A Dissertation Entitled Molecular Imprinting Technology Towards the Development of a Novel Biosensor by Abraham Avalos Submitted

A Dissertation

Entitled

Molecular Imprinting Technology Towards the Development of a Novel Biosensor

Abraham Avalos

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in Biomedical Engineering

______Dr. Arunan Nadarajah, Committee Chair

______Dr. Sarit Bhaduri, Committee Member

______Dr. Dong-Shik Kim, Committee Member

______Dr. Joseph G. Lawrence, Committee Member

______Dr. John D. Dignam, Committee Member

______Dr. Stephen Callaway, Committee Member

______Dr. Patricia R. Komuniecki, Dean College of Graduate Studies

The University of Toledo December 2014

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author.

An Abstract of

Molecular Imprinting Technology Towards the Development of a Novel Biosensor

Abraham Avalos

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biomedical Engineering

The University of Toledo

December 2014

Current advances in molecular imprinting has many drawbacks such as the lack of specificity, this is the inability to selectively detect one single species of biomolecule. In addition, it is very difficult to produce a device that accounts with high sensitivity to detect relevant biomolecules and generate reproducible data from complex samples. The present project aims to offer an alternative by developing a technique to synthesize a polymeric matrix with molecularly imprinted cavities that excludes non-targeted species and binds to the targeted molecule, this synthetic platform in combination with proven efficient surface plasmon resonance technology achieve high sensitivity, for real time monitoring of molecular binding.

Polyacrylamide is a hydrophilic and biocompatible synthetic polymer used in industry and in the lab. It is used in chromatography, electrophoresis, and recently it has been used in molecular imprinting. However conventional techniques for molecular imprinting using acrylamide as the functional monomer are not yet robust enough due to the high swelling ratio in the presence of water.

In the present study, molecularly imprinted polyacrylamide was produced in nano- thin films on top of the gold surface of a surface plasmon resonance sensor. The model

iii template used in this study was lysozyme and bovine serum albumin because they are relatively big and complex proteins that easily solubilize in water. The system was evaluated by comparing signals when exposing the imprinted polyacrylamide to a solution containing lysozyme. Uv-Vis spectrophotometry, scanning electron microscopy (SEM), matrix assisted laser desorption ionization coupled with time of flying mass spectrometry

(MALDI-TOF MS), and surface plasmon resonance (SPR) were used to characterize the novel biosensor.

The results obtained from this project showed that it is possible to prepare a molecularly imprinted system with high sensitivity and selectivity that is stable and offers reproducibility in the detection of proteins.

Dedicated to my beloved family

Acknowledgements

I want to start by thanking God for all his blessings. I also would like to express my sincere gratitude to Dr. Arunan Nadarajah for his continued guidance, support and encouragement during this entire research. His knowledge, commitment and patience have contributed to my formation as a scientist. I am also grateful to the members of my committee, Dr. Joseph Lawrence, Dr. Sarit Bhaduri, Dr. Dong-Shik Kim, Dr. John Dignam, and Dr. Stephen Callaway for their suggestions and guidance. A special thanks to Tamara

Phares and Denise Turk for their support and assistance.

I am truly thankful to my parents Abraham A. Avalos and Rosa E. Postigo for their endless love and continued support throughout my life. This dissertation would not be possible without them. To my brothers and sisters Paul, Maria, Alexander, Mayra, Maggie,

Martin, and Silvia for their encouragement and friendship. And to my wife Lidia Rodriguez and daughter Adriana Avalos for their support and love and because they are the engine that motivates me to be a better person every day. Finally thanks to the Bioengineering

Department and the Center of Material and Sensor Characterization for giving me the opportunity to pursue my Ph. D. degree at the University of Toledo.

Table of Contents

Abstract ...... iii

Acknowledgements ...... vi

Table of Contents ...... vii

List of Tables ...... xi

List of Figures ...... xii

List of Abbreviations ...... xvi

1 Introduction ...... 1

2 Literature Review...... 8

2.1 Molecular imprinting (MI) ...... 8

2.2 Molecular imprinting applications ...... 10

2.3 Molecular imprinting in molecular sensing applications ...... 11

2.4 Molecular imprinting techniques ...... 12

2.5 Consideration in the selection of the molecularly imprinted system ...... 15

2.6 Molecular imprinting of proteins ...... 15

2.7 Acrylamide ...... 15

2.8 Polyacrylamide ...... 17

2.9 Selection of the model protein ...... 18

2.10 2D and 3D molecular imprinting ...... 18

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2.11 Sensing of molecular rebinding ...... 20

2.12 Coupling Molecular Imprinting with Surface Plasmon Resonance ...... 21

3 Experimental Methods ...... 23

3.1 UV/Vis Spectrometry ...... 23

3.2 Scanning Electron Microscopy analysis (SEM) and Energy Dispersive X-ray

Spectroscopy (EDS) ...... 25

3.3 Sputter Coating ...... 27

3.4 Physical Vapor Deposition (PVD) ...... 28

3.5 Surface Plasmon Resonance (SPR) ...... 30

3.6 Freeze Drying ...... 34

3.7 Matrix-Assisted Laser Desorption Ionization-Time of Flying Mass Spectrometry

(MALDI-TOF MS) ...... 36

3.8 Atomic Force Microscopy (AFM) ...... 37

4 Synthesis of polyacrylamide hydrogel ...... 39

4.1 Introduction ...... 39

4.2 Glassware preparation before polyacrylamide synthesis ...... 40

4.3 Materials for synthesis of polyacrylamide hydrogel ...... 41

4.4 Bulk synthesis of polyacrylamide hydrogel ...... 41

4.5 Synthesis of hemoglobin imprinted hydrogel ...... 43

4.5.1 Materials ...... 44

4.5.2 Hemoglobin imprinting procedure ...... 44

4.5.3 Protein removal ...... 45

4.6 Tuning of the concentration of monomer and crosslinker ...... 46

viii

4.7 From bulk to thin films of polyacrylamide hydrogel ...... 51

4.7.1 Coating of glass slides with bind-silane...... 52

4.7.2 Synthesis of thin films of polyacrylamide ...... 53

4.8 Discussion ...... 54

5 Affinity characterization of protein imprinted hydrogel ...... 55

5.1 Introduction ...... 55

5.2 Use of charged monomer ...... 55

5.3 Protein template and a proposed mechanism for signal intensification ...... 57

5.4 β-galactosidase as protein template ...... 58

5.5 Test to determine relative enzyme abundance in the imprinted hydrogel...... 59

5.5.1 Materials ...... 60

5.5.2 Thin film synthesis of β-galactosidase imprinted polyacrylamide ...... 61

5.5.3 β-galactosidase removal ...... 62

5.5.4 Rebinding assay of β-galactosidase to imprinted hydrogel ...... 63

5.5.5 Test with ONPG ...... 64

5.6 Affinity test results ...... 65

5.7 Discussion ...... 69

6 Integration of molecular imprinting and SPR ...... 71

6.1 Introduction ...... 71

6.2 Gold surface of the SPR sensor ...... 72

6.3 Thiol as adhesive layer for the attachment of the nano-thin film of

polyacrylamide ...... 74

6.4 Design and preparation of the gold chip for the SPR sensor ...... 80

6.4.1 Material and thickness specification of the layers onto the gold chip ...... 81

6.4.2 Fabrication of the gold chip coated with SiO2 ...... 85

6.5 In-situ synthesis of the nano-thin layer of polyacrylamide ...... 86

6.6 Integrity of the SiO2 and bind-silane adhesion system ...... 88

6.7 SPR affinity test of the imprinted films ...... 89

6.7.1 Regeneration of the imprinted films ...... 90

6.7.2 SPR monitoring of the interaction protein-imprinted matrix ...... 91

6.8 Stability of the imprinted platform ...... 94

6.9 Discussion ...... 95

7 Other steps for the improvement of the MI platform ...... 97

7.1 Method to control the thickness of the film of polyacrylamide ...... 97

7.1.1 Procedure for mold preparation for the casting of the nano-thin film ...... 98

7.1.2 In-situ synthesis of a nano-thin film of polyacrylamide ...... 99

7.1.3 Characterization of the thickness of the nano-thin film ...... 99

8 Conclusions ...... 108

9 Future work ...... 112

References ...... 115

List of Tables

Table 4.1 Different compositions for the synthesis of hydrogels. Samples 1 to 6...... 48

Table 4.2 Swelling results of samples 3, 5, and 6 ...... 48

Table 4.3 Variation of the amount of crosslinker for the synthesis of the hydrogels.

Samples 3, 6, 7, and 8...... 49

Table 4.4 Swelling results of samples 3, 6, 7, and 8 ...... 50

Table 4.5 Variation of the amount of water used in the preparation of the hydrogels. .... 50

Table 4.6 Swelling results of samples 6, 9, 10, and 11 ...... 51

Table 5.1 Description of the samples prepared to test the effectiveness of the use of positive functional monomer in the imprinting of β-galactosidase...... 62

Table 5.2 Absorbance readings at 420nm ...... 65

Table 6.1 Depth of penetration of the evanescent field yeff for various multilayered systems...... 85

Table 6.2 Sample description of the inclusion of template protein and charged monomer in the preparation of the imprinted films for SPR test...... 87

Table 7.1. Recorded thicknesses of the thin films of polyacrylamide synthesized using molds that were prepared with 5, 7, 15, 30, 60, and 120 seconds of coating time with a sputter coater...... 101

List of Figures

Figure 2-1. Molecular imprinting of proteins ...... 9

Figure 2-2. Covalent MI. Imprinted region is homogeneous to all imprinted cavities. .... 13

Figure 2-3. Non-covalent MI. Imprinted recognition sites are non-homogeneous or different for each imprinted cavity...... 14

Figure 2-4. 2D molecular imprinting of proteins ...... 19

Figure 2-5. 2D molecular imprinting of crystallized proteins ...... 20

Figure 3-1. Scattering and reflection losses in a typical quartz cell used for UV-Vis spectroscopy ...... 24

Figure 3-2. Electron bean interaction with the sample ...... 27

Figure 3-3 Physical vapor deposition schematics ...... 30

Figure 3-4. Disturbance of the resonant plasmon by adsorption of protein on the gold surface ...... 31

Figure 3-5. Antibody immobilized onto the gold surface of an SPR sensor ...... 32

Figure 3-6. SensiQ Discovery SPR instrument...... 33

Figure 3-7. Schematics of the internals of an SPR sensor from SensiQ Technologies Inc.

Note its size is comparable to a quarter dollar coin ...... 33

Figure 3-8. Gold chip mounted on the prism of the SPR sensor...... 34

xii

Figure 3-9. Final setup of the gold surface coupled with the SPR sensor and mounted on the microfluidic channel...... 34

Figure 3-10. Phase diagram of water ...... 35

Figure 3-11. Schematics of an AFM in tapping mode ...... 38

Figure 4-1. Dichlorodimethylsilane also called repel-silane is used for hydrophobic coating of glass surfaces...... 40

Figure 4-2. Bulk synthesis of polyacrylamide hydrogel ...... 43

Figure 4-3. Bulk polyacrylamide hydrogel imprinted with hemoglobin. A twin bulk imprinted hydrogel was washed with SDS and AcOH 3%...... 46

Figure 4-4. 3-(trimethoxysilyl) propyl methacrylate molecule, also called bind-silane used to strongly attach polyacrylamide to glass surfaces...... 52

Figure 4-5. Thin film synthesis onto a solid support...... 54

Figure 5-1. Positively charged monomer (3-acrylamidopropyl) trimethylammonium chloride (APTAC) used in the synthesis of imprinted hydrogel...... 56

Figure 5-2. Inclusion of a positively charged monomer in the synthesis of molecularly imprinted hydrogel ...... 57

Figure 5-3. β-galactosidase from Escherichia coli. The picture shows charges on the surface of the enzyme ...... 59

Figure 5-4. Hydrolysis of ONPG by enzyme β-galactosidase ...... 59

Figure 5-5. The imprinted films are placed in glass beakers...... 64

Figure 5-6. Sample A, absorbance versus time...... 65

Figure 5-7. Sample B, absorbance versus time ...... 66

Figure 5-8. Sample C, absorbance versus time...... 66

xiii

Figure 5-9. Sample D, absorbance versus time...... 67

Figure 5-10. Comparison of the ratios ΔAbs420/ΔTime that represent the relative amount of β-galactosidase present in the films after the rebind experiment...... 68

Figure 6-1. SensiQ Discovery SPR instrument from SensiQ Technologies Inc...... 72

Figure 6-2. Schematics of the SPR sensor that is loaded to the SensiQ Discovery instrument. The sensor utilizes the Kretschmann SPR geometry ...... 73

Figure 6-3. Treatment with aqua regia to remove the original gold surface of the SPR sensor...... 73

Figure 6-4. Schematics of the new gold chip-SPR sensor setup...... 74

Figure 6-5. Parts of a self-assembled monolayer (SAM)...... 75

Figure 6-6. AFM images of the surface morphology of gold prepared by physical vapor deposition ...... 76

Figure 6-7. Representation of some intrinsic and extrinsic defects found in SAMs ...... 77

Figure 6-8. N,N’-bis(acryloyl)cystamine (BAC) used in SAM coating of gold as adhesive layer between gold and polyacrylamide...... 78

Figure 6-9. 2-propene-1-thiol used in SAM coating of gold as adhesive layer between gold and polyacrylamide...... 78

Figure 6-10. SEM images of two areas of the top surface of the polyacrylamide film partially attached to the gold chip ...... 79

Figure 6-11. SEM image of the cross-section at a wrinkle ...... 80

Figure 6-12. Thicknesses of the layered design of the gold chip...... 86

Figure 6-13. In-situ synthesis of the thin film of polyacrylamide...... 88

xiv

Figure 6-14. Gold chip with the attached thin film is mounted in the SPR sensor by means of a drop of index matching fluid ...... 88

Figure 6-15. SEM image of the cross-section of the film of polyacrylamide hydrogel attached to the gold surface by means of the SiO2 and bind-silane coatings ...... 89

Figure 6-16. Setup of the imprinted chip and SPR sensor coupled to the microfluidic system...... 90

Figure 6-17. Sensorgram of the regeneration cycle ...... 91

Figure 6-18. Sensorgram of the interaction between the injected bovine serum albumin protein and the imprinted film loaded in the SPR system...... 92

Figure 6-19. Sensorgram of the interaction of lysozyme with the matrix ...... 94

Figure 6-20. Test of reproducibility over a time lapse of seven days ...... 95

Figure 7-1. Preparation of a mold by creating a frame of a thin layer of gold on a glass slide...... 98

Figure 7-2. Casting of thin films of polyacrylamide...... 99

Figure 7-3. SEM images of films synthesized on top of glass slides ...... 100

Figure 7-4. SEM images of glass particles found on the surface of the glass slides...... 102

Figure 7-5. SEM image of glass particles trapped in the film of polyacrylamide...... 102

Figure 7-6. SEM image of the cross-section of a freeze dried bulk piece of polyacrylamide...... 103

Figure 7-7. Two-stage synthesis for molecular imprinting of proteins...... 104

Figure 7-8. MS spectrum of the prepolymer solution in polymerization stage one ...... 107

List of Abbreviations

Å ...... Angstroms ACS ...... Meets the specifications of the American Chemical Society AFM ...... Atomic force microscopy AMPS ...... 2-acrylamido-2-methyl-1-propanesulfonic acid APS ...... Ammonium persulphate APTAC ...... (3-acrylamidopropyl) trimethylammonium chloride

BAC ...... N,N’-bis(acryloyl)cystamine BAS ...... Bovine serum albumin Bhb ...... Bovine hemoglobin Bind-silane ...... 3-(trimethoxysilyl)propyl methacrylate

ϲ ...... Light speed in vacuum

Ɛ...... Dielectric constant EDS ...... Energy Dispersive X-ray Spectroscopy

KBr ...... Potassium bromide KDa ...... Kilo Daltons k...... Wave vector

LED ...... Light-emitting diode Lysz ...... Lysozyme

MALDI-TOF...... Matrix-Assisted Laser Desorption Ionization-Time of Flying MAPTAC ...... [3-(Methacryloylamino)propyl] trimethylammonium chloride MI ...... Molecular imprinting m/z...... Mass-to-charge ratio

NaCl ...... Sodium chloride Neg ...... Negative monomer

OD ...... Optical density ONPG ...... o-nitrophenyl β-D-galactopyranoside

xvi

PAGE ...... Polyacrylamide gel electrophoresis pI ...... Isoelectric point Pos ...... Positive monomer PVD...... Physical vapor deposition

Repel-silane...... Dichlorodimethyl silane RRU ...... Relative response units RU ...... Relative units

SAM ...... Self-assembling monolayer SDS ...... Sodium dodecyl sulfate SEM ...... Scanning electron microscopy SiO2 ...... Silicon dioxide SPR ...... Surface plamon resonance

TEMED ...... N,N,N′,N′-tetramethylethylenediamine TES ...... N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid TFA ...... Trifluoroacetic acid

UV-VIS ...... Ultraviolet-visible spectroscopy v/v ...... Volume/volume percent

ω ...... Angular frequency w/v...... Weight/volume percent y ...... Penetration depth

λ ...... Wavelength

xvii

Chapter 1

Introduction

The development of methods for analysis of proteins and in general of biomolecules has become central in the life sciences. Biomarkers are biomolecules that indicate an alteration of the physiological state of an organism relative to the health or disease state. In fact, proteins are one group of biomolecules that carry this alteration information [1].

Therefore, development of instruments and techniques for biomarker analysis has had an important impact on the diagnoses of diseases such as cancer. One example is the detection of the biomarker carcinoembryonic antigen (CEA) which aids in the diagnosis, prognosis, and monitoring of therapy for colon, breast, lung, and pancreatic cancers. Moreover, new biomarkers are being discovered using these advances in technology, which also plays a role in drug discovery and personalized medicine. With increasing biomarker discovery, it seems that expensive technologies such as proteomics mass spectrometry (MS) and multiplex immunoassays could dominate the medical diagnosis field in the future [2-6].

