surface functionalization of Au NRs following a ligand exchange process.
Figure 5.3.2.2. Z-potential results for the Au NRs before and after ligand
exchange from CTAB to m-PEG-SH.
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A study for the effect of capping ligand in the PTT conversion capacity of the Au
NRs was performed by placing solutions of 200 ppm of Au-CTAB and Au-PEG-SH respectively, in eppendorf tubes (Figure 5.3.2.3). This time, the experiments were performed in a closed system and a thermal camera was used to collect the changes in temperature (∆T). The purpose of this set-up was to determine the maximum ∆T that can be obtained from each sample without having the effect of heat loss (characteristic of the open system set-up). The FLIR EX series thermal camera was used to determine ∆T produced by each sample. Results show comparable ∆T values, with slightly higher values for Au NRs-PEG-SH in saline. These results may be attributable to the presence of salts in saline solution that tend to promote aggregation in Au NRs-CTAB.
Figure 5.3.2.3. Infrared thermal images showing the photothermal conversion capacity
for 200 ppm of Au NRs with different surface chemistries. Data n value equals 3.
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5.3.3 Excitation Method Effect on Au NRs PTT Heat Production
Taking into account the intended application of this work, we were interested in studying how the PPTT conversion capacity of Au NRs-PEG-SH is affected when the treatment is performed on a Titanium (Ti) implant (as is one of the most commonly used implants for knee replacement surgeries). Figure 5.3.3.1 shows the custom plate Ti implant used for these experiments. Scanning electron microscopy (SEM) images were obtained in order to understand the surface of the Ti implant. This is important, since the formation and attachment of a bacterial biofilm is dependent on the surface roughness, as explained in our previously published review Applications and challenges of using 3D printed implants for the treatment of birth defects.16 Moreover, we were interested in determining the optimum experimental conditions for the application of PPT in order to produce the maximum heat conversion in an open system. The study of the experimental condition variables is important in order to achieve complete nosocomial bacterial eradication in biomedical applications. Considering that this material will be incorporated in to a composite, and the final treatment time will be dependent on all the components, we established a maximum PPT treatment time with Au NRs of 45 min. This will support a method designed for real biomedical applications. There is a maximum gap of 45 min to 2 h to apply the antibacterial treatment. To design a method for Au NRs PTT on implants, it was important to understand how much heat is created and how much is dissipated through the sample (distance of field decay). Due to the promising results obtained from the Au
NRs-PEG-SH sample, we were interested in studying the PPTT heat conversion capacity
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of this sample on a Ti implant, and in evaluating the influence of excitation position and time on the creation and dissipation of the heat (Figure 5.3.3.2 and Figure 5.3.3.3).
Figure 5.3.3.1. Ti implant used for the Au NRs PPTT conversion studies. (a) Ti implant
dimensions. (b) SEM images showing significant roughness of the Ti implant surface.
For the PPTT experiments, a custom made plate with the Ti implant was divided into four quadrants. The excitation laser was applied continuously during 45 min, and the temperature was recorded on each quadrant for 11.25 min on each (while keeping the extraction laser stationary). Results showed ∆T values of 11 ˚C for 500 ppm of Au NR-
PEG-SH on this open system set-up. Moreover, the data showed that the heat produced by the Au NRs-PEG-SH is higher at the direct position of the applied laser and the dispersed heat can be from 2-3 ˚C lower in adjacent quadrants (2 and 4), and up to 4 ˚C in the opposite
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quadrant number 3. These result suggest the need for rotating the position of the excitation
field, in order to increase heat generation and enhance heat distribution through the area of
interest (Figure 5.3.3.2). Infrared thermal images showing the photothermal conversion
were also obtained to show the initial and final heat distribution through the implant (Figure
5.3.3.3).
Figure 5.3.3.2. PPTT heat conversion capacity of 500 ppm of Au NRs in 1 mL of saline solution. The gap between the laser and the sample was kept at 4 cm. The excitation source (808 nm) was kept stationary at quadrant #1, while the temperature was recorded on successive quadrants during 11.25 min each. The heat produced was obtained using a temperature probe. 143
Figure 5.3.3.3. Infrared thermal images showing the photothermal conversion for 500 ppm of Au NRs in saline solution on top of the Ti implant.
5.3.4 Treatment of Bacteria with Au NRs-PEG-SH and IR Laser
Biofilms of S. Aureus were grown for 3 days on Ti implants, and treated with 500
ppm of Au-PEG-SH PTT for 45 min. Pictures were taken before and after PTT treatment
to show the disruption of the biofilm on top of the Ti implant surface caused by the PTT
(Figure 5.3.4.1). The swabs, showed the presence of some viable bacteria after the
treatment (Figure 5.3.4.1). Confocal microscopy experiments using LIVE/DEAD bacterial
cell viability kit showed that an approximate of 60% of the bacteria was killed after the
PTT with 500 ppm of Au-PEG-SH (Figure 5.3.4.2).
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Figure 5.3.4.1. Au NRs-PEG-SH (500 ppm) 45 min PTT in S. Aureus biofilm on Ti implants. (a) Positive control, (b) Treated S. Aureus, (c) Negative control.