The advent of biomarkers is expected to facilitate earlier detection of diseases. This approach would help physicians in delivering a more efficient health care, to patients, in addition, to monitor activity and therapeutic response of many diseases. Screening

1 technologies for early detection of diseases especially in the case of cancer provides decreased mortality and better expectations of rapid recovery [7,8].

In industry, the conventional process of biomarker discovery is labor intensive and expensive as the cost increases from stage to stage. This process involves several steps such as the discovery phase in which many technologies are applied to identify biomarker candidates; the prototype developmental phase in which immunological assays are established and validated; and the product development phase with assay formats suitable for automated platforms [9]. In addition, in clinical practice, biomarker analysis demands significant resources; it requires the use of expensive equipment with a large footprint that also implies labor intensive sample preparation. The need for a faster, robust, and cost effective analysis of proteins and the need for equipment with a smaller footprint encourage us to develop a new biosensor technique that will satisfy most requirements. In this project we will attempt to develop such a biosensor employing molecular imprinting (MI) combined with surface plasmon resonance (SPR).

MI is a polymerization technique that allows us to produce a stable polymer matrix with 3D cavities of specific affinity for the protein of interest. This synthetic platform is the equivalent of a molecular receptor that is relatively inexpensive, easy and quick to produce for a multitude of applications [10-12]. For example, MI can be used in analytical separations, drug delivery, enzyme like catalysis, and chemical sensors. In the fabrication of MI polymers, intermolecular interactions such as hydrogen bonding, electrostatic interaction, hydrophobic interactions, π-π stacking, and van der Waals forces are used to determine the spatial arrangement of monomers around the protein or template molecule.

This spatial arrangement is then affixed by polymerization of the monomers and

2 crosslinker. Later, the template molecule is removed, leaving an empty imprinted cavity with specific affinity for the target molecule [13].

Many groups were successful when imprinting small molecules; however, it becomes more challenging when imprinting macromolecules such as proteins, DNA, etc.

Firstly, the size of macromolecules does not allow them to easily diffuse in and out through the polymer. This leads to a slow kinetics in removal of the template and rebinding to the imprinted cavities, and may lead to physically entrapping the template molecules in the polymer. Secondly, it is difficult for macromolecules to specifically bind in the cavities due to the natural shape complexity of macromolecules. As crosslinking density is decreased, the diffusion of the molecules is improved. However, this improvement comes at the expense of, the specificity of the recognition cavities [13,14].

Many materials and solvents can be used in the fabrication of polymers with specific memory for the target molecules. However, protein imprinting imposes special requirements. Since proteins are very sensitive to harsh conditions, it is optimal to work with aqueous mixtures and mild pH. This will protect proteins from denaturation and in general will prevent specific binding to the imprinting cavities [15]. This requires the polymer to be hydrophilic and biocompatible. For this reason, acrylamide is a popular choice for MI polymers and was selected for this study.

Acrylamide is a colorless crystalline solid that is formed from the hydration of acrylonitrile. Acrylamide is soluble in water, acetone, and ethanol, and has a high mobility in soil and groundwater. It is also biodegradable. However, because of the exposure to this carcinogenic chemical, the effect of acrylamide in cells, tissues, animals, and humans have been extensively studied. Acrylamide is used worldwide as the monomer in the synthesis

3 of polyacrylamide [16]. The networks of polyacrylamide chains are highly hydrophilic, and they can be found as hydrogels which are highly absorbent for water, and swells during the water adsorption. Polyacrylamide has found various applications as a soil conditioner in wastewater treatment, in the cosmetic, paper, and textile industries, and in the laboratory as a solid support for the separation of proteins by electrophoresis [17]. Recently polyacrylamide is also being used as the structural matrix for molecularly imprinted (MI) hydrogels in various molecular sensing applications.

Synthesis of MI polyacrylamide hydrogel implies casting of bulk pieces of hydrogel containing the imprinted cavities. As it is difficult to measure in real time the binding of proteins into the cavities, indirect methods are often preferred. UV-Vis spectrometry is frequently used to measure changes in protein concentrations in the supernatant solutions after rebinding to the imprinted cavities [18]. However, protein depletion in the supernatant solution can also be caused by other phenomena such as adsorption on the walls of the test tube [13,19]. In addition, as protein and water diffuses into the MI hydrogel, there is an amount of protein that does not bind to the imprinted cavities but remains in solution inside the pores of the MI hydrogel. It is possible to correct for this artifacts; however, it demands precious time and resources.

Since relevant biomolecules are found in low concentrations, in some cases below the detection limits of the instrument, in this research a technique to multiply a signal due to protein rebinding was studied. This method utilizes a color reporter that is produced due to enzymatic activity, therefore, high enzyme concentrations in the imprinted matrix produces high rates of photometrically measurable product. This technique lacks of real time monitoring and would only be useful when the protein of interest is an enzyme.

Therefore, a direct and real time response method of measurement of the rebinding process needs to be developed.

Some groups have managed to produce two dimensional (2D) films of imprinted polymer where only one face of the proteins is imprinted in the matrix. This imprinted film, attached to the gold surface of a surface plasmon resonance (SPR) sensor, allows for a real time response of the rebinding phenomena. This technology integration is not trivial and difficult to achieve [20-23], and lacks of reproducibility and statistical strength for a high confidence identification of targeted proteins. Additionally, this requires relatively large amounts of crystallized protein and for practical clinical applications; most proteins are difficult to crystallize or to find in relevant quantities. However, with the development of three dimensional (3D) and more sophisticated recognition systems, this technology is likely to be robust enough for clinical use.

Advances in Surface Plasmon Resonance (SPR) allow for the fabrication of flexible sensing devices with much smaller footprint as compared to that of mass spectrometry. It is also possible to produce SPR systems with some degree of portability without compromising robustness. SPR has shown to be a powerful tool in sensing molecules adsorbed onto gold surfaces. The presence of materials such as proteins or other molecules adsorbed in the immediate vicinity of the surface, within 200 nm approximately will change the refractive index on the material. The change of this physical property allows for sensing of the adsorbed molecules. However, as this sensing is non-specific an integration with molecular imprinting is required for discrimination of the species of biomolecules. Other non-imprinting techniques can be employed to overcome the lack of specificity, an example is the immobilization of affinity biomolecules, such as antibodies,

5 onto the gold surface. However this immobilization is often unstable and may not provide reproducible results. Moreover, for some biomolecules the corresponding monoclonal antibodies are expensive and difficult to find or to produce.

In comparison with MI hydrogel, proteins have the advantage that exhibit a high specificity for their target biomolecules. On the other hand, these proteins lack of stability and robustness, and usually, can only be used once in an assay [24]. Therefore the development of a synthetic biosensor will require improvement on both specificity and sensitivity of the molecular imprinting platform. In this study, we propose the development of a new biosensor based on the integration of MIP and SPR technologies for protein detection, which is comparatively smaller and easier to use than spectrometric instruments.

In this project polyacrylamide was selected as the matrix for molecular imprinting.

It’s hydrophilic and biocompatibility characteristics make it an excellent candidate for MI of proteins. Acrylamide and bisacrylamide participate as the monomer and crosslinker building blocks in polymerization. After synthesis was complete, the template molecule was removed. In the case of MI of proteins, their substantial large size prevent their diffusion. A method to facilitate protein removal and rebinding was to use low crosslinked polyacrylamide; however, due to the hydrophilic nature of the polymer, this also allows for greater swelling undermining the integrity of the imprinted cavities. A study to optimize the amount of crosslinker for the least swelling is shown. In addition, positively and negatively charged affinity monomers were included to enhance the affinity towards the target protein. The 3D imprinted platform has been produced in bulk and in nano-thin films, the later the most useful form for coupling with SPR sensors. Moreover, a method for in- situ synthesis and coupling of the thin films to inert gold surfaces was studied. Self-

6 assembling monolayers (SAM’s) were tested as an adhesive layer between the thin films and the gold surface; however, a proposed new technique proved to be more robust than

SAM’s. After coupling the film to the SPR sensor, data was recorded and plotted in sensorgrams. The nano-thin films were fabricated by a casting technique and thickness reproducibility was studied. Finally a molecularly imprinted synthesis in two stages was explored.

In the process of accomplishing the objectives of this research, challenges and obstacles were encountered, especially when working with films in the nanometer range and when coupling the imprinted platform to the SPR sensor. Many approaches have been utilized to accomplish a successful selective MI system. The fabrication process, characterization of the imprinted films, and coupling with the SPR sensor are presented in this study.

Chapter 2

Literature Review

2.1 Molecular imprinting (MI)

Polymers, the most versatile materials, have changed our daily lives over the past decades. Its application in the synthesis of molecularly imprinted matrices goes back to the decade of the 70's with the pioneering work by G. Wulff et al. [25] , and by O. Norrlöw et al. [26] with the aim to design artificial receptors to use in the separation of enantiomers of a chiral molecule.

Molecular imprinting is a technique that allows us to produce a stable polymer matrix with cavities that also includes synthetic recognition moieties of specific affinity for the molecule of interest, similar to the lock and key model for the enzyme and substrate system. This synthetic platform is the equivalent of a molecular receptor that is relatively robust, inexpensive, easy and quick to produce for a multitude of applications [10-12].

In the synthesis of MI polymers, forces such as hydrogen bonding, electrostatic interaction, hydrophobic interactions, π-π stacking, and van der Waals forces are used to determine the spatial arrangement of the functional monomers around the template molecule. This spatial arrangement is then affixed by polymerization of the monomers and

8 crosslinker. Later, the template molecule is easily removed, leaving an empty imprinted cavity with affinity for the target molecule. See figure 2-1.

Free monomer

Free crosslinker

Template protein

Polymerization

Monomer unit Crosslinker unit

Template protein

Protein removal

Imprinted cavity (recognition site)

Figure 2-1. Molecular imprinting of proteins

2.2 Molecular imprinting applications

Over the past years, many groups have published important advances in the fabrication and application of these synthetic platforms. For example, MI technology can be used in:

a) Analytical separations; where imprinted polymers are produced in bulk, ground into particles, and loaded into high pressure liquid chromatography columns for use in the separation of a target molecule from a mixture of similar molecules. An outstanding example is the separation of quercetin, which is a pigment found in plants [27,28].

b) Enzyme-like catalysis; where there is a strong interest in producing artificial enzyme analogues that possess high catalytic activity and at the same time have better accessibility, stability, and catalyze a larger variety of reactions. These enzyme mimics are obtained by the inclusion of catalytically active groups such as imidazole, OH, and COOH during copolymerization. One example is the synthesis of an enzyme-like polymer that mimics the hydrolyzing action of the serine protease chymotrypsin onto L- phenylalaninamide [29].

c) Drug discovery; in this case, it is interesting that absolute specificity is not required for this application. In drug discovery, it is more important to find new compounds of similar shape and functionality, not necessarily from the same chemical class, which will bind and exert an effect at the same imprinted site [30]. An example of this application is the synthesis of a MI testosterone matrix using methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the crosslinker. Overall, the synthesized sensor recognized the testosterone, and showed cross-reactivity to four other similar steroids: β-estradiol, progesterone, testosterone propionate, and estrone [31].

d) Drug delivery systems; although they have not reached clinical application, these systems are needed to administer drugs, particularly if there are absorption barriers, dosage limitations, and protect them from metabolic degradation. Ideally, this delivery system must ensure that the drug is released at the right dosage, place, and time frame; it also needs to be biocompatible, since they are meant to be used in vivo. The drug, coupled to the MIP, would be released when the complex drug-MIP binds to its target in vivo, for example on the surface of a cell [31-34].

e) Molecular sensing; which is the main focus of this study, has proven to be an important application in the detection of relevant molecules for diagnosis of diseases, therapeutic monitoring, and prognosis. Also, in the detection of organisms and toxins, including bioterror agents [35].

2.3 Molecular imprinting in molecular sensing applications

Current technologies such as proteomics mass spectrometry and multiplex immunoassays are extensively used in the diagnosis and prognosis of diseases, even in the monitoring of response to therapy [2-6]. These technologies are very accurate and well developed. However, they require an exhaustive and time consuming sample preparation.

In most cases, sample preparation implies purification through the use of chromatographic columns or with the use of affinity molecules like antibodies that are frequently unstable under chemical and biological agents, expensive and in some cases difficult to find or to produce. MI on the other hand promises to be robust enough to withstand most of these limitations. Some advantages this novel technology offers in sensing applications are the ease, rapid, and inexpensive production, long storage life, reusability, and when coupled

11 with the appropriate technology for signal transduction, it can be used as a portable device for fast detections in remote places.

2.4 Molecular imprinting techniques

In the fabrication of MI polymers, two approaches are widely used, each with its own advantages and disadvantages. Based on the complex formation between the functional monomers and the template molecule during synthesis, molecularly imprinted polymers can be classified into “covalent molecular imprinting” and “non-covalent molecular imprinting”.

Covalent molecular imprinting implies that the template and the functional monomer are covalently bond. After polymerization, the bond is hydrolyzed to remove the template, leaving a functional group that is intended to interact later with the target molecule See figure 2-2. Although this technique facilitates the synthesis of homogeneously imprinted cavities, the template is often difficult to remove. In addition, the rebinding process is slow due to the necessary formation of covalent bonds between the target molecule and the imprinted cavity. Furthermore this technique implies the production of modified template which requires strict synthetic conditions [36,37].

Covalent bond

Bond cleavage and protein removal

Similar imprinted region (recognition site)

Figure 2-2. Covalent MI. Imprinted region is homogeneous to all imprinted cavities.

In non-covalent molecular imprinting, non-covalent forces such as hydrogen bonding, electrostatic interaction, hydrophobic interactions, π-π stacking, and van der

Waals forces are used to determine the spatial arrangement of the functional monomers around the template molecule. After polymerization, the template molecule is easily removed, leaving an empty imprinted cavity with specific complementary shape and affinity for the target molecule [13]. Due to the relatively weak interactions involved in creating the binding sites and the association-dissociation equilibrium that occurs during synthesis between the template and affinity monomers, the imprinted cavities would be of

13 non-homogeneous formation and have different degrees of affinity to its target [38-40], see figure 2-3.

Protein removal

Imprinted region (recognition site)

Figure 2-3. Non-covalent MI. Imprinted recognition sites are non-homogeneous or different for each imprinted cavity.

Although the covalent method produces more homogeneous imprinted cavities and better rebinding than the non-covalent technique, it is limited by the chemistry of the template molecule and the availability of compatible, easily cleavable, functional monomers. Therefore, non-covalent molecular imprinting is more widely used due to the ease of synthesis. Also, there is a wide availability of functional monomers compatible with a multitude of template molecules.

2.5 Consideration in the selection of the molecularly imprinted system

A variety of monomers, crosslinkers and solvents can be used in the fabrication of polymers with specific memory for the target molecule. Molecular imprinting requires the affinity molecules to spatially arrange around the template during polymerization. Since the forces involved are mainly hydrogen bonding and electrostatic interactions, the spatial arrangement is enhanced when the solvent used is a hydrophobic media; therefore it is preferable that the MI be done with organic solvents. However, in the special case of protein imprinting, strict requirements need to be met.

2.6 Molecular imprinting of proteins

Proteins are very sensitive to harsh conditions. The overall charge is easily modified by changing the pH. In the presence of hydrophobic interaction, they denaturate and lose their conformation. Therefore, it is optimal to work with aqueous mixtures and mild pH to protect them from denaturation. These conditions will also prevent non- specific binding to the imprinted cavities [15]. Additionally MI of proteins includes the use of water soluble monomers, functional monomers, and crosslinkers with some degree of biocompatibility.

For this reason, acrylamide and bisacrylamide being a stable water soluble mixture of monomer and crosslinker respectively, are popular choices in the fabrication of MI polymers for proteins and were selected in this study.

2.7 Acrylamide

Acrylamide (C3H5NO, molar mass 71.08g/mol) is a colorless crystalline solid that is formed from the hydration of acrylonitrile. Its IUPAC name is prop-2-enamide.

Acrylamide is soluble in water up to 216g/100ml at 30°C. It is also soluble in methanol

(155g/100mL), ethanol (86g/100mL), acetone (63.1g/100mL), and only minimally soluble in benzene or heptane [41]. It has a high mobility in soil and groundwater, and is also biodegradable. The polymers of acrylamide are called polyacrylamides.

Acrylamide polymerization is a free radical polymerization and extends in a chain reaction fashion. It occurs in the presence of a free radical initiator. A popular choice is ammonium persulfate (NH4)2S2O8, and its free radical formation is depicted in the following equations:

2- -* S2O8 2SO4

-* - * SO4 + H2O HSO4 + OH

After the formation of the free radical, it transfers its activity to the vinyl group of an acrylamide molecule, which then reacts with the vinyl group of a second acrylamide molecule. This process repeats thousands of times and is catalyzed by the presence of

TEMED under an oxygen free environment. End group studies have shown that termination happens with both sulfate and hydroxide groups at the end of the chains [42-

46].

N,N′-methylenebis(acrylamide) (C7H10N2O2), is a popular crosslinker used in the synthesis of polyacrylamide, because it facilitates the formation of networks rather than only the linear polyacrylamide chains. Networks of polyacrylamide are more structurally stable, and its physical stability is highly influenced by the ratio acrylamide/ bisacrylamide in the prepolymer solution.

In 2002, acrylamide was discovered by the Swedish National Food Authority to be present in some cooked foods. It was found that during high temperature processing (fried

16 and oven cooked) of food containing amino acids such as asparagine and reducing sugars, acrylamide is produced during the Maillard reaction that generates flavor and color [47,48].

Due to the carcinogenicity of acrylamide, that many studies have prompted, its interaction with cells, animals, and humans has been extensively studied.

2.8 Polyacrylamide

Polyacrylamides are widely used in industry [16]. They are highly hydrophilic and can be found as highly water adsorbent hydrogels. Polyacrylamide has various applications: as water soluble thickener; as a soil conditioner for agriculture; in wastewater treatment; in the cosmetic industry; in paper-making; in oil recovery; in the textile industry; and in ore processing. To a lesser extent, it is found in molecular biology laboratories as a solid support for the separation of proteins by gel electrophoresis.

Among the different protocols available to synthesize polyacrylamide, there are two major formulations that differ in the type of initiator used. The most used protocol is called chemical polymerization which employs ammonium persulfate and TEMED as initiators. The second most popular initiator recipe, a photochemical polymerization, includes the use of ultraviolet light and the photoinitiator riboflavin-5’-phosphate. In the presence of light and oxygen, riboflavin is converted to its active form and initiates the polymerization.

Polyacrylamide appearance is primarily transparent and elastic. However, its mechanical resistance to deformation and flow depends on the ratio and concentration of acrylamide and bisacrylamide. At high amounts of bisacrylamide, polyacrylamide is highly crosslinked and exhibits a hard, inelastic structure, also could show an opaque white color.

At lower amounts of crosslinker, is it highly elastic and sticky. In addition, its physical properties are also impacted by the amount of initiator used during polymerization. For example at high amounts of ammonium persulfate, the chains of polyacrylamide are short, and visible polymerization would not be obvious.

2.9 Selection of the model protein

A wide variety of proteins can be used as the template in the synthesis of the new sensor platform. After synthesis and removal of the template from the imprinted site, the same model protein is used as a target protein in the rebinding experiments.

One purpose of this study focuses on the understanding of the parameters that affect the recognition and specificity of the imprinted polymer towards the targeted protein. For this reason, proteins that are related in shape, size and overall charge can be selected. For example, hemoglobin, myoglobin, and cytochrome c oxidase are heme proteins, containing the heme group in their structure. They participate in the transport, storage, and reduction respectively of molecular oxygen and are related in primary structure. More strikingly, the secondary structures of myoglobin and the subunits of hemoglobin are virtually identical

[49-51]. Therefore, they are widely used in competitive studies, taking advantage that these proteins are inexpensive and easy to find.

In this study, hemoglobin, β-galactosidase, lysozyme, and bovine serum albumin

(BSA) were used in the synthesis and testing of the sensing platform.

2.10 2D and 3D molecular imprinting

Molecular imprinting of small molecules has been widely practiced. Small

18 molecules easily diffuse through the polymer and bind in the imprinted cavity [14].

However it becomes more difficult in the case of large molecules such as proteins, DNA, and even in the case of cells. Complex three dimensional (3D) shapes and size prevent diffusion through the polymeric network and binding in the imprinted cavity. Some groups have worked around this issue by imprinting on the exterior surface of a film of polymer.

This is two-dimensional (2D) MI [20-23] promises accurate sensing results where the protein template is available in relatively large amounts.

The main drawback of this 2D MI technology is that it requires the template to be immobilized on a support material, which could lead to protein denaturation. In some approaches, the protein is first crystallized on top of the substrate (see figures 2-4 and 2-

5), but this technique requires the use of relatively large amounts of protein, and in some cases the protein cannot be crystallized.

Protein Sugar coating Plasma film deposition

Mica (support)

Peel off and protein 2D protein imprinted Glued to support removal surface

Figure 2-4. 2D molecular imprinting of proteins. Adapted from Turner, N.W., et al. [14]

Sensor Weight Pre-polymerization chip mixture Lysozyme crystals

Cellulose ester membrane

2D protein imprinted After polymerization chip

Removal of cellulose Removal of protein ester membrane crystals

Figure 2-5. 2D molecular imprinting of crystallized proteins. Adapted from Takeuchi, T. and T. Hishiya [21]

3D MI is more flexible in terms of amount of protein needed and ease of synthesis.

In addition there is no need for the template to be immobilized or crystallized. However, when the polymer is highly crosslinked, the diffusion of protein towards the imprinted cavity or out of it, is very slow or even impossible. The porosity of the matrix can be increased by using low amounts of crosslinker, but this comes at the expense of structural stability of the matrix and loss of the imprinted cavity. The lower the amount of crosslinker, the higher the swelling and stretching of the matrix in the presence of the solvent. Both 2D and 3D MI present advantages and disadvantages. In this study we suggest a new 3D molecular imprinting technique that might place it in line with 2D molecular imprinting.

2.11 Sensing of molecular rebinding

Molecular imprinting techniques offer the discriminatory capability in identification of a single targeted molecule among diverse molecules that vary in

20 concentration and species. However, MI is not designed to produce a signal after molecular rebinding. Coupling with a transduction system is required in order to generate a signal that can be perceived in real time or indirectly after molecular binding. In various publications, the use of a transduction system has been applied to small molecules and takes advantage of special chemical properties of the analytes, properties that are not commonly found in proteins [52]. For example, IR, UV-Vis, Raman spectroscopy, fluorescent, or electrochemical are some of the transduction techniques used in sensing.

Other reagentless techniques, such as change in thickness and refractive index, utilize the changes in physical properties of the polymer to detect binding. These techniques are frequently subject to bias and errors that are integral to indirect sensing methods.

Direct sensing is more practical and provides more information in less time. Some groups coupled molecularly imprinted polymers to surface plasmon resonance. Due to the advantages offered by this pair of technologies, their combined use is becoming very popular but there are still challenges to be overcome.

2.12 Coupling Molecular Imprinting with Surface Plasmon Resonance

Surface plasmon resonance (SPR) has shown to be an extremely sensitive tool that can be used in sensors with a very small foot print. Although this technology lacks of any discrimination capability needed to identify a single molecular species, researchers have been able to overcome this by the inclusion of affinity molecules such antibodies and aptamers. As discussed before, these affinity molecules show high specificity towards its complementary target molecules however they lack the robustness and reusability that synthetic platforms can offer.

Coupling of MI polymers and SPR technology is versatile in many ways. SPR not only works as a transduction system, but also allows monitoring in real time of the molecular rebinding. In addition, SPR technology is highly sensitive, relatively cheap, and offers the possibility of portability. However, its implementation imposes special requirements. SPR will only detect changes in the near vicinity of its gold surface, up to

200nm. This implies that the imprinted polymer has to be first fabricated in very thin films and second attached to the gold surface of the SPR sensor. In this study we have overcome these special requirements.

Chapter 3

Experimental Methods

This chapter presents a description of the main experimental procedures and characterization techniques used in the completion of this project.

3.1 UV/Vis Spectrometry

In UV/Vis spectrometry, absorbance of light in the visible and adjacent to the near infrared and ultraviolet ranges are used for the quantitative determination of a large number of inorganic, organic and biological species. Molecules containing π-electrons or non- bonding electrons can absorb ultraviolet or visible light energy to excite these electrons to higher anti-bonding molecular orbitals. When the electrons are more easily excited, they absorb light from a longer wavelength. The measurement is based on the transmittance “T” or the absorbance “A” of a solution contained in a transparent quartz cell having a path length of “b” centimeters. The concentration of the analyte is linearly related to the absorbance as given by Beer’s law (see equation below).

푃0 퐴 = − log 푇 = log = 휀푏푐 푃

Where: A= Absorbance

T= Transmittance

P0 = Incident radiant power

P = Transmitted radiant power

ε = Molar absorptivity

b = Path length of sample

c = Concentration of absorber

Transparent Cuvette Reflection losses at interfaces

Scattering losses in solution

Incident Emergent beam beam

Reflection losses at interfaces

Figure 3-1. Scattering and reflection losses in a typical quartz cell used for UV-Vis spectroscopy. Adapted from L. Rodriguez [34]

All the analyses were conducted in the dual beam UV-2450 UV/Vis spectrophotometer from Shimadzu Scientific Instruments. The UV/Vis spectrum wavelength range from 190 to 900 nm. The use of a dual beam spectrophotometer allows compensation for the reflection and scattering losses of the beam when passing through a typical quartz cuvette, solvent and cuvette-air interfaces. In dual beam spectrophotometer, the power of the beam transmitted by the analyte solution is compared with the power of the beam transmitted by an identical cell containing only solvent [53,54]. In this study, phosphate and TES buffer are used to dissolve the protein of interest. Additionally, the analyte concentration ranges over which the absorbance of the analyte was found to be linear with its concentration. This ensures the applicability of the Beer’s law in that range, and a linear correlation that helped to determine a characteristic of the system.

3.2 Scanning Electron Microscopy analysis (SEM) and Energy Dispersive X-ray

Spectroscopy (EDS)

Scanning electron microscopy provides information about the topography (surface features of the sample), morphology (shape and size of the film making up the sample). In combination with the energy dispersive spectroscopy (EDS) accessory, it also provides information on the elemental composition of the sample.

SEM uses a beam of highly energetic electrons to produce an image of the sample at high resolution and wide depth of field. Images are produced by detecting secondary electrons which are emitted from the surface of the sample due to excitation by the primary electron beam. In this electron microscope, the electron beam is scanned across the surface of the sample, with detectors building up an image by mapping the detected signals with

25 beam position [55-57], see figure 3-2. Samples must be coated with a heavy metal nano- film in order to reduced sample charging by increasing electrical conductivity, improve secondary electron emission and protect the sensitive beam. The maximum resolution of

SEM is near 10 nm. Therefore, images produced by SEM when compared with those produced by light microscopy with equal magnification are 10 times clearer and sharper, giving a perfectly defined tridimensional view of the sample’s surface [56].

In this research, samples were coated with a thin layer of gold under argon atmosphere using a sputter coater, model 108auto from Cressington Scientific Instruments

Inc., and then placed in the scanning electron microscope for analysis. A Hitachi S-4800

High Resolution Scanning Electron Microscope (Hitachi High-Technologies Corp., Japan) was used in this project to characterize the molecularly imprinted nano-films of hydrogels.

The instrument used is located at the Center for Material and Sensor Characterization of

The University of Toledo (Toledo, OH, USA).

EDS is a technique for localized chemical analysis used in conjunction with SEM.

The EDS technique detects X-rays emitted from the sample during bombardment by the

SEM electron beam to characterize the elemental composition of the analyzed sample [58].

The EDS analysis was performed in a Hitachi S-4800 SEM fitted with an Oxford

Instruments EDS detector. An accelerating voltage in the range of 5 to 20 KV can be used for data collection.

Electron beam

Characteristic Backscattered X-rays electrons

Cathodoluminescence

Secondary electrons Auger electrons

Sample Specimen current

Transmitted electrons

Figure 3-2. Electron bean interaction with the sample. Adapted from reference [55]

3.3 Sputter Coating

Sputter coating is a widely used technique for sample preparation in scanning electron microscopy. It is a physical deposition process used to cover a specimen with a thin layer of a conducting material, such as gold, or a metal alloy such as gold/palladium.

On non-conducting materials typically polymers, organic tissue, and glass samples, this deposition forms a conductive coating which is necessary to prevent charging of the specimen due to electron saturation during conventional SEM mode at high vacuum

[59,60]. The sputtering process is driven by the bombardment of the target with energetic argon ions or plasma. Due to the momentum transfer during the molecular collisions

27 between the ions and the metal atoms of the target, the atoms at the surface are ejected, and transported to the surface of the substrate to be coated [61,62].

In this study, a sputter coater model 108auto from Cressington Scientific

Instruments Inc. was used in the deposition of a thin film of gold onto the samples before loading into the scanning electron microscope. It was necessary due to the non-electrical conductivity nature of the glass substrate and polymer samples. Thickness of the gold coating was not measured since the sputter coater was not equipped with a thickness control device, and it was solely controlled by the time of coating, ten seconds of coating ensured enough deposition to avoid charging on imaged samples.

In addition, the sputter coater was also used in the synthesis of a nano-thin film of polyacrylamide. The deposited gold coating was used to create a spacer between two flat glass surfaces, for the casting of a thin film of polyacrylamide. The center of a glass slide was masked in order to leave a cavity for the synthesis of the nano-thin film of polyacrylamide. The thickness of the spacer was controlled by time of deposition.

3.4 Physical Vapor Deposition (PVD)

In physical vapor deposition, an electron beam is used to evaporate metals such as,

Au, Ti, Cr, Al, and Pt. The electrons created by a filament are accelerated onto the top surface of the metal source or target. As a consequence, the top surface of the metal is heated, causing evaporation of the material which will condense onto the surface of the substrate to be coated. Evaporation is done in a high vacuum chamber to allow the evaporated material a clear path to the substrate and to reduce impurities in the film [63], see figure 3-3.

The electron bean uses a small point source and a long deposition distance. This allows the deposition of material to be in a directional fashion; therefore the evaporated films do not coat tall or deep features on the substrate. Also the heat transfer to the substrate is limited by the long deposition distance in vacuum. The thickness of the deposited metal layer is controlled with a quartz crystal monitor feedback.

The controller pre-heats the source materials with the shutter closed. Then, during deposition, the controller uses the feedback from the crystal monitor and varies the electron beam power to maintain a preset deposition rate and time to achieve the programed total thickness.

The PVD equipment at Laurie Nanofabrication Facility located in the University of

Michigan in Ann Arbor was used for the coating with titanium and gold of a glass coverslip in the preparation of a gold surface for Surface Plasmon Resonance. An adhesion layer of

10Å of titanium was first deposited onto the glass coverslip, then 500Å of gold.

Rotating mechanism Substrate holder

Vacuum chamber

Source material

Figure 3-3 Physical vapor deposition schematics

3.5 Surface Plasmon Resonance (SPR)

Surface plasmon resonance is used in the study of biomolecular interactions where one molecule is immobilized onto a gold surface and the other is free in solution. This technique is very sensitive and produces signal in real time.

The study of biomolecular interactions refers to protein-protein, DNA-protein, and lipid-protein interactions, and does not require the use of labels for signal transduction.

Signal versus time from the SPR is plotted arbitrarily in a sensorgram which is used to determine the interaction between species of molecules, to characterize the strength and equilibrium of this interaction, and to measure the concentration of the analyte [64].

In SPR systems, plasmon on the surface of noble metals are induced by the incidence of polarized light, which will only occur at a specific angle of incidence φ called resonance angle. The metal surface acts as a mirror, and at the resonance angle, the reflected light will achieve a minimum intensity because photons from the polarized light interact with the free electrons of the metal surface, causing a wave oscillation of the electrons and therefore reducing the intensity of the reflected light. Any disturbance in the immediate vicinity, even a few hundred nanometers, will cause a change in the refractive index of the metal surface. This causes a shift in the angle of reflection for the minimum intensity. This change is commonly used to detect molecular deposition of the analyte on the metal surface of the SPR sensor [65-68], see figure 3-4.

Mobil phase running through Solution containing protein is Protein adsorption and the channel injected into the flow channel accumulation on the gold surface

Flow Channel Surface Plasmon

Gold Layer φ

Optical Photodiode Reflected array light RRU Buffer

Buffe r

Protein injection Time

Figure 3-4. Disturbance of the resonant plasmon by adsorption of protein on the gold surface. The signal change due to the shift in the angle of reflected light is represented in the sensorgram by the green line.

The first use of SPR to study biomolecular interactions was accomplished by

Liedberg in 1983 [69]. Since then, numerous groups have applied SPR in biomolecular sensing. Although SPR is very sensitive to the accumulation of molecules in its vicinity, it does not discriminate among species of molecules. To circumvent this deficiency, affinity molecules are immobilized onto the surface of the metal film of the SPR sensor. Gold is the standard metal used in the SPR technology due to its chemical stability. However, as it is chemically inert, it is difficult to covalently immobilize affinity molecules onto its surface. For this reason, the gold surface is usually coated with a self-assembled monolayer

(SAM) that works as a bridge between the gold and the affinity molecule [70,71], see figure

3-5.

Complementary Immobilized antibody antigen

Monolayer

Figure 3-5. Antibody immobilized onto the gold surface of an SPR sensor. Only complementary antigen will bind with the antibody, other non-specific molecules are washed away by the mobile phase.

The immobilization of affinity molecules imply the use of antibodies; however in this study, we coupled SPR technology with the synthetic molecularly imprinted platform developed in our lab. The SensiQ Discovery SPR instrument from SensiQ Technologies

Inc. was used, (see figure 3-6 and 3-7). We modified the setup by removing the gold surface

32 from the sensors with the use of aqua regia, a highly corrosive mixture of 1 part of nitric acid and 3 parts of hydrochloric acid. Additionally a new gold surface was prepared and used; it was manufactured by physical vapor deposition of gold onto a glass cover slip. The cover slip was attached to the prism of the SPR sensor using an index matching fluid

(refractive index 1.515) [72], similar to that of the glass cover slip and the prism inside the

SPR sensor, see figure 3-8 and 3-9.

Figure 3-6. SensiQ Discovery SPR instrument located at the Center of Material and Sensor Characterization in the University of Toledo

Figure 3-7. Schematics of the internals of an SPR sensor from SensiQ Technologies Inc. Note its size is comparable to a quarter dollar coin. Picture taken from the SensiQ user’s manual.

Figure 3-8. Gold chip mounted on the prism of the SPR sensor.

Sample Microfluidic Sample Outlet Channel Inlet

Sensor chip (Gold coated cover slip)

Optical LED Photodiode medium array (Prism)

Figure 3-9. Final setup of the gold surface coupled with the SPR sensor and mounted on the microfluidic channel.

3.6 Freeze Drying

Freeze-drying also known as lyophilization, is a popular dehydration process used to preserve perishable materials. It removes water from usually unstable products to preserve them in a dried stable formulation. Freeze-drying is accomplished by reducing the pressure on frozen samples to allow water in the material to sublimate directly from the solid to the gas phase. The freeze drying process is typically divided into four stages. The

34 pretreatment stage, where the material is treated to reduce unstable components, increase its surface area, or to include additives such as stabilizers, flavor and texture enhancer. In the freezing stage, the material can be either fast or slowly frozen in the lab. Dry ice or liquid nitrogen is used to reduce the temperature of the material to below its triple point

(the temperature and pressure at which liquid, solid, and gas phases of a substance coexist).