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After + Control Au NRs-PEG-SH PTT
Figure 5.3.4.2. Confocal micrographs showing live (green) and dead (red) S. Aureus.
(a) Positive control, (b) after PTT treatment with 500 ppm of Au NRs-PEG-SH. (b) Data showing fraction of LIVE/DEAD bacteria.
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5.4 Conclusion
This study shows a systematic work for the development of the optimum conditions for the use of Au NRs for treating bacterial biofilm (S. Aureus) through PPTT. Au NRs of various sizes were successfully synthesized, and their plasmonic photothermal heat conversion ability was evaluated. The results showed a relation between the aspect ratio of the nanostructure, the excitation field wavelength and the heat conversion capability.
Moreover, modification of the surface chemistry of Au NRs with m-PEG-SH ligand produced comparable ∆T values as the synthesized Au NRs-CTAB. The proposed method showed an approximate 60% of bacteria killing. These results are promising considering that this Au NRs will be incorporated in a composite material. These results are part of a project that aims to develop a novel, less invasive treatment that can be used as an alternative to antibiotics to prevent implant associated infections.
5.5 References
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Chapter 6. Research Outlook
6.1. Cortisol Sensing
Cortisol is an important biomarker that have been correlated to multiple medical conditions like chronic stress, adrenal insufficiency, Cushing’s disease, neurological diseases, and central serous retinopathy.1-2 The importance of monitoring cortisol levels biological fluids has motivated the development of multiple sensing technologies .3
However, there are very few reports that propose detection techniques to monitor cortisol for point-of-care applications. This is mostly due to the difficulty of the sample processing of biological fluids. This problem can be avoided by considering cortisol sensing in biological fluids that require minimal to zero sample preparation, such as the case in exhaled breath condensate. However, cortisol can be found in very small concentrations in this biological sample, which require the development of sensor systems with high sensitivity. The work presented in this thesis provide a promising approach that utilizes tailored nano-alloy composites.4 Future work can be conducted on the sensor system developed in this thesis work by coupling the unit into an exhaled breath condensate collection tube with a portable electrochemical measurement set-up. This work can significantly contribute to the development of biomedical detection tools for point-of-care applications.
6.2 Pb2+ Sensing
The extensive use of lead (Pb2+) in agricultural, domestic, and industrial applications promotes multiple routes of exposure of vulnerable groups like children to this toxic heavy metal. To date, several studies have reported that even small concentrations of
Pb2+ can cause serious organ failure and health problems especially for children younger
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than 6 years. Even though this has been a persistent problem for decades, the current treatment for lead poisoning lacks selectivity. Consequently, conventional approaches can lead towards further complications like anemia or Ca2+ deficiency. The work presented in this thesis showcases a systematic methodology that uses magnetic particle spectrometry to sensitively and selectively detect lead by using a chemically surface modified iron oxide nanoparticles. Further work in the development of new selective ligands for different small analytes of interest (like lead) can be design and this assay can be used to test their chelation ability and further propose the design of new magnetic chelation therapies for heavy metal poisoning.
6.3 Environmental Monitoring of Magnetic Nanoparticles
During the last decades the use of magnetic nanomaterials in a variety of technologically relevant applications have increased, however, very little is known about their fate in environmental systems.5 This is an issue of concern, since their uptake and translocation in plants have a direct way into our food chain. Here, were present the design of a magnetic particle spectrometry assay that allows the direct monitoring of the uptake and translocation of magnetic nanoparticles inside plants, without the need of any kind of sample pre-treatment. Moreover, we explored the effect of TMS-EDTA modified iron oxide nanoparticles on plant growth and development. Future work can use the developed methodology in this report to further investigate the effect of other types of magnetic nanoparticles in environmental system. This study also provides a platform approach to design and explore the potential of new magnetic nanomaterials for agricultural fertilizer applications.
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6.4 Au Nanorods for Photothermal Therapy
The acquisition of post-surgery infections is one of highest risks associated with surgeries.6 This is an alarming issue especially for immunocompromised patients, children, and geriatric patients. Several reports have shown that the most common hospital-acquired infections are multi-drug-resistant nosocomial pathogens like Staphylococcus aureus.
Currently, periprosthetic infections are regarded as one of the main causes of failure in total knee replacement arthroplasty (TKRA) which is one of the most commonly performed surgical procedures in the US. However, even though multiple control and sterilization methods are employed, the risk of acquiring an infection is still high. So far, the treatment of TKRA infections involve the surgical removal of the focus infection and multiple doses of antibiotic treatment against the biofilm producing bacteria. The results presented in this work illustrate how heat generation from the light excitation of Au nanorods in photothermal therapy (PTT) can be used for the disruption of pre-formed bacterial biofilms.
Current results showed an approximate killing of 60% of Staphylococcus aureus using PTT with Au NRs. Ongoing work in the Samia lab are underway towards the development of composite based materials and methodologies that can achieve complete eradication of bacteria. This work can lead to a novel, non-antibiotic based treatment that can prevent the onset of hard to treat resistant bacterial strains.
6.5 References
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