In the primary drying stage, most of the water is removed by sublimation due to the decrease of pressure; in this stage, it is important to control the pressure and temperature conditions in order to guide the system around the triple point, avoiding the liquid-gas transition seen in ordinary drying process, see figure 3-10. In addition, heat is allowed mainly by radiation to provide enough energy and guaranty the sublimation of water.

Finally in the secondary drying stage, more heat is allowed into the system in order to remove water that is physicochemical bonded to unfrozen material [73-75].

Pressure Solid Phase Liquid Phase Critical Point Freezing point at Boiling point at 1atm 1atm Ordinary drying route 1 atm

Freezing drying route Triple Vapor Phase Point

0°C 100°C Temperature

Figure 3-10. Phase diagram of water. Freeze drying is depicted by the green arrow.

In this project, freeze drying was used to study the swelling properties of different formulations of polyacrylamide in the presence of water. The samples were frozen in a freezer, immersed in liquid nitrogen, then were freeze-dried in a system that consisted of a container connected to a vacuum pump via a dry ice cold trap. The cold trap was used to prevent the moisture from being drawn into the vacuum pump. After freeze-drying, sample thickness was determined using a caliper.

3.7 Matrix-Assisted Laser Desorption Ionization-Time of Flying Mass

Spectrometry (MALDI-TOF MS)

During MALDI analysis, the analyte is mixed with a matrix that converts ultra violet light to heat energy. The matrix heats up rapidly and is vaporized with the ionized analyte. TOF is a mass spectrometry technique that is widely used due to its large mass range and utilizes the difference in mass to charge ratio (m/z) to resolve the ionized analyte.

The ionized species of various sizes, are released from the solid phase, and are driven by a constant potential difference between ground and the sample holder. Using the law of conserved energy and the ionized analyte path, it is possible to determine their velocity.

Ions with smaller m/z value and highly charged ions, move faster through the drift space until they reach the detector. Therefore, the time that ions spend during travel differs according to the m/z value of the ion [76-80].

In this study, the UltrafleXtreme MALDI-TOF/TOF mass spectrometer from

Bruker Daltonics at the Instrumentation Center of the University of Toledo was used in the investigation of specific polymeric molecules that are synthesized during molecular imprinting on polyacrylamide.

3.8 Atomic Force Microscopy (AFM)

AFM is a technique used to characterize the surface of a material at the atomic level. It provides a 3D topography of the surface. The prime advantage of AFM is its ability to image non-conducting samples without any specific treatment, thus allowing imaging of delicate biological and polymer nanomaterial. AFM requires minimal sample preparation and can be performed in ambient conditions. The technique involves imaging a sample with the use of a probe, or tip, with a radius of approximately 20 nm. The tip is supported by a flexible cantilever and held very close to the surface of the sample (0.2-10nm). When the AFM tip touches the surface, the small Van der Waals force between the tip and the sample surface is monitored during scanning. Compiling the tip-sample interactions provides the image of the sample surface. The AFM can be operated in static contact mode, dynamic non-contact or tapping mode depending on the type of application.

In this research, AFM tapping mode was the technique used to evaluate the topography of the gold chips at a scan rate of 0.50 Hz. The analysis was done at the Center for Material and Sensor Characterization at The University of Toledo. The instrument used for this analysis was the Veeco Multimode Nanoscope IIIa AFM from Bruker Corporation.

In tapping mode, the cantilever is oscillated at its resonant frequency, see figure 3-11. The tip lightly touches on the sample surface at constant oscillation amplitude during scanning.

With this mode, it is possible to achieve high resolution images with lessen damage to the surface of the sample. This mode is also recommended for soft and fragile samples. In addition, Tapping Mode incorporated with Phase Imaging can be used to detect variations in composition, adhesion, friction, and viscoelasticity of the sample. Applications include identification of contaminants, mapping of different components in composite materials,

37 and differentiating regions of high and low surface adhesion or hardness. Phase Imaging is a powerful technique for producing contrast on heterogeneous samples. This mode is used commonly for mechanical and composition characterization of sample surfaces. Phase imaging is the mapping of the measure phase of the cantilever’s periodic oscillations, relative to the phase of the periodic signal that drives the cantilever. Changes in this measured phase often correspond to changes in the properties across the sample surface.

The phase and topography images of a material are usually collected simultaneously [81-

84].

Photo diode Laser beam

Sample Cantilever

Piezo z oscillator x y

Controller box

Figure 3-11. Schematics of an AFM in tapping mode. The cantilever drives in specific amplitude onto the surface of the sample. The piezo oscillator moves the samples in the x,y, and z direction.

Chapter 4

Synthesis of polyacrylamide hydrogel

4.1 Introduction

Polyacrylamide hydrogel is highly hydrophilic and protein compatible. It is synthesized in a free radical polymerization reaction of acrylamide and bisacrylamide, and its physicochemical properties are highly influenced by the experimental conditions set for its preparation. Bisacrylamide participates as the crosslinker in the polymeric network of polyacrylamide. In the prepolymer mixture, the monomer and crosslinker are mixed with the initiator and accelerator of the reaction in water media. In addition, nitrogen gas is used to prevent oxygen from entering the system and undermining the reaction.

In this chapter it is shown how the variation in component ratios (acrylamide, bisacrylamide, and water) impact the degree of crosslinking and furthermore the structural stability and physical behavior of the polymeric matrix. In addition, it is common to store the gels in water to prevent drying and shrinkage. However, this leads to a negative effect due to the hydrophilic nature of the polymer. The hydrogel will swell causing, the chains that define its structure to stretch in order to contain the intake of water. Therefore swelling studies had to be performed in order to determine the best synthesis conditions for an optimum imprinted hydrogel. 39

After synthesis is completed, the hydrogel adopts the shape of the mold it was casting and has to be stripped from the container. In this study, dichlorodimethylsilane

(repel-silane) was used as a hydrophobic coating onto the glass surfaces to prevent the hydrogel from sticking to the surface of the container.

4.2 Glassware preparation before polyacrylamide synthesis

An important step in the synthesis of bulk hydrogels is the ease of detaching the bulk hydrogel from the mold in which it was synthesized. For this purpose, a stable hydrophobic coating on glassware is used prior to the initiation of the polyacrylamide synthesis. The lab grade glassware is treated with dichlorodimethylsilane, also called repel- silane, which forms a monolayer on the glass surface, exposing the hydrophobic methyl groups outward and preventing hydrophilic molecules from contacting and adhering to the glass surfaces of the mold.

3HC CH3 Si

Cl Cl

Figure 4-1. Dichlorodimethylsilane also called repel-silane is used for hydrophobic coating of glass surfaces.

In a fume hood, dichlorodimethylsilane was dropped into pre-cleaned dry glass petri dishes and vacuum flasks. One drop is sufficient to coat the inside and outside surfaces. After 2 minutes, the glassware was rinsed with toluene twice, and then rinsed with methanol three times. It is extremely important to take special care during this cleaning process in order to effectively remove any unbound dichlorodimethylsilane from 40 the container. The coating procedure was performed after three uses of the glassware to prevent the polyacrylamide from sticking to the glass surfaces.

4.3 Materials for synthesis of polyacrylamide hydrogel

Acrylamide and bisacrylamide were obtained with 99.9% purity from Polysciences

Inc. Ammonium persulfate and TEMED were obtained from Sigma-Aldrich. Ultra high purity nitrogen 99.999% was used for degasing of the prepolymer solution and as a blanketing gas during polymerization. Purified water with a minimum resistivity of 18.0

MΩ was obtained from an ultrapure water system, and was used in the preparation of all solutions and mixtures. Dichlorodimethylsilane 99.5% purity was purchased from Sigma-

Aldrich. Toluene and methanol ACS reagent grade were purchased from Fisher Chemical.

All glassware was of lab grade.

4.4 Bulk synthesis of polyacrylamide hydrogel

Amounts of acrylamide and bisacrylamide described in the tables of section 4.6 were combined in a 100ml vacuum flask. A volume of 10ml of ultrapure water was added to the container and a PTFE coated magnetic stirring bar was used to facilitate dissolution on a magnetic stir plate at 80rpm at room temperature. After 5 minutes, the stirring bar was retrieved from the vacuum flask, and the container was placed on the benchtop with a rubber stopper to seal the top. A vacuum pump was used to degas the prepolymer solution for 5 minutes or until no air bubbles came from the solution. After degassing, the vacuum hose was disconnected and a nitrogen stream blanket was applied to the system to prevent oxygen from contaminating the prepolymer solution. Ammonium persulfate was injected

41 using a micro pipette, followed by an injection of TEMED via a 10ul micro syringe.

Shaking was performed by hand. The prepolymer solution was poured into a petri dish coated with repel-silane. It is crucial to maintain the nitrogen atmosphere at all times, because, a small amount of oxygen may lead to irreproducible results. Visible polymerization occurs at 20 minutes and continues up to 2 hours at room temperature. After synthesis is complete, the nitrogen stream is stopped. The bulk hydrogel can then be removed from the petri dish and placed into a container with ultrapure water for storage at room temperature. Figure 4-2 shows graphically the steps and the setup of the experiments for the synthesis of bulk polyacrylamide hydrogels.

Mix at room temperature for 5 minutes Acrylamide

Bis-acrylamide UP H2O

Ammonium persulfate and Nitrogen Degas to eliminate TEMED are dissolved oxygen injected

Vacuum pump

Mix to start polymerization. Blanket with nitrogen gas

Nitrogen

Pour the solution into the Allow polymerization for silane coated petri dish and at least 2 hours under blanket with nitrogen gas nitrogen gas

Figure 4-2. Bulk synthesis of polyacrylamide hydrogel

4.5 Synthesis of hemoglobin imprinted hydrogel

The imprinting of proteins requires the presence of the template protein in the prepolymer mixture before synthesis. The template, bovine hemoglobin (Bhb), is an

43 oxygen transport protein with a molecular weight of 64500g/mol and an isoelectric point

(pI) of approximately 6.8. After synthesis is completed, removal of the template is necessary in order to test further for rebinding of hemoglobin.

A washing solution composed of a mixture of sodium dodecyl sulfate (SDS) with and acetic acid is popularly used to denaturate the template protein and help to remove it, leaving empty imprinted cavities in the hydrogel. SDS works as an anionic detergent for protein solubilization. It is commonly found in PAGE-SDS systems for the measurement of molecular weight of proteins.

4.5.1 Materials

Acrylamide and bisacrylamide were obtained with 99.9% purity from Polysciences

Inc. Ammonium persulfate and TEMED were obtained from Sigma-Aldrich. Ultra high purity nitrogen 99.999% was used as a blanketing gas. Purified water with a minimum resistivity of 18.0 MΩ was obtained from an ultrapure water system, and was used for the preparation of all solutions. Bovine hemoglobin (BHb), sodium dodecylsulphate and acetic acid were purchased from Sigma-Aldrich and used as received.

4.5.2 Hemoglobin imprinting procedure

An amount of 600mg of acrylamide and 60mg of bisacrylamide were mixed with

4ml of water in a 100ml vacuum flask. In another vacuum flask, 140mg of bovine hemoglobin was dissolved in 6ml of water. Both solutions were degased with the use of a vacuum pump and then blanketed with a stream of nitrogen following degassing, the hemoglobin solution was added to the monomer solution and 10mg of ammonium

44 persulfate and 6ul of TEMED were added to the system to initiate synthesis. The prepolymer solution was poured into a repel-silane coated petri dish and polymerization was allowed under nitrogen environment at room temperature for 2 hours. After the completion of the synthesis, the samples were stored in a refrigerator or used immediately in further experiments.

4.5.3 Protein removal

After synthesis, it is noticeable that the bovine hemoglobin produced an intense red-brown color in the hydrogel. The hydrogels were rinsed with copious amounts of water, and subsequently a series of washes were performed using a mixture solution containing

3% (v/v) of acetic acid and 3% of SDS (w/v). This washing solution did not completely remove the bound protein, even when using 5% or 10% concentrations of the washing solution. The template removal was never one hundred percent efficient; this is supported by observation on the color of the hydrogel when imprinted with hemoglobin (rust-brown color) as shown in figure 4-3. This strongly suggests limitations in protein diffusivity and entrapment into bulk hydrogels for MI applications. Most of the proteins are trapped in the hydrogel due to the large size of the protein, the degree of crosslinking, and the fact that some protein could be covalently immobilized during polymerization.

Figure 4-3. At the right, bulk polyacrylamide hydrogel imprinted with hemoglobin. At the left, a twin bulk imprinted hydrogel was washed with SDS and AcOH 3%. Both disks of hydrogel were synthesized in a 5cm Ø diameter petry dish.

4.6 Tuning of the concentration of monomer and crosslinker

Many groups reported the challenges that protein molecular imprinting imposes in the development of a novel biosensor. One such problem is the large size and complex shape of proteins that hinder its diffusion through the network of the polymeric matrix.

Moreover, if the matrix is highly crosslinked, it can be impossible to remove the template.

A way to circumvent this complication could be by using low amounts of crosslinker during the synthesis.

Exposed to high volumes of water, the hydrogel swells. This behavior is limited by the crosslinking concentration. The higher the amount of crosslinker, the less the swelling.

At low crosslinking concentration, porosity is high and the template protein easily diffuses out of the imprinted cavity. However, because this removal of template process is performed in water, at low crosslinking the swelling is high, promoting stretching of the imprinted cavity and undermines its recognition properties.

Therefore, a successful molecular imprinting requires precise tuning of the amounts of the components set for the synthesis. In this study, the objective is to produce a hydrogel

46 that shows the lowest swelling at the lowest crosslinking concentration.

The following tables show eleven different recipes used to synthesize the hydrogels. No protein was used in these syntheses. The amounts of acrylamide, bisacrylamide, and water where changed for each sample in order to test the swelling behavior. All hydrogels were synthesized in flat bottom petri dishes following the procedure described in section 4.4. After synthesis, a portion of the hydrogels was frozen in the freezer, then it was immersed in liquid nitrogen for two minutes and placed in the freeze drying setup and leave overnight. After the drying process was completed, using a caliper, the thickness of the cross section was measured and averaged for each sample. A second piece of each hydrogel was immersed in ultra-pure water and stored overnight at room temperature. After 24 hours in water, the swollen hydrogels are fast frozen and then thicknesses were recorded and averaged. Swelling percentages were calculated by the following formula:

푉푓 − 푉표 푆푤푒푙푙푖푛푔 % = × 100% 푉표

Where : Vo is the initial thickness after synthesis and before exposure to water

Vf is the final thickness after 24 hours immersed in water

The following table 4.1 shows the concentrations of monomer and crosslinker used in the synthesis of 6 hydrogels. The amounts of initiator and accelerator of the reaction were constant for all the samples. From samples 1, 2, and 3, it is observed that as the concentration of acrylamide is increased, the physical appearance of the hydrogel changes 47 from a transparent viscous oil to a consistent gel. A similar observation occurs with an increase in the concentration of bisacrylamide in samples 4, 5, and 6. Note that only in the cases where the hydrogel maintains its physical shape, it was possible to characterize its swelling behavior. As seen in table 4.2, it was possible to measure the swelling percentage for samples 3, 5, and 6 only. Sample 6 was determined to have the minimum swelling with an 11.3% increase with respect to its initial state, before exposing it to fresh water.

Table 4.1 Different compositions for the synthesis of hydrogels. Samples 1 to 6.

Component Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6

Acrylamide 200 400 800 200 400 800 (mg)

Bisacrylamide 1 1 1 2 4 6 (mg)

APS 2.5 2.5 2.5 2.5 2.5 2.5 (mg)

TEMED 3 3 3 3 3 3 (µl)

Water 9.5 9 8 9.5 9 8 (ml)

Note: The concentration of ammonium persulfate (APS) and TEMED were constant across all samples.

Table 4.2 Swelling results of samples 3, 5, and 6

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6

Physical Viscous oil Viscous glue Gel Viscous glue Gel Gel appearance

Initial average - - 5.6 - 5.5 6.0 thickness (mm)

Final average - - 8.4 - 6.7 6.7 thickness (mm)

Swelling (%) - - 48.7 - 22.4 11.3

Another set of samples 7 and 8 were synthesized. Table 4.3 shows that the concentration of acrylamide was maintained constant and only the bisacrylamide concentration was changed. Again, as the concentration of the crosslinker is increased, the hydrogel is physically more stable. It maintains its shape and shows low swelling after being exposed to water during 24 hours. Samples 3 and 6 from the previous table were included to compare with the new set of samples 7 and 8. It is clear that the most influential component of the hydrogel that impacts its physical appearance is the concentration of the crosslinker. Sample 6 is still the best hydrogel in terms of stability in the presence of water.

Table 4.3 Variation of the amount of crosslinker for the synthesis of the hydrogels. Samples 3, 6, 7, and 8.

Component Sample 8 Sample 3 Sample 7 Sample 6

Acrylamide 800 800 800 800 (mg)

Bisacrylamide 0 1 2 6 (mg)

APS 2.5 2.5 2.5 2.5 (mg)

TEMED 3 3 3 3 (µl)

Water 8 8 8 8 (ml)

Note: The concentration of ammonium persulfate (APS) and TEMED were constant across all samples.

Table 4.4 Swelling results of samples 3, 6, 7, and 8

Sample 8 Sample 3 Sample 7 Sample 6

Physical Viscous glue Gel Gel Gel appearance

Initial average - 5.6 5.1 6.0 thickness (mm)

Final average - 8.4 7.0 6.7 thickness (mm)

Swelling (%) - 48.7 37.3 11.3

The last set of samples 9, 10, and 11was prepared to test the influence of the amount of water used in the synthesis. Table 4.5, shows that as the water content is increased, the physical stability decreases. Also, high amounts of water also impact its swelling. When the amount of water for the synthesis is doubled, the swelling percentage is three times higher. This is suggested by the swelling percentage results from samples 6 and 9 in table

4.6.

Table 4.5 Variation of the amount of water used in the preparation of the hydrogels.

Component Sample 6 Sample 9 Sample 10 Sample 11

Acrylamide 800 800 800 800 (mg)

Bisacrylamide 6 6 6 6 (mg)

APS 2.5 2.5 2.5 2.5 (mg)

TEMED 3 3 3 3 (µl)

Water 8 15 30 40 (ml)

Note: All other components were maintained at constant concentration.

Table 4.6 Swelling results of samples 6, 9, 10, and 11

Sample 6 Sample 9 Sample 10 Sample 11

Physical Very viscous Gel Gel Viscous glue appearance glue

Initial average 6.0 5.3 - - thickness (mm)

Final average 6.7 7.1 - - thickness (mm)

Swelling (%) 11.3 33.6 - -

4.7 From bulk to thin films of polyacrylamide hydrogel

An important factor that affects the experiments on molecular imprinting is the time the protein takes to diffuse out of the polymeric matrix during the protein removal process, as well as back into the imprinted cavity during the rebinding test. This intricacy is highly influenced by the size of the bulk hydrogel, because the protein has to travel through relatively long distances (several millimeters to a few centimeters) in order to be completely removed from or fully cover all imprinted cavities in the matrix, during removal or rebinding respectively. However, when the size of the hydrogel is small, such as in thin films of 1mm or less, the protein diffuses basically through one dimension. In addition, protein removal after synthesis is faster and more efficient that in bulk synthesis.

For this reason, thin films of polyacrylamide were produced, with only one drawback to this design. The films are more difficult to handle, are delicate and tend to break under manipulation. A way to overcome this is by attaching the film to a solid support. Therefore, in this study, in-situ synthesis was chosen to fabricate the films directly attached to one surface of glass slides. 51

When the films were synthesized on the glass support, the films partially detached after manipulation. For this reason, a pre-treatment was needed to enhance the attachment.

Bind-silane, 3-(trimethoxysilyl) propyl methacrylate is a chemical used to attach polyacrylamide onto glass for media support in isoelectric focusing. Bind silane is also used to attach cells and microscopic sections of organs to slides and other glass surfaces for in situ hybridization purposes. After coating with bind-silane, the vinyl groups are exposed outward and are able to copolymerize with acrylamide and bisacrylamide, forming a strong covalent bond that prevents detachment.

CH3

O O H2 H2 2HC C C C Si O CH3 C O C H2 O CH3 CH3

Figure 4-4. 3-(trimethoxysilyl) propyl methacrylate molecule, also called bind-silane used to strongly attach polyacrylamide to glass surfaces.

4.7.1 Coating of glass slides with bind-silane

All glass slide surfaces were deeply cleaned with piranha solution a mixture of sulfuric acid and hydrogen peroxide in 7:3 proportions respectively. Piranha solution is prepared fresh and used immediately following strict precautions since this mixture is extremely reactive and corrosive. The glass slides are immersed in a glass beaker, containing piranha solution and left for 4 hours to remove any organic contaminant from the glass surfaces. Following the cleaning the substrate (glass slides) are rinsed in ultrapure water followed by 200 proof ethanol. The substrates are then dried in a drying oven at 60°C for 2 hours. 52

Bind-silane was obtained from Sigma-Aldrich and used as received. All glass slides were treated with bind-silane solution as follows:

A volume of 50ul of bind-silane is added to a tube containing 10 mL of 200 proof ethanol. Immediately prior to use, 300ul of 10% glacial acetic acid in water is added to the mixture; the methyl groups of bind-silane hydrolyzes at acidic pH and the oxygen molecules bind with silicon molecules on the surface of the glass support. The glass slides are then immersed in the mixture and left for 3 minutes. Finally the substrates are thoroughly rinsed with ethanol to remove any residual reagent, and allowed to dry in nitrogen at room temperature.

4.7.2 Synthesis of thin films of polyacrylamide

A procedure similar to that described in section 4.4 was followed, but instead of pouring the prepolymer solution into the petri dish, a degased drop of the mixture was set onto the surface of a glass slide previously coated with bind-silane. Spacers with a thickness of 0.5mm were used as a frame set close to the borders of the slide to create enough separation for the casting of the film. A second glass slide coated with repel-silane

(see section 4.2) was set on top of the drop and a 200 grams weight was used to maintain contact between the second glass slide and the spacers. After 2 hours under nitrogen environment at room temperature, the repel-silane coated glass slide and the spacers were striped from the system, leaving a 0.5mm thin film attached to the bind-silane coated glass slide, see figure 4-5.

Repel-silane Prepolymer coated glass 0.5mm thin solution Spacer slide film

Bind-silane coated After 2hrs the repel- glass slide silane coated glass slide is striped

Figure 4-5. Thin film synthesis onto a solid support.

4.8 Discussion

Bulk polyacrylamide hydrogels were successfully synthesized, and in order to finely tune their swelling behavior in the presence of excess of water, a swelling test was performed over different recipes for polyacrylamide synthesis. It was found that variations in the amount of monomer, crosslinker, and water added to the prepolymer mixture highly influence the hydrogel swelling characteristic. A swelling of 11.3% was the lowest result suitable for molecular imprinting experiments. Thin films of polyacrylamide hydrogel of approximately 0.5mm thickness were successfully prepared using a casting technique that included spacers and glass slides coated with-bind silane and repel-silane. The delicate films were also attached to the glass slides in order to protect them during handling.

Chapter 5

Affinity characterization of protein imprinted hydrogel

5.1 Introduction

In the previous chapter, a method for polyacrylamide synthesis and characterization of its swelling properties were explored in order to fine-tune the best monomer and crosslinker concentrations for the minimum swelling/stretching of the matrix. In this chapter, a protein model is included during the synthesis to allow the formation of protein imprinted cavities. In addition, a charged monomer was included in the synthesis which would help to enhance the affinity of the imprinted cavity towards the target protein. Also a method to characterize the affinity of the imprinted hydrogel towards the target protein is developed. Emission of a signal due to protein rebinding is based on the use of a reporter that enhances the signal in cases where the signaling is lower than the detection limit of the instrument used for the detection.

5.2 Use of charged monomer

Two charged monomers can be used to test for enhancement of the affinity characteristic of the imprinted matrix towards the target protein. Positively charged monomers effect attraction of negatively charged molecules, and negatively charged 55 monomers have a similar effect on the positively charged molecules. These functional monomers, once copolymerized with acrylamide and bisacrylamide, would facilitate the attraction and orientation of the target protein into the imprinted cavity, due to attraction of the oppositely charged regions on the surface of the target protein. (3-acrylamidopropyl) trimethylammonium chloride (APTAC), see figure 5-1, was included as a positively charged functional monomer and its vinyl group can copolymerize with acrylamide and bisacrylamide. During the synthesis process, the APTAC interacts with the negatively charged regions on the template and copolymerization is then initiated, see figure 5-2. After the synthesis is complete and the template is removed, the imprinted cavities would have enhance attraction towards the protein, and aid in the orientation of the structure of the protein to fit into the imprinted cavity.

O CH3 H2 H2 2HC C C C N CH3

C N C Cl H H H2 CH3

Figure 5-1. Positively charged monomer (3-acrylamidopropyl) trimethylammonium chloride (APTAC) used in the synthesis of imprinted hydrogel.

Since this charged monomer would also attract other non-specific negatively charged molecules, the highest purity chemicals and media were used in the imprinting process. Moreover, a low ratio charged monomer/protein template was used to assure that none of the charged monomer is found in a branch outside of an imprinted cavity.

Another concern that needs to be addressed is that positively charged monomers can be attarcted by negatively charged glass surfaces of the container where it is handled 56

[85]. For this reason, all glass containers were treated with repel-silane which could help to prevent polar molecules from contacting the glass surface. Repel-silane also helps to prevent adsorption and denaturation of the proteins on the glass surfaces.

Positively charged monomer

Negatively charged region

Figure 5-2. Inclusion of a positively charged monomer in the synthesis of molecularly imprinted hydrogel. Red color corresponds to a negatively charged region on the surface of the protein. Blue color corresponds to the positively charged region on the imprinted cavity.

5.3 Protein template and a proposed mechanism for signal intensification

A wide variety of proteins can be used in the synthesis of the new sensor platform.

In the detection of relevant proteins for diagnosis applications, protein markers are found in low amounts or are expensive and difficult to obtain. Assay development, testing, and protein detection itself requires that the technology be capable of detecting low amounts of available protein. Otherwise, a different approach is necessary in order to detect such low levels. One option is the use of protein concentration methods, but this introduces

57 additional steps and complications. Another approach is the use of a mechanism for enhancement of the signal due to the presence of target protein bonded into the imprinted cavity. However, this requires a special type of template. In this research, signal amplification is achieved by the use of an enzyme as the protein template, and the breakdown of its substrate is used for signal amplification related to the amount of protein bonded to the imprinted cavities.

5.4 β-galactosidase as protein template

For the reasons mentioned before, β-galactosidase was selected as a protein model.

β-galactosidase is an enzyme with molecular weight of 465KDa, see figure 5-3, it catalyzes the hydrolysis of β-glycosidic bonds. The advantage of β-galactosidase is that it assists in the degradation of a synthetic analog of lactose o-nitrophenyl β-D-galactopyranoside

(ONPG) to galactose and o-nitrophenol, see figure 5-4. The product, o-nitrophenol exhibits a yellow color, and its rate of formation can easily be monitored in a UV-vis spectrophotometer with a peak absorbance at 420nm wavelength. Hence high rates of o- nitrophenol formation correlates with relatively high amounts of β-galactosidase. The detection of this enzyme bound in the imprinted polymer, below the detection limit of the

UV-vis spectrophotometer, is addressed by this indirect method. One disadvantaged of this technique is the protein template is limited to enzymes.

Figure 5-3. β-galactosidase from Escherichia coli. The picture shows charges on the surface of the enzyme, negative charges in red color and positive charges in blue color. The image was produce with the software Cn3D, version 4.1 by the National Center for Biotechnology Information (NCBI).

CH2OH CH2OH O OH O B-galactosidase OH OH O + OH OH HO

H2O NO2 OH OH NO2 ONPG (colorless) Galactose (colorless) o-nitrophenol (yellow)

Figure 5-4. Hydrolysis of ONPG by enzyme β-galactosidase

5.5 Test to determine relative enzyme abundance in the imprinted hydrogel

ONPG is colorless, but the product o-nitrophenol exhibits a yellowish color, so that as the reaction progresses, more o-nitrophenol is produced, and the yellow color of the solution is intensified. By measuring the rate at which the o-nitrophenol concentration increases, the amount of β-galactosidase can be calculated.

Absorbance, also called optical density (OD), is a measure of the amount of light absorbed by a sample. O-nitrophenol absorbs light maximally at 420nm. Thus the increase of OD420 with respect to time in this experiment correlates with the amount of o-nitrophenol being produced in the sample and can be used to calculate the activity of β-galactosidase, bound in the imprinted cavity [86]. The correlation below helps to illustrate this statement.

Δ푀표−푛𝑖푡푟표푝ℎ푒푛표푙 ∆퐴푏푠420 [β-galactosidase] ∝ Activity of β-galactosidase ∝ ∝ Δ푡 ∆푡

Where: Mo-nitrophenol = Molar concentration of o-nitrophenol

Abs420 = Absorbance reading at 420nm wavelength

t = time

The activity of β-galactosidase is measured in units. One unit will hydrolyze 1µmol of ONPG per minute at a pH 7.3 and at 37°C. To determine the relative concentration of

β-galactosidase in a hydrogel sample with respect to other samples, the rates of formation of o-nitrophenol is divided by the mass of the sample, given that all samples are prepared under the same conditions.

5.5.1 Materials

Acrylamide and bisacrylamide were obtained with 99.9% purity from Polysciences

Inc. Ammonium persulfate, TEMED, and APTAC were obtained from Sigma-Aldrich.

Ultra high purity nitrogen 99.999% was used for degasing of the prepolymer solution and as a blanketing gas. Purified water with a minimum resistivity of 18.0 MΩ was obtained 60 from an ultrapure water system, and was used in the preparation of all solutions and mixtures. Sodium dodecyl sulphate, acetic acid ACS reagent grade, β-galactosidase from

E. coli, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES buffer), and

ONPG were purchased from Sigma-Aldrich and used as received. All other reagents were of ACS grade. All glassware was of lab grade and was coated with repel-silane following a procedure described in chapter 4, section 2.

5.5.2 Thin film synthesis of β-galactosidase imprinted polyacrylamide

Synthesis of 0.4mm thin films of imprinted hydrogel onto glass slides was done by mixing 500mg of acrylamide, 1mg of bisacrylamide, 1mg of APS, 5ul of TEMED, 0.2mg of β-galactosidase, and 10ml of water. The mixture was degased in a vacuum flask coated with repel-silane, and the synthesis was performed onto bind-silane coated glass slides as described in chapter 4, section 7.2. Also, a film was synthesized with the inclusion of the

APTAC positive monomer with a molar ratio APTAC to enzyme of 60:1. Four sets of hydrogel films of 0.4mm thickness were synthesized in this experiment.

Sample A: Synthesized without the presence of protein as template, and without the presence of charged monomer. This sample serves as a blank to test for non-specific affinity of the protein to the polymer matrix.

Sample B: The target protein β-galatosidase was used as the template. Charged monomer was not included in this sample. Results from rebinding performance will be compared to sample D to test whether the presence of a charged monomer enhances the affinity for the target protein.

Sample C: Synthesized without the presence of protein as template, but the positively charged monomer APTAC was included in the hydrogel. Performance of this sample will help to understand whether the presence of charged monomer attached to the polymer matrix effects any non-specific affinity for the target protein.

Sample D: The protein β-galatosidase was included as the template. The positively charged monomer APTAC was also included in the MIP at the same concentration as in the sample C. See table 5.1 for a description of the components in the preparation of samples A, B, C, and D. The amount of monomer, crosslinker, initiator, accelerator, and water are constant across all samples.

Table 5.1 Description of the samples prepared to test the effectiveness of the use of positive functional monomer in the imprinting of β-galactosidase. Non-imprinted Imprinted Non-imprinted Imprinted non-charged non-charged charged charged

Sample ID A B C D

β-galactosidase x √ x √

APTAC (+) x x √ √

Note: Check mark refers to the inclusion either of β-galactosidase or APTAC. Cross mark refers to the absence of the specified component.

5.5.3 β-galactosidase removal

After synthesis, the glass slides containing the films are rinsed with water and

w placed in glass beakers. A solution of sodium dodecyl sulphate (SDS) 3% /w is used to denature the enzyme and help to remove it from the hydrogel films. The films were exposed to the cleaning solution for one hour.

SDS is an effective surfactant and is widely used as a component in detergents. It can also be used to aid in lysing cells during DNA extraction and in the preparation of proteins for electrophoresis in the SDS-PAGE technique.

SDS is an anionic molecule that denatures proteins. It disrupts non-covalent bonds and wraps around the polypeptide backbone, causing them to unfold and lose their native conformation. At concentration of 0.1 mM, SDS binds to proteins, and provides an overall negative charge to the polypeptide that is proportional to the length of the backbone. The electrostatic repulsion that is created by the binding of SDS causes proteins to unfold into a rod-like shape [87,88] . This is effect on proteins is used to eliminate differences in shape as a factor for separation in the SDS-PAGE.

After the protein removal, all samples were rinsed with water, followed by three rinses with a solution of NaCl 3M, which helps to remove any trace of SDS. Finally all samples were thoroughly rinsed with UP water and TES buffer.

5.5.4 Rebinding assay of β-galactosidase to imprinted hydrogel

After removal of the template from the imprinted cavities, the samples were tested for rebinding of the enzyme back into the imprinted cavities. The samples were set in clean repel-silane coated beakers with 10ml of TES buffer at pH 7.3. 500ul of β-galactosidase

(0.4mg/ml) in TES buffer is added and rebinding is allowed for one hour in a shaker at room temperature. Finally, after the rebinding, all samples are washed with 20ml of buffer three times to remove any loose enzyme molecule.

5.5.5 Test with ONPG

β-galactosidase conversion of ONPG into o-nitrophenol at is optimum at 37°C and pH 7.3. For this reason an incubator and TES buffer (pH 7.3) were used to achieve optimal conditions during the activity test. 50ml of TES buffer was added to each sample and incubated at 37°C for 5 minutes to equilibrate to optimum temperature and pH. Following equilibration 600ul of ONPG 4mg/ml is added. An initial aliquot of 750ul is taken from the mixture immediately after the addition of the ONPG. Initial absorbance readings at

420nm are obtained with the UV-Vis spectrophotometer. Aliquots are taken every three minutes to test for absorbance. The system is maintained at 37°C with continuous agitation during the entire process, see figure 5-5. After collection of the absorbance information, data is normalized by mass to correct for differences in the amount of film used for the

ONPG conversion test.

TES ONPG buffer A 750ul aliquot is taken every 3 minutes for absorbance Film attached reading at 420nm to a glass slide wavelength

Figure 5-5. The imprinted films are placed in glass beakers, Buffer and ONPG are added and the system is incubated in a water bath shaker model C76 from New Brunswick Scientific at 37°C and 60rpm.

5.6 Affinity test results

The absorbance data at times 0, 3, 6, and 9 minutes is shown in table 5.2. The absorbance/time ratio is calculated from the equation of the trendline. The coefficient of x represents the velocity of the reaction, this is the rate at which ONPG is converted to o- nitrophenol by the enzyme. See equations in the figures 5-6, 5-7, 5-8, and 5-9.

Table 5.2 Absorbance readings at 420nm Time (minutes) Sample ID 0 3 6 9 Absorbance at 420nm

A 0.000 0.003 0.005 0.007 B 0.001 0.007 0.014 0.021 C 0.001 0.006 0.013 0.021 D 0.001 0.006 0.013 0.016

0.025 Sample A

0.020

0.015 420nm

Abs 0.010

0.005 y = 0.000767x + 0.000300 R² = 0.988785 0.000 0 3 6 9 Time (minutes) Figure 5-6. Sample A, absorbance versus time. The mean absorbance/time ratio is calculated from the trendline, the equation is also showed below.

0.025 Sample B

0.020

0.015 420nm

Abs 0.010

0.005 y = 0.002233x + 0.000700 R² = 0.998665 0.000 0 3 6 9 Time (minutes) Figure 5-7. Sample B, absorbance versus time. The mean absorbance/time ratio is calculated from the trendline, the equation is also showed below.

0.025 Sample C

0.020

0.015 420nm

Abs 0.010

0.005 y = 0.002233x + 0.000200 R² = 0.989857 0.000 0 3 6 9 Time (minutes)

Figure 5-8. Sample C, absorbance versus time. The mean absorbance/time ratio is calculated from the trendline, the equation is also showed below.

0.025 Sample D

0.020

0.015 420nm

Abs 0.010

0.005 y = 0.001733x + 0.001200 R² = 0.979710 0.000 0 3 6 9 Time (minutes)

Figure 5-9. Sample D, absorbance versus time. The mean absorbance/time ratio is calculated from the trendline, the equation is also showed below.

The rates of conversion are practical representations of the amount of enzyme. Even if the amount of enzyme is below the detection limit of the instrument, the progress of the conversion will eventually reach values above the detection limit. However to accurately determine the relative concentration of enzyme among all samples, it is necessary to account for variations on the mass of sample used in the test. The absorbance/time ratio is normalized by dividing by the dry mass of film used in the test. This is shown in table 5-3 and figure 5-10.

Table 5.3 Normalized ΔAbs420/ΔTime ratio Normalized Mean Mass of film Sample ID ΔAbs420/ΔTime ΔAbs /ΔTime (mg) 420 x1000 A 0.000767 13 0.059 B 0.002233 27 0.083 C 0.002233 51 0.044 D 0.001733 15 0.116

0.15

Time)x1000 0.10 Δ Abs420/

Δ 0.116 0.05 0.083 0.059 0.044 Normalized Normalized ( 0.00 A B C D Non-imprinted Imprinted Non-imprinted Imprinted Non-charged Non-charged Charged Charged Sample ID

Figure 5-10. Comparison of the ratios ΔAbs420/ΔTime that represent the relative amount of β-galactosidase present in the films after the rebind experiment.

There is a dependence of the resultant activity in the imprinted film of hydrogel at the four conditions of polymerization. As the film adsorbs more targeted enzyme, the specific activity in the film increases proportionally.

From figure 5-10, it is evident that the imprinted polyacrylamide hydrogels adsorb more the target molecule than their non-imprinted versions. This is sample B compared to sample A, and sample D compared to sample C. Moreover, the presence of the charged

68 monomer in the matrix of polyacrylamide, enhances the adsorption for the protein, this is sample D compared to sample B.

5.7 Discussion

It is peculiar that sample A shows enzymatic activity. After exposure to the solution containing the enzyme, the sample was thoroughly rinsed with water. The purpose of this procedure is to eliminate any lose enzyme and leave only those that are re-bound to an imprinted cavity. However, sample A is neither imprinted with the enzyme, nor was the charged monomer present during synthesis. Any activity in this sample must be related to the presence of enzyme non-specifically adsorbed into the matrix of the hydrogel, and/or due to a bad rinsing process. In addition, if there is a non-specific adsorption, samples A and C should similarly adsorb the enzyme; however, sample C shows a lower activity.

There are many possible reasons for this decrease in activity. In sample C, the positively charged monomer in theory should be randomly distributed in the polymer matrix. Since glass surfaces from the container may be negatively charged, the positively charged monomer may diffuse and accumulate near the negatively charged surfaces, even with the presence of the repel-silane coating as a barrier between the prepolymer solution and the glass surfaces. When the film is exposed to the enzyme solution, the enzyme begins diffusing through the outer surface of the film. Accumulation on the outer surface could later block any pores on that surface. This could hinder diffusion of any other enzyme molecules into the inner part of the film of hydrogel, dramatically decreasing the amount of enzyme adsorbed into the hydrogel.

From figure 5-10, higher efficiency of adsorption on sample D with respect to sample B is apparent. This correlates with the inclusion of the positively charged monomer in the polymer. The charged monomer is mainly located in the imprinted cavities. The addition of a charged affinity ligand enhances the sensitivity of the imprinted hydrogel for the target molecules.

However, more testing needs to be done to confirm that the inclusion of this charged monomer will help to discriminate a single protein among a mixture of other species. This method of utilizing a color reporter would only be useful when the protein of interest is an enzyme and has a spectrophotometric measurable product. Since more relevant applications demand the detection of non-enzyme proteins at very low concentration, other techniques needs to be developed. The next section details a technique to overcome some of the difficulties mentioned above.

Chapter 6

Integration of molecular imprinting and SPR

6.1 Introduction

This section describes the advantages of the integration of both SPR and MI technologies to overcome some of the difficulties mentioned in chapter 5. In the integration of these technologies, the MI platform provides specificity for a particular protein, while

SPR technology provides high sensitivity in the sensing of molecular binding in real time.

In addition, SPR allows for better detection limits compared to UV-Vis spectrophotometer, and advanced SPR systems surpass detection limits of quartz crystal microbalances

[89,90]. However, this technology integration is not trivial, because it is necessary to produce and attach a nano-thin film of imprinted polymer to the gold surface of the SPR detector. The fabrication of the nano-thin film is a necessity that also allows an advantage to arise. In nano-thin films, a high ratio area: volume is achieved, therefore the target protein does not need to diffuse deep into the hydrogel to completely reach all imprinted cavities. Also removal of imprinted protein is faster. In this chapter, a proposed techniques for the fabrication of the new gold chip; a technique to perform an in-situ synthesis of the imprinted thin film; a method to bind the film to the gold surface; and SPR tests of two different imprinted proteins are explored and the benefits and disadvantages are discussed. 71

6.2 Gold surface of the SPR sensor

For the SPR test, a SensiQ Discovery SPR instrument from SensiQ Technologies

Inc, (see figure 6-1) was used with modification of the SPR sensors. The sensors are originally designed by Spreeta from Sensata Technologies. Because the shape and small size of the sensor limit the ease and handling, it was not feasible to produce the nano-thin film of polymer attached to its gold surface (see figure 6-2). Therefore, the nano-thin film had to be in-situ synthesized on the surface of an individual gold coated cover slip. In addition, to attach the new gold chip to the original SPR sensor, the original gold surface had to first be removed from the sensor. This is accomplished by dissolving it with aqua regia (nitro-hydrochloric acid solution of freshly mixed concentrated nitric acid and hydrochloric acid in 1:3 volume ratio). A drop of this mixture is set on the surface and allowed to interact with the gold for 30 seconds. The sensor is then rinsed with deionized water. The procedure is repeated twice or until all the metallic material is removed from the sensor leaving only the glass surface from the prism inside the sensor, see figure 6-3.

Figure 6-1. SensiQ Discovery SPR instrument from SensiQ Technologies Inc. This instrument belongs to the Center of Material and Sensor Characterization at the University of Toledo

Gold surface to be removed from the sensor

Figure 6-2. Schematics of the SPR sensor that is loaded to the SensiQ Discovery instrument. The sensor utilizes the Kretschmann SPR geometry. Image adapted from the SensiQ Discovery user manual.

Aqua regia

Gold surface

Optical LED Photodiode medium array (Prism)

Clean surface

Optical LED Photodiode medium array (Prism)

Figure 6-3. Treatment with aqua regia to remove the original gold surface of the SPR sensor. 73

After the gold surface is removed, a specially designed gold chip is set onto the surface of the sensor using an index matching fluid [72]. The index matching fluid provides good contact and continuity of the glass media between the light source and the gold surface. See figure 6-4. The gold coated glass cover slip is also designed to reproduce the surface plasmon found in the original gold surface. The preparation of the gold coated cover slip is discussed in detail in section 6.4. In addition, the top of the gold surface has to be coated with an adhesive that helps to attach the nano-thin film of polymer to the gold chip.

Gold chip

Optical LED Photodiode medium array (Prism)

Figure 6-4. Schematics of the new gold chip-SPR sensor setup. The index matching fluid is sandwiched between the glass surface of the gold coated chip and the glass surface from the prism of the SPR sensor.

6.3 Thiol as adhesive layer for the attachment of the nano-thin film of

polyacrylamide

An option that was explored in this research is the use of affinity ligands that strongly interact with gold and can form a covalent bond with polyacrylamide. For this purpose thiolated and disulfide compounds with a vinyl terminal functional group were

74 selected. Thiols and disulfides, with a long backbone length of carbon atoms, form SAMs on gold surfaces. These monolayers are widely use in the antibody and aptamer immobilization on the surfaces of SPR sensors for the study of molecular interactions [91].

For example, thiolated compounds account for a carbon-bonded sulfhydryl group that shows high affinity for gold. In addition, when these molecules with a long alkane chain,

(10 to 18 carbon atoms), its SAM forms readily with a crystalline or semicrystaline structure, conferring the monolayer with stability under normal conditions from a few days to few weeks [92]. Moreover, the particular terminal functional groups on SAMs determine its surface properties. The surfaces can be made hydrophobic, hydrophilic, electroactive, or biologically active. However, on highly packed SAMs, it can be very difficult to form a covalent bond on the terminal functional group, see figure 6-5.

Terminal functional group Self-assembling monolayer Alkane chain

Ligand group

Gold Substrate

Figure 6-5. Parts of a self-assembled monolayer (SAM).

An approach to covalently bond acrylamide to the vinyl terminated SAM uses cross-metathesis reactions; however the high temperature required on this technique limits the applicability to proteins [93]. As mentioned previously, densely packed areas in SAMs are not suitable places for covalent bonding. Gold surfaces are not perfectly flat, see figure 75

6-6, therefore SAM formations on gold surfaces contain a variety of intrinsic and extrinsic defects that allow areas where vinyl end groups are exposed. Any bonding between acrylamide and the vinyl end groups of the SAMs might occur mainly in these areas, see figure 6-7. However this interaction is limited to the amount of defected area present in the

SAM. Additionally, the extent of the surface coverage is unknown.

Figure 6-6. AFM images of the surface morphology of gold prepared by physical vapor deposition. This gold substrate was used in the fabrication of the nano-thin film of polyacrylamide. The bottom image is a 3D representation of the top image.

Exposed vinyl Exposed vinyl Densely groups at gold groups at grain packed area step edges boundaries Surface Metal impurity impurity

Gold layer

Glass layer

Figure 6-7. Representation of some intrinsic and extrinsic defects found in SAMs. Covalent coupling is not feasible at densely packed areas; any covalent bonding would be regarded only to defect sites. Adapted from [92].

On the other hand, vinyl terminated thiol and disulfide compounds with a backbone of 3 and 5 carbons respectively would not form highly packed SAMs, therefore covalent coupling is possible almost at any site, allowing a large extent of the surface coverage [94-

99].

It can be expected that the organic surface, created by the formation of vinyl terminal group surfaces of SAMs, tends to repel the water based prepolymer mixture that is set in contact before polymerization. However, since acrylamide and bisacrylamide are molecules that also contain the hydrophobic vinyl group, it can easily interact and copolymerize with the vinyl terminal groups of the SAM.

In this study, a disulfide and a thiolated compound with a vinyl group as the terminal group were used to produce an adhesive layer. Either the disulfide or the thiol group provides adhesion to the gold substrate and the vinyl group provides the bond with the polyacrylamide matrix. In-situ polymerization was performed to manufacture the thin

77 layer of polyacrylamide, in summary, in the nano-thin film synthesis a small drop of the prepolymer mixture is sandwiched between the gold substrate and a repel-silane coated glass slide. Polymerization is allowed for two hours in nitrogen atmosphere. This process is described in detail in section 6.5. N,N’-bis(acryloyl)cystamine (BAC) a disulfide chemical was tested to produce the SAMs, see figure 6-8. In addition the thiolated compound 2-propene-1-thiol (see figure 6-9) was also tested in the fabrication of the bonding layer.

O H2 H2 H H 2HC C C S C N C C N C S C C CH2 H H H2 H2 O Figure 6-8. N,N’-bis(acryloyl)cystamine (BAC) used in SAM coating of gold as adhesive layer between gold and polyacrylamide.

H2 2HC C C SH H

Figure 6-9. 2-propene-1-thiol used in SAM coating of gold as adhesive layer between gold and polyacrylamide.

In the coating of the gold substrate with either the disulfide or the thiol compounds,

200 proof ethanol is used as the solvent to prepare a fresh 5% solution. The gold chip is immersed in the solution and leaved to interact at room temperature in a nitrogen environment overnight. The chip is rinsed with copious amounts of 200 proof ethanol and dried in nitrogen. Finally the gold chip coated with the thiol or disulfide is used for the in-

78 situ synthesis of the thin film. It is important to mention that after polymerization is completed, the repel-silane coated glass slide is striped, leaving a thin film of imprinted matrix attached to the gold surface. However, visual examination shows that the film is not totally attached to the gold chip. SEM imaging confirms this statement. Figure 6-10 shows wrinkles at the top of the polyacrylamide film suggesting that it is partially attached to the gold surface. Moreover an image of the cross-section also helps to demonstrate the partial attachment of the thin film at the wrinkle, see figure 6-11. An explanation for this could be that after polymerization is complete the film is attached to the gold through the thiol; however this adhesion is not strong enough to withstand the step of striping of the repel- silane coated glass slide. Finally the striping would stretch the film, leaving wrinkle-like features on the surface. A different approach needs to be developed to successfully bind the polymeric imprinted matrix to the bare gold chip.

Figure 6-10. SEM images of two areas of the top surface of the polyacrylamide film partially attached to the gold chip. On the right image, dark areas represent the polyacrylamide film; bright areas represent the uncoated gold surface.

Polyacrylamide film Gold layer

Glass substrate

Figure 6-11. SEM image of the cross-section at a wrinkle. Thickness of the film is 280nm.

6.4 Design and preparation of the gold chip for the SPR sensor

Since it is easier to synthesize the nano-thin film directly onto a flat gold chip than synthesizing it onto the actual sensor, a gold coated coverslip was specially prepared.

Important requirements in the design of this gold chip are the assurance of formation of the surface plasmon produced similarly in the original gold surface. In addition the chip has to provide adherence to the imprinted film of polyacrylamide.

Physical vapor deposition (PVD) technique was used to produce the layer of gold.

The optimum thickness of the noble metal layer is dependent on the wavelength used but typically is between 40 and 50nm [100]. Also, it is important to use an adhesive layer to bind the gold to the glass cover slip. Chromium and titanium are popular adhesive layers for this application; however, titanium was selected since leaching through the gold layer is less frequent than in chromium [101,102].

Additionally, it is known that highly inert materials such gold do not facilitate the

80 binding of the polyacrylamide film. As previously mentioned self-assembling monolayers

(SAM’s) of thiol can be used, but this is often unstable and not strong enough to sustain integrity during the handling. Therefore a new technique was proposed to successfully maintain the polyacrylamide layer attached to the gold chip. A thin layer of SiO2 was deposited onto the gold surface. Bind-silane is used to form a covalently bonded layer onto the SiO2 surface, ultimately providing a strong attachment between the glass and the polyacrylamide, see figure 6-11. However, it was necessary to do some calculation to determine the number and composition of possible layers that can be set onto the gold surface, since it is necessary to prevent the surface plasmon from being critically attenuated.

6.4.1 Material and thickness specification of the layers onto the gold chip

The electromagnetic plane wave that propagates in medium in the near vicinity of the gold surface is composed of two wave vectors. The wave vector parallel to the plane of the surface is denoted by the kx component. The other component is denoted by the ky and represents the wave vector perpendicular to the surface, and exponential decays along the y-direction. ky also represents the evanescent field that extends up to the near vicinity of the metallic surface. Only a changing dielectric property (this is refractive index) in this region, will influence the electric field. The following equations (1) and (2) are derived from Maxwell’s equations, using Snell’s law and Fresnel’s equations, applied to metal- dielectric interfaces [103].

The penetration depth of the evanescent field can be calculated from the following equations:

2 휔 휀푖 푘푦𝑖 = √ (1) 푐 |휀푔표푙푑+휀퐻2푂|

1 푦퐻2푂 = (2) 푘푦퐻2푂

Where ω is the angular frequency, ϲ is the propagation velocity in vacuum, ɛi is the dielectric constant of the media where the plasmon extends, y is the penetration depth of the evanescent field, and ky is the wave vector perpendicular to the interface.

For a multilayered system, it is more complex to calculate the depth of penetration.

An intuitive approach to calculating it is by first finding an effective dielectric constant ɛeff.

This constant is an average of all dielectric constants weighted by the penetration depth in water of the surface plasmon evanescent field. Equation (3) is useful to find the effective dielectric constant.

−2푦 2 ∞ 푦 휀푒푓푓 = ∫ 휀𝑖푦푒 퐻2푂 푑푦 (3) 푦퐻2푂 0

Therefore, to calculate the effective yeff for a multilayered system, first calculate kyH2O for water media using equation (1)

2 휔 휀퐻2푂 푘푦퐻2푂 = √ 푐 |휀푔표푙푑 + 휀퐻2푂|

Then using equation (2), calculate the characteristic distance of the evanescent field into water media:

1 푦퐻2푂 = 푘푦퐻2푂

To calculate the effective penetration depth yeff into the compound multilayer system, replace yH2O in equation (3), calculate the ɛeff. Replace ɛi from equation (1) with the ɛeff value and find kyeff. Using kyeff calculate yeff using equation (2) for the multilayer

82 system.

This system of equations enables calculating of the depth of penetration of the evanescent field even with the presence of additional layers on the gold surface. Since the goal is to produce and bind a nano-thin film of imprinted polyacrylamide to an extremely inert gold surface, an adhesive medium is needed. In this study, the use of a monolayer of bind-silane is proposed. It is proven that bind-silane does not interact with gold but does interact with glass and acrylamide. Therefore a very thin coating of glass or preferably SiO2 is needed. However, as previously mentioned an adhesive layer between glass and gold is also required. Titanium and chromium, according to the equations (1), (2), and (3), dramatically undermine the extent of the evanescent field. For this reason, it was necessary to apply the SiO2 coating without the adhesive layer, relaying only on a mechanical force of adhesion between gold and SiO2. The equations mentioned above, help to determine that a thin layer of SiO2 with a dielectric constant of 3.6 would not greatly affect the penetration depth of the evanescent field. In addition, the dielectric constant for polyacrylamide varies between 4.8 and 9 [104] and the exact value strongly depends on the composition of the polymer. Since the density of the polymeric network used in this study is comparably lower than the polyacrylamide used commercially, it was estimated that the synthesized film would not greatly affect the evanescent field.

The following procedure shows how to use equations (1), (2), and (3): It is known that λ=700nm for many SPR setups, and the speed of light in vacuum c=299 792 458m/s.

Replacing these values in the equation below to find the value for ω:

2휋 휔 = λ 푐

Using equations (1) and (2) to find yH2O, use ɛH2O = 1.77 and ɛgold =-16 83

2 휔 휀퐻2푂 푘푦퐻2푂 = √ 푐 |휀푔표푙푑 + 휀퐻2푂|

1 푦퐻2푂 = 푘푦퐻2푂

Replacing yH2O in equation (3) and solving for a system comprised of a 5nm layer of SiO2 and infinite extent of water.

2 ∞ −2푦 푦 휀푒푓푓 = ∫ 휀𝑖푦푒 퐻2푂 푑푦 푦퐻2푂 0

2 5푛푚 −2푦 푏 −2푦 푦 푦 휀푒푓푓 = [∫ 휀푆𝑖푂2푦푒 퐻2푂 푑푦 + lim ∫ 휀퐻2푂푦푒 퐻2푂 푑푦] 푦퐻2푂 0 푏→∞ 5푛푚

Using equations (1) and (2), find the effective depth of penetration on the evanescent field into the multilayered system.

2 휔 휀푒푓푓 푘푦푒푓푓 = √ 푐 |휀푔표푙푑 + 휀퐻2푂|

1 푦푒푓푓 = 푘푦푒푓푓

The following table describes the calculated penetration of the evanescent field for various materials and thicknesses used in the preparation of the gold chip.

Table 6.1 Depth of penetration of the evanescent field yeff for various multilayered systems. Layer/Material

Case TiO2 Glass Polyacrylamide Water yeff (nm)

Thickness (nm)

1 5 0 0 ∞ 56 2 5 5 0 ∞ 55 3 0 5 0 ∞ 227 4 0 10 0 ∞ 218 5 0 5 200 ∞ 91

Note: The dielectric constant value used for TiO2 is 110. Since water is present in the evanescent field and extends beyond, it is considered as infinite in the multilayered system.

From table 6.1 case 1, it is clear that the presence of a single 5nm layer of TiO2 would profoundly reduce the extent of the evanescent field. Case 5 describes the thickness and materials used for the fabrication of the actual gold chips used in this research.

6.4.2 Fabrication of the gold chip coated with SiO2

The coverslips were cleaned with piranha solution, rinsed thoroughly with ultrapure water, and dried in nitrogen atmosphere at room temperature. Then by means of the

PVD instrument, a 1nm layer of TiO2 was deposited onto the surface of the glass as a bonding layer between the glass coverslip and the gold layer. This was followed by a 50nm layer of gold deposited onto the TiO2 and a 5nm layer of SiO2 deposited on the gold surface to create an appropriate substrate for bonding the polyacrylamide film to the gold chip, see figure 6-10. The gold chip is either stored or coated with bind-silane to provide the necessary bonding layer and strongly attach the polyacrylamide nano-thin film. The bind- silane coating procedure is discussed in detail in chapter 4, section 7.1

Polyacrylamide nano-thin film

Bind-silane

SiO2 (5nm layer)

Gold (50nm layer)

TiO2 (1nm layer) Glass coverslip

Figure 6-12. Thicknesses of the layered design of the gold chip.

6.5 In-situ synthesis of the nano-thin layer of polyacrylamide

After a fresh coating with bind-silane on the SiO2 layer, the chip is ready for the in- situ synthesis of the nano-thin film of imprinted polyacrylamide. The prepolymer mixture is prepared by combining 400mg of acrylamide, 2mg of bisacrylamide, 0.4mg of APS, 2µl of TEMED, and water in a total volume of 2.1ml. Additionally 4.4µmols of either of bovine serum albumin (BSA) or lysozyme (Lysz) were included as template proteins. Moreover, to enhance the binding efficiency and specificity of the imprinted film towards the target protein, [3-(Methacryloylamino)propyl] trimethylammonium chloride (MAPTAC) was included as the positively charged monomer, and 2-Acrylamido-2-methyl-1- propanesulfonic acid (AMPS) was included as the negatively charged monomer. Charged

86 monomers were added in a molar ratio 10:1 with respect to the template protein. Table 6.2 summarizes the composition of a set of four prepolymer mixtures prepared for the synthesis of the imprinted films.

Table 6.2 Sample description of the inclusion of template protein and charged monomer in the preparation of the imprinted films for SPR test.

Sample ID 1 2 3 4

BSA √ √ x x Template Protein Lysozyme x x √ √

MAPTAC (+) √ x √ x Charged monomer AMPS (-) x √ x √

Note: Check mark corresponds to the inclusion of the component in the film. Cross mark indicates the absence.

In the preparation of the nano-thin film, a small drop of the prepolymer mixture was placed on the top surface of the gold chip freshly coated with bind-silane. A repel- silane coated glass slide was placed on top and a 200 gram weight maintained close contact between the two pieces. All steps were performed in a nitrogen atmosphere to prevent oxygen from inhibiting polymerization. After the synthesis was complete, the repel-silane coated glass slide was stripped , successfully leaving a very thin film of hydrogel attached to the gold chip. The imprinted chip was then dried at room temperature and using a diamond scribe, the glass substrate was cut in small rectangles designed to fit on the SPR sensor surface. The small rectangular chips were later mounted on the SPR sensor using index matching fluid, see figure 6-13 and 6-14.

The glass slide is Repel silane stripped leaving a nano- coated glass slide thin film of imprinted polyacrylamide

Drop of prepolymer mixture

Gold chip with The glass slide is The chip is ready coating of SiO2 preset onto the gold for SPR tests layer and bind- chip to cast the nano- silane thin layer of polyacrylamide

Figure 6-13. In-situ synthesis of the thin film of polyacrylamide. A drop of solution containing the building blocks of polymerization is sandwiched between the gold chip and the glass slide creating a thin film of imprinted matrix attached to the gold chip.

Figure 6-14. Gold chip with the attached thin film is mounted in the SPR sensor by means of a drop of index matching fluid with refractive index 1.515

6.6 Integrity of the SiO2 and bind-silane adhesion system

It is notable that the proposed adhesion system is an improvement over the thiol or disulfide systems. In addition the SiO2 coating is easy to apply and much more stable than the thiol system. This is shown in SEM images from figure 6-15. Therefore, this proposed technique was used to attach the polyacrylamide film to the gold surface.

Polyacrylamide film Gold layer

Glass substrate

Figure 6-15. SEM image of the cross-section of the film of polyacrylamide hydrogel attached to the gold surface by means of the SiO2 and bind-silane coatings. Thickness of the film is 185nm

6.7 SPR affinity test of the imprinted films

The SPR sensor with a dried films attached to its gold surface was loaded on the

SensiQ Discovery microfluidic device, see figure 6-16. Then normalization of the sensor was performed to capture a reference signal for the sensor when there is no water present of the surface of the sensor, and therefore no plasmon resonance. This helps to correct for differences among the signal from the sensor for channels one and two. The normalization is controlled by the software and it is performed every time that a new imprinted chip was loaded in the instrument. After normalization, the protein removal cycle and stabilization were performed before the injection of the target protein solution. After stabilization of the imprinted matrix, the protein solution is injected to test for real-time interaction with the imprinted film. Data for the sensorgram was recorded using the SensiQ Discovery software from Nomadics Inc.

Micro Injection Connector flow Imprinted port for data channel chip acquisition

Mobil phase

To SPR waste sensor

Figure 6-16. Setup of the imprinted chip and SPR sensor coupled to the microfluidic system.

6.7.1 Regeneration of the imprinted films

The cleaning or regeneration of the imprinted matrix is a critical step before it is used for analysis. The regeneration procedure consists of the dissociation and elimination of the protein, and should not cause irreversible damage to the matrix. Regeneration is accomplished by the injection of sodium dodecyl sulfate (SDS) as an ionic detergent that denaturates and removes any protein adsorbed on the polyacrylamide film. NaCl is then injected to remove clean any trace of SDS. The removal of the template protein produces a decrease in the response units (RU) which is proportional to the amount of protein removed.

The test was performed at a flow rate of 20µl/min. A volume of 100µl of SDS at

5% w/v concentration was injected in the two channels of the SPR device between 100 and

400 seconds. The sensorgram in figure 6-15 indicates a decrease in 300 response units (RU)

90 at the completion of this step. After a brief rinse with ultra-pure water, 100µl of sodium chloride 3M was injected between 500 and 800 seconds to remove any remaining SDS that would potentially effect non-specific rebinding of proteins. This resulted in a change of

1450 RU. The system was allowed to stabilize for twelve minutes with a steady 20µl/min flow of ultra-pure water. This procedure was performed prior to any rebinding test. Figure

6-17 depicts changes in the RU due to the regeneration procedure.

40000

38000

36000

34000

32000

30000 Relative Respomse Units (RRU) Units Respomse Relative

28000 SDS injection NaCl injection Stabilization with UP water

26000 0 200 400 600 800 1000 1200 1400 1600 Time (Seconds) Figure 6-17. Sensorgram of the regeneration cycle. This is performed to remove any bond protein from the imprinted film. Continuous and dotted lines represent micro channels 1 and 2 respectively that can be used in parallel.

6.7.2 SPR monitoring of the interaction protein-imprinted matrix

After stabilization of the imprinted hydrogel, a fresh 5μM solution of BSA in water was injected into the flow cell of the SPR device. Data was recorded for 680 seconds.

Figure 6-18 shows that the BSA did not effect a significant response in the sensor, either with the presence of a positive or negative functional monomer. Moreover, this behavior 91 indicates that the size of the protein (66KDa) is too large to diffuse into the polymer.

Protein would accumulate only on the surface of the film, far from the reach of the surface plasmon. The imprinted matrix had to be modified in order to reduce the crosslinking, increase the pore size of the matrix and effectively allow the diffusion of the protein into the polymer film.

31400 BSA, Positive BSA, Negative 31200 Lysz, Positive Lysz, Negative 31000 RRU

30800

30600

30400 0 100 200 300 400 500 600 700 Time (seconds)

Figure 6-18. Sensorgram of the interaction between the injected bovine serum albumin protein and the imprinted film loaded in the SPR system.

Another set of films prepared with the same amounts of building molecules, as described in section 6.5 and table 6.2, with a reduced amount of 50% of crosslinker was prepared. After loading the new imprinted films between the SPR sensor and the flow channel, a 4.8µM solution of lysozyme in water was injected in the instrument. Since lysozyme has a molecular weight of 14.3KDa, it is a smaller molecule with respect to BSA, it diffused and interacted successfully with an area of the imprinted matrix close to the

92 surface plasmon. This produced a change in the response units and their normalized sensorgrams are shown in figure 6-19.

It is expected that lysozyme would bind only on lysozyme imprinted films.

However since the isoelectric point of lysozyme is 11.35, and the pH of the mobile phase was approximately 8, lysozyme is positively charged and rapidly binds to the negatively charged matrices. This is confirmed by the rapid increase in the response units shown in the sensorgrams for samples 2, 4, and 5. In addition, if the film is positively charged, it takes more time for the protein to be adsorbed into the imprinted cavities. This is shown from the sensorgrams for samples 1 and 3 which show a delay in the change of response.

The same response is seen even if the film is imprinted with a different protein such as

BSA (samples 2 and 5). The fact that the imprinted film in sample 2 is specific to BSA did not prevent the lysozyme protein to be adsorbed. Moreover, in sample 1 adsorption did not reach a plateau due to the limited exposure (300 seconds) of the imprinted film to the protein solution, and the presence of the positive monomer in the matrix that delayed the protein diffusion.

31600

4. Lysz, (Neg) 31400

31200 2. BSA, (Neg) 5. BSA, (Neg)

31000 3. Lysz, (Pos) 1. BSA, (Pos) RRU 30800

30600

30400

30200 0 100 200 300 400 500 600 700 Time (seconds)

Figure 6-19. Sensorgram of the interaction of lysozyme with the matrix. 4.8µM solution of dissolved protein was injected to test affinity interaction with different imprinted films. Lysz and BSA indicate the protein used for imprinting. (Pos) and (Neg) indicate when a positive or negative monomer was included during polymerization respectively.

6.8 Stability of the imprinted platform

A test similar to those performed in the previous sections, was conducted to test the stability of the new sensor over time. The test was performed when a lysozyme imprinted film was freshly made and after storage for seven days at room temperature coupled to the microfluidic channels of the SensiQ Discovery instrument. Normalization was only performed when the sensor was loaded to the SensiQ Discovery instrument. Then regeneration cycle was performed each time before the tests. Figure 6-20 shows sensorgram data for the injection of lysozyme in the two channels of the SPR instrument,

94 a small change in the final amount of protein adsorbed in the films is observed. Since the

SPR instrument did not account with any temperature controller or correction system, the explanation to the small shift in the RU could be the different temperature conditions at which the test was performed on time cero and after seven days.

30400

30200

30000

29800 RRU 29600 7 days (Ch1)

29400 7 days (Ch2) 0 days (Ch1) 29200 0 days (Ch2) 29000 0 100 200 300 400 500 600 700 Time (seconds)

Figure 6-20. Test of reproducibility over a time lapse of seven days. The injection of lysozyme effect a similar response in time cero and after seven days. The test was performed in duplicate.

6.9 Discussion

An important observation questions the validity of this test. If imprinted chips 2 and 5 were prepared with the same conditions, then both should show similar sensorgrams.

However, in sample 5 the adsorption of protein is faster and greater than in sample 2. An explanation to this relies on the fact that the films are not of equal thickness. It is assumed that both samples are of equal porosity and structure, the concentration of injected protein is constant at the surface of the film, and that the thickness of sample 2 is greater than of

95 sample 5. If these conditions are used in the solution of the Fick’s second law of diffusion, it can be clearly establish that in a thicker film, the time required for the protein to diffuse from the surface of the film to the proximity of the gold surface is longer. More testing needs to be done to support this explanation and it is critical to effectively have control over the thickness of the synthesized films before continuing future experiments.

Results shown in figure 6-19 shows that the positively charged protein is attracted and adsorbed in all films. This adsorption is delayed or enhanced when the films were charged with a positive or negative monomer respectively. Also, the effectiveness of the imprinting factor is not clear. It is possible that the charged films effect a strong ionic attraction/repulsion towards the protein. It is important to state that the overall charge of the target protein is positive; however it contains both positively and negatively charged areas on its surface. Therefore its negative areas interact with the positively charged sites of the matrix, and its positive areas interact with the negatively charged sites of the matrix.

Then it is suggested that a ratio of 10:1 charged monomer:protein used in the imprinting process, is too excessive to test whether the inclusion of the charged monomer effects an enhancement of the specificity for the target protein.

Chapter 7

Other steps for the improvement of the MI platform

This chapter presents approaches for the improvement of the sensing platform. A technique to control the thickness of the synthesized film and consistently reproduce films is investigated. In addition, a method to better build the imprinted cavities and synthesize the polyacrylamide film with improved diffusion characteristic is proposed and explored.

7.1 Method to control the thickness of the film of polyacrylamide

After the preparation of the gold chip, including a fresh coating with bind-silane on top of the SiO2 layer, the chip is ready for the in-situ synthesis of the nano-thin film of imprinted polyacrylamide, and testing in the SPR instrument. In order to obtain sensorgram data that can be accurately reproduced over batches synthesized with the same conditions, the thickness of the films has to be consistently reproduced. To achieve such control, a spacer had to be created especially for this application. The purpose of this spacer is to create a standard cavity thin enough to cast a 200nm layer of polyacrylamide. After the mold is made, it is used in the in-situ synthesis of the film as described in chapter 6, section

7.1.1 Procedure for mold preparation for the casting of the nano-thin film

There are many methods to setup molds for the casting of thin films. In chapters 4 and 5 methods for the respective preparations of bulk and thin films of polyacrylamide hydrogels were discussed. In chapter 6 a technique to prepare films with thickness in the nanometer range was established with limited reproducibility.

In this chapter, a technique is explored for the casting of nano-thin films with reproducible thickness. The mold is prepared by the deposition of a metal layer, the thickness of which is controlled by time of deposition. The substrate is a glass slide that was previously cleaned with piranha solution and then coated with repel-silane as described in chapter 4, section 7.1. The substrate is loaded into a sputter coater and a smaller glass slide is used to mask the central area and protect it from the coating. A layer of gold is deposited for the production of a frame of thin metal on the glass slide. After this procedure is complete, the mold is ready to be used in the casting of the polyacrylamide film, see figure 7-1.

Remove masking layer after sputtering

Pre-cleaned glass Frame of a slide, coated with thin layer of repel-silane gold particles

Uncoated Masking layer Sputter coating with glass surface gold particles

Mold (face up) Clip

Figure 7-1. Preparation of a mold by creating a frame of a thin layer of gold on a glass slide.

7.1.2 In-situ synthesis of a nano-thin film of polyacrylamide

For the final step in the synthesis of the thin film of polyacrylamide, the mold prepared in the previous section was used. A small drop of the prepolymer mixture, without template protein or charged monomer, was placed on the top surface of the glass cover slip that was freshly coated with bind-silane. The mold was placed on top of the cover slip facing downwards. A 200 gram weight was used to force a close contact of the two pieces.

All steps were done in nitrogen atmosphere to prevent oxygen from inhibiting polymerization. Synthesis was allowed for two hours at room temperature, see figure 7-2.

After the polymerization is completed, the mold was striped leaving a very thin film of hydrogel attached to the glass slide.

Mold (face down) Nano-thin film of imprinted polyacrylamide

Prepolymer mixture

Gold chip with Mold is preset onto The chip is ready coating of SiO2 the gold chip to cast for SPR tests layer and bind-silane the nano-thin layer of polyacrylamide

Figure 7-2. Casting of thin films of polyacrylamide. A drop of prepolymer solution is sandwiched between the repel-silane coated mold and the bind-silane coated glass slide.

7.1.3 Characterization of the thickness of the nano-thin film

Sputtering times for 5, 7, 15, 30, 60, and 120 seconds were used to coat the masked glass slides, maintaining the same coating distance of 25mm between the gold target and the surface of the glass slide for all molds. The molds were used in the fabrication of thin

99 films of polyacrylamide as described in the previous section. After synthesis, the films attached to the glass slide were dried and cut in cross-sections using a diamond scribe for

SEM for imaging, see figure 7-3. Thicknesses were recorded from different points from each sample. All data is shown in table 7.1.

Figure 7-3. SEM images of films synthesized on top of glass slides. The thicknesses are: (A) 313nm, (B) 134nm, (C) 389nm, (D) 1244nm, (E) 1146nm, and (F) 1254nm

100

Table 7.1. Recorded thicknesses of the thin films of polyacrylamide synthesized using molds that were prepared with 5, 7, 15, 30, 60, and 120 seconds of coating time with a sputter coater.

Sample ID A1 A2 A3 B1 B2 B3 CDEF

Time of sputtering of gold for mold 5 secs 7 secs 15 secs 30 secs 60 secs 120 secs preparation 245.1 918.6 123.9 405.0 265.8 147.2 508.6 949.1 1081.0 936.9 231.3 801.4 219.0 150.3 285.0 114.3 493.8 698.3 1065.0 1121.0 206.0 656.9 218.1 169.9 305.3 134.3 353.2 686.3 1127.0 1118.0 246.3 590.9 303.2 188.2 244.3 103.1 389.8 1029.0 1091.0 1200.0 226.5 583.3 299.8 155.3 229.1 141.8 596.1 1147.0 1320.0 1259.0 Thicknes of film of 212.7 508.5 268.9 151.1 277.9 168.5 494.3 1232.0 1109.0 1391.0 polyacrylamide (nm) 551.5 129.0 693.0 650.1 1244.0 1504.0 1325.0 550.9 114.9 530.4 1292.0 1509.0 1314.0 397.8 514.2 1206.0 1747.0 1254.0 313.3 1267.0 1226.0 545.5 632.1

Average thickness 228.0 583.5 238.8 183.0 267.9 214.6 503.4 1075.1 1283.7 1161.5 (nm)

Std deviation (nm) 16.5 167.9 67.5 92.5 27.8 212.0 91.3 228.6 249.1 214.6

Average thickness of 350.1 221.8 ---- sample group (nm) Std deviation of the 202.2 42.9 --- - group (nm)

Sample B1 exhibits the lowest average thickness of film (183nm average thickness) created with the use of a mold with 7 seconds of coating time. Sample group A exhibits an average thickness of films of 350.1nm, 0.57 times larger than that on sample group B.

Moreover, the standard deviation for sample group A is 202.2nm noticeably higher than the standard deviation of 42.9nm of sample group B. Finally, coating times of 30, 60, and

120 seconds did not produced significant variation in the averaged film thickness

The significance of this data suggests that other factors are affecting the reproducibility of the thickness. Careful examination of SEM images of the top surfaces of bare glass substrates and synthesized films of polyacrylamide show glass particles of different shapes and sizes were found. This is shown on figure 7-4 and 7-5. It was found 101 that the source of the glass particles is the edges of the glass substrate. These glass chips may break from the edges and contaminate the surface during manipulation. Therefore it is imperative to establish a protocol to prevent this contamination

Figure 7-4. SEM images of glass particles found on the surface of the glass slides.

Figure 7-5. SEM image of glass particles trapped in the film of polyacrylamide.

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7.2 New approach for the synthesis of imprinted films

Figure 7-6 shows SEM images of the cross-section of a freeze dried hydrogel prepared using the protocol described in chapter 4, table 4.1, for sample 6. The freeze drying procedure was described in chapter 3, section 7. The images show a highly crosslinked hydrogel network that the protein has to diffuse through in order to bind to an imprinted cavity in the proximity of the surface plasmon. The degree of crosslinking of the network can be reduced. However, from chapter 4, section 6 and in chapter 6, section 7.2, it was hypothesized that low amounts of crosslinker help to enlarge the pore size allowing for improved protein diffusion, but affinity and recognition moieties are lost.

Figure 7-6. SEM image of the cross-section of a freeze dried bulk piece of polyacrylamide.

Therefore, in this chapter it is proposed to produce a highly crosslinked imprinted cavity inside a low crosslinked matrix by performing a two-stage synthesis of polyacrylamide. In the first stage, the template protein is mixed with the charged monomer in a media that allows their interaction. A solution that is composed of a high ratio crosslinker/monomer is added, and synthesis is initiated by the addition of the initiator and accelerator of polymerization. A particularity in the design of this synthesis is the amount 103 of crosslinker and monomer used. It is small enough to prevent from the formation of branching and building of networks that are typically found in hydrogels; and it is large enough to allow the formation of highly crosslinked structures with complementary shape for the template protein. These structures initiate formation by the addition of a bisacrylamide molecule to the charged monomer that is in interaction with the template.

As more crosslinker is added, the structure grows adopting a complementary shape due to the close contact with the protein. The second stage is characterized by the formation of large networks by the abundant addition of monomer. After the synthesis is complete, the template is removed. See figure 7-7.

First Stage Highly Charged crosslinked monomer recognition structure Oppositely charged region in the Template surface of protein the template Charged monomer is added to the system A high ratio crosslinker/monomer is added to and allowed to interact with the template allow for the formation of the imprinted sites

Second Stage

Network Network

Recognition site

A high ratio monomer/crosslinker is added Template protein is removed to allow for the formation of the network

Figure 7-7. Two-stage synthesis for molecular imprinting of proteins.

104

7.2.1 Two-stage synthesis procedure

A procedure similar to that outlined in chapter 4, section 4 was followed to synthesize the highly crosslinked imprinted sites. In a vacuum flask, 4mg of acrylamide was mixed with 2mg of bisacrylamide. This represents a molar ratio crosslinker/monomer of 0.23. A volume of 6ml of water containing 0.036mg of ammonium persulfate, and 6µl of TEMED are added to initiate synthesis. Polymerization is allowed to proceed for two hours in nitrogen atmosphere. No protein or charged monomer was added to the system because the purpose of this test was to prove the efficacy of the new proposed technique of molecular imprinting. After polymerization was complete, no hydrogel was formed from the mixture as expected. A sample of this solution was taken for analysis by MALDI-TOF

MS to scan for the formation of high molecular weight polymeric structures.

The second polymerization stage would proceed for two additional hours maintaining the nitrogen atmosphere during the entire process. It would include the addition of 600mg of acrylamide, 1mg of bisacrylamide, 2.5mg of ammonium persulfate,

3µl of TEMED and 3ml of water to the vacuum flask after the first polymerization stage is completed.

7.2.2 MALDI-TOF MS analysis of highly crosslinked structures

The solution obtained from the first stage was taken for analysis to scan for the presence of polymeric structures from the molecular addition of acrylamide and bisacrylamide. 1μl of sample was mixed with 2μl of the MALDI matrix α-cyano-4- hydroxycinnaminic acid (4mg/ml in acetonitrile/methanol/0.1% TFA in water, 84/14/2 v/v/v). 1μl of the mixture was spotted on a MALDI target and allowed to dry in a vacuum

105 chamber. The MALDI target was loaded and analyzed in the mass spectrometer

UltrafleXtreme MALDI TOF/TOF from Bruker Daltonics at the Instrumentation Center at the University of Toledo. Data was acquired in the reflection mode in the range of 140 –

2000 Da. Mass calibration was performed with a peptide calibration standard. Figure 7-8 shows MS data in the range of 140-1000. No spectrum was observed in the range 1000-

2000. Spectrum from the MALDI matrix was also collected in order to compare with the spectrum from the samples. MS spectrums were generated from samples collected at the beginning of polymerization, and after one and two hours. It was observed that besides peaks from the matrix, no additional peaks appear in the spectrums from the samples. This could indicate that the components in the solution are not polymerizing, or that the reaction allowed the formation of structures with molecular weight beyond the scope of the instrument. Another possibility is that the instrument is not sensitive enough to detect the polydisperse structures. More tests will determine whether the proposed two stage polymerization allows for the synthesis of an improved molecularly imprinted system.

106

13800 MALDI Matrix 11800

9800

7800 RU 5800

3800

1800

-200 140 190 240 290 340 390 440 490 540 590 640 690 740 790 840 890 940 990 M/Z 13800 Prepolymer 11800 solution t=0 9800

7800 RU 5800

3800

1800

-200 140 190 240 290 340 390 440 490 540 590 640 690 740 790 840 890 940 990 M/Z 13800 Prepolymer 11800 solution t=1hr 9800

7800 RU 5800

3800

1800

-200 140 190 240 290 340 390 440 490 540 590 640 690 740 790 840 890 940 990 M/Z 13800 Prepolymer 11800 solution t=2hr 9800

7800 RU 5800

3800

1800

-200 140 190 240 290 340 390 440 490 540 590 640 690 740 790 840 890 940 990 M/Z Figure 7-8. MS spectrum of the prepolymer solution in polymerization stage one. Data was collected for a 140 - 1000Da range for time 0, 1, and 2 hours of polymerization.

107

Chapter 8

Conclusions

As a molecularly imprinted platform, polyacrylamide is a popular choice for imprinting of proteins because of its excellent biocompatibility, robustness, affordability, and ease of synthesis. In addition, since their physicochemical characteristics are highly influenced by the synthesis conditions set for its preparation, its characteristics can be customized for different applications. One drawback of polyacrylamide for imprinting applications is its hydrophilic nature that allows its matrix to easily swell in presence of water. This behavior can be corrected by increasing the crosslinking degree in its matrix; however it was shown that this tends to negatively affect the protein diffusion.

Other synthesis protocols were studied in order to understand the influence of the amount of monomer, crosslinker and water in hydrogel swelling. It was found that a composition of 8% monomer (w/w) with 0.06% crosslinker (w/w) were sufficient to achieve a minimum swelling of 11.3% with respect to its initial state of synthesis. A bulk polyacrylamide hydrogel was synthesized with the inclusion of bovine hemoglobin

(molecular weight: 64500Da) to test for the effectiveness of the template protein removal protocol. It was found that due to the high crosslinked characteristic of the hydrogel, the protein could not be removed from the matrix. 108

Films with thicknesses in the millimeter range were successfully prepared by a casting technique. It was possible to in-situ synthesize the films leaving them attached to glass slides by the use of bind-silane. The use of repel-silane was also critical to prevent adhesion of the casted films in the mold. The immobilization system also proved to be essential to maintain the integrity of the film during handling. Additionally, films with thicknesses in the nanometer range were possible to produce with the same technique. They were attached to gold substrates by using SiO2 and bind-silane as an adhesive layer; but it is still necessary to adjust the protocol to prevent contamination that affects the reproducibility of the film thickness. Another advantage that the use of nano-thin films offers is that the protein removal protocol works efficiently and when loaded to the microfluidic device it requires very small amounts of cleaning solution, mobile phase, and protein solution for the experiments.

As previously mentioned for practical applications, relevant proteins are found in very low amounts. In some cases their concentrations are below detection limits of conventional instruments such as UV-Vis spectrophotometer. A technique to generate an enhanced signal due to protein binding to the imprinted cavity was proposed but could only be applied to enzymes that produce a spectrophotometric measurable metabolite.

The results obtained by coupling the nano-thin films of MI hydrogel to the SPR technology are very encouraging. It is clear that this integration is essential to achieve real time detection and improved sensitivity in the monitoring of protein binding to the MI hydrogel. There is still a need to clarify the extent of this sensitivity and stability of the system under complex conditions, and identify variables that affect it.

Removal of the original gold surface was required for the coupling of the imprinted

109 platform to the SPR sensor. This could be performed without damaging the sensor.

Moreover, a new gold surface had to be prepared by deposition of 1nm of titanium and

50nm of gold on glass cover slips with 0.145mm of thickness. This new gold chip proved to effectively work once coupled to the SPR sensor by the use of an index matching fluid of 1.515 of refractive index. Additionally, since the nano-thin film had to be adhered to the gold surface, thiol and disulfide SAMs were tested for this purpose but did not produce a strong enough system to withstand the striping step after the casting process. The proposed technique of using a SiO2 and bind-silane coating as adhesion layers is an easier alternative that showed better performance. However, there are still limitations in the strength of this system. Any new approach has to include calculations of the depth of penetration of the evanescent field in the multilayered system in order to prevent a drastic inhibition of its extent.

Positively and negatively charged monomers were successfully included in the imprinted matrices and they enhanced the adsorption for oppositely charged proteins. The effectiveness of the inclusion of these charged monomers still has to be explored in more detail. It is not clear whether the inclusion of the charged monomers produced enhancement in a specific fashion, or just effects an electrostatic attraction regardless of the three dimensional characteristics of the imprinted cavity. Moreover, it is possible that differences of the thickness of the imprinted films influenced the rate at which protein was adsorbed.

More test needs to be done to support this statement and it is critical to effectively have control over the thickness of the synthesized films.

In addition, a critical factor that undermines the specificity of the imprinted films is the necessity of low crosslinked matrices to allow protein diffusion. As discussed in

110 chapter 4, and 6, reduction of the crosslinker implies the loss of specificity by stretching of the imprinted cavity in presence of abundant water. Therefore it is necessary to develop new techniques of molecular imprinting where the imprinted cavities are not affected by the swelling.

The overall goal of this research was to develop a highly sensitive and specific system that allows reproducible detection of proteins for biomedical applications, using technologies that are affordable and allow the fabrication of portable systems. This research shows that with the use of SPR technologies it is possible to achieve such a high sensitivity.

In terms of recognition capabilities, more test need to be done to achieve better insight about how to fine-tune the composition and amount of building blocks to allow for a better selectivity towards the target protein. In addition, it is necessary to test other non- conventional synthesis processes, and the inclusion of other affinity ligands and monomers in the polymer matrix.

111

Chapter 9

Future work

Previously stated in chapter 8, coupling MIP with SPR technology was possible, and a high sensitivity of detection could be achieved. However, protein diffusion through the matrix and selectivity of the imprinted cavity for a particular protein needs to be improved for the successful molecular imprinting.

The main molecular imprinting technique used by most if not all research groups is the free radical polymerization which extends in a chain reaction fashion. In this technique the chain propagation is much faster than the chain initiation, meaning that chain initiation occurs at different points of time during synthesis. In addition, chain termination also occurs at any time due to the coupling of two growing chains or to a process called disproportionation. This characteristic leads to a matrix composed of a wide range of molecular weights, also called a high polydispersity.

A successful molecular imprinting of proteins would have to take into account control over the formation of the imprinted cavities around the template. In addition it must be possible to control the number and type of the affinity ligands that are included in the imprinted sites. To a great extent such control is not possible with current techniques.

Future work will require the use of new chemistry to optimize the selectivity of the 112 imprinted site by controlling the adequate formation of the imprint.

Polymerization techniques similar to reversible-deactivation radical polymerization (RDRP), also known as living radical polymerization or controlled radical polymerization have been brought to the attention to the scientific community [105]. Their applications range from self-healing materials used in space equipment to the easy design of copolymers for ion-exchange membranes in fuel cells or nanoscale lithography etc

[106,107]. These new polymerizations techniques are still not widely used in industry.

However, the field is rapidly growing, as well as the list of applications.

Ideally, RDRP allows control over the initiation, propagation and termination of the chains. In principle, all chains are initiated at the same time, they grow at the same rate, and termination is not allowed during the process. This is only possible with the inclusion of reactants that interfere with the propagating radicals by a reversible deactivation.

Overall, a proportion of the chains are maintained in a dormant state. Equilibration with the active form allows an intermittent growth that ensures a uniform propagation. This allows for a narrow molecular weight distribution, or a very low polydispersity [108,109].

Some groups have developed systems of RDRP with the participation of acrylamide in aqueous conditions [110]. More recently a group has applied this technique in the production of imprinted polymers[111]. However, all approaches in MI of proteins are still in some way rather conventional. Bulk three dimensional MI and two dimensional MI (also called surface imprinting) are the only approaches. As stated in the previous chapters, one of the main problems is the diffusivity of the protein and the degree of crosslinking of the matrix. In this respect, both 3D and 2D MI techniques have their advantages and disadvantages. In addition, creating a MI polymer in a presentation compatible with SPR

113 sensors is an additional complication that can be circumvent by new ingenious techniques.

Future developments will require the design of a new chemistry that allows an effective control over the synthesis of the imprints by using a technique such as RDRP compatible with proteins. In addition, in this document it is proposed that an intricate polymeric network is not necessary for the creation of the imprinting site. The highly selective imprinted cavities are the only focus of MI. Therefore future studies should center efforts to design and synthesize bodies of imprinted cavities alone, preventing the formation of the network. This will provide a way to circumvent the complications with protein diffusion, non-specific binding to the matrix, and the use of molds in the synthesis of the nano-thin layer of polymer attachment to the gold surface of the SPR sensor. Finally the inclusion of functional monomers that allow hydrophobic and electrostatic interactions can be effectively added, controlled and studied.

114

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