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thesis entitled

DEVELOPMENT OF A BIOMIMETIC SENSOR THROUGH
MOLECULAR IMPRINTING

presented by
Lisa Marie Kindschy

has been accepted towards fulfillment
of the requirements for the

MS. degree in Biosystems Engineering

 

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DEVELOPMENT OF A BIOMIMETIC SENSOR THROUGH MOLECULAR
IMPRINTING

By

Lisa Marie Kindschy

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Biosystems and Agricultural Engineering

2005

ABSTRACT

DEVELOPMENT OF A BIOMIMETIC SENSOR THROUGH MOLECULAR
IMPRINTING

By

Lisa Marie Kindschy

Molecular imprinting is a technique for creating synthetic receptor sites in a
polymer. In this research, a molecularly imprinted polymer (MIP) biomimetic sensor was
formed for theophylline using a copolymer of two monomers, methacrylic acid and
ethylene glycol dimethacrylate. The presence of theophylline in the biomimetic sensor
was measured using cyclic voltammetry, specifically, the corresponding peak currents on
the voltammograms. The peak currents of the MIP sensor in the presence and absence of
theophylline were compared to the blank sensor (non-imprinted polymer).

In the initial measurements, the peak current for the MIP sensor on indium tin
oxide (ITO) increased by a factor of 5.3 upon addition of theophylline compared to the
blank non-imprinted MIP. The ratio of peak currents increased by a factor of 7.5 for the
MIP sensor on silicon compared to the blank. The sensitivity of the MIP on ITO was
between 2 - 4 mM theophylline. The concentration of theophylline that resulted in the
best signal was 3 mM. The MIP sensor showed no cross reactivity to caffeine, which has
a similar chemical structure. This research will provide the foundation for future work in
biomimetic sensors by developing durable sensors with longer shelf lives when exposed

to rugged and harsh environments.

Copyright by
Lisa Marie Kindschy

2005

This thesis is dedicated to my family, Steven,

Judith, Brad, and Lori Kindschy for their love, support, and encouragement.

iv

ACKNOWLEDGEMENTS

I would like to thank all the people who helped me through this degree. First, I
would like to send my deepest thanks to my family, my mom and dad, Steve and Judy,
and to my siblings, Lori and Brad, for their love and support. Brad, for your unsolicited
and “expert” advice, Lori for those late night slurpee runs, mom for always listening
when I needed to vent, and dad for trying solve my problems and offering your great
opinions.

I would also like to thank Dr. Evangelyn Alocilja for being a great mentor,
advisor, and fiiend, and for always offering advice and positive thinking when I needed
it. Without her constant encouragement and advice, this thesis would not have been
possible.

I would like to thank my labmates, Finny, Maria, Zarini, and Stephen who not
only helped me in the lab, but also helped to shape the person I have become through this
degree. Thanks to Stephen for being a good arguing buddy and for never making the lab
a dull place; F inny for being the “handyman” of the lab and helping me with all number
of problems from research questions to computer issues; Maria for giving me
encouragement (and distractions) when I needed it; and Zarini for giving me useful
advice and helping me through the last two grueling weeks. The conversations I have
had about biosensors and life will stay with me forever. Without all of you, this degree

would not have been accomplished!

TABLE OF CONTENTS

 

 

 

 

 

 

LIST OF TABLES - _- - - - -- - -VIII
LIST OF FIGURES _ X
LIST OF ABBREVIATIONS XIII
CHAPTER 1: INTRODUCTION _ - - 1
1 .1 SIGNIFICANCE ............................................................................................................. 1
1.2 HYPOTHESIS ................................................................................................................ 2
1.3 SPECIFIC ACTIVITIES ................................................................................................... 2
1.3.1 Confirmation of Imprinted Polymer .................................................................... 2
1.3.2 Attachment to Electrode for Formation of Biomimetic Sensor ........................... 2
1.3.3 Sensitivity of the Biomimetic Sensor ................................................................... 3
1.3.4. Selectivity of the Biomimetic Sensor .................................................................. 3
1.3.5. Biomimetic Sensor on Silicon ............................................................................ 3
CHAPTER 2: LITERATURE REVIEW - - -- -- -4
2.1 MOLECULARLY IMPRINTED POLYMERS ....................................................................... 4
2.1.] Introduction ......................................................................................................... 4
2.1.2 Molecular Imprinting Technique ........................................................................ 5
2.1.3 Chemical Modification of the Polymer ............................................................... 9
2.1.4 Methods for Pre-Screening Polymers ............................................................... 12
2.1.5 Physical Modification of the Polymer ............................................................... I 3
2.1.6 Membrane Imprinted Polymers ........................................................................ 14
2.1. 7 Transducer Selection ........................................................................................ I 6
2.1.8 Food and Agricultural Applications of MIPs ................................................... 19
2.1.8.1 Food Applications ...................................................................................... 19
2.1.8.2 Agricultural Applications — Pesticides, Toxins, and Biowarfare
Agents ........................................................................................................ 20
2.1.9 Potential Applications ....................................................................................... 22
2.1.10 Limitations and Challenges ............................................................................ 23
2.2 CYCLIC VOLTAMMETRY ............................................................................................ 24
CHAPTER 3: METHODS AND MATERIALS - 29
3.1 OVERVIEW OF PROCEDURE FOR RESEARCH .............................................................. 29
3.2 CHEMICALS ............................................................................................................... 30
3.3 PREPARATION OF ELECTRODES .................................................................................. 30
3.3.1 IT 0 electrode .................................................................................................... 30
3.3.2 Silicon electrode ................................................................................................ 30
3.4 ELECTROCHEMICAL CELLS ........................................................................................ 31
3.5 PREPARATION OF THE THEOPI—IYLLINE IMPRINTED BIOMIMETIC SENSOR .................. 34
3.6 EXTRACTION OF TEMPLATE ...................................................................................... 36
3.7 BASELINE MEASUREMENT ........................................................................................ 36

vi

3.8 REBINDING AND MEASUREMENT OF THEOPHYLLINE ANALYTE ................................ 37

 

 

 

 

3.9 SELECTIVITY EVALUATION ....................................................................................... 38
3.10 LIGHT ABSORBANCE MEASUREMENTS .................................................................... 39
3.1 1 STATISTICAL ANALYSIS .......................................................................................... 39
3.11.1 Determination of Peak currents on Cyclic Voltammograms .......................... 39
3.1 1. 2 Evaluation of Blank and MIP Sensors ............................................................ 40
3.12 CHARACTERIZATION OF SURFACE ........................................................................... 40
CHAPTER 4: RESULTS AND DISCUSSION -- - - 41
4.1 THEORY OF DETECTION ............................................................................................ 41
4.2 CONFIRMATION OF IMPRINTED POLYMER ................................................................. 42
4.3 CHARACTERIZATION OF SURFACE ............................................................................. 43
4.4 INITIAL MEASUREMENTS — CELL A ........................................................................... 45
4. 4. 1 MIP-ITO Sensor ................................................................................................ 45
4. 4. 2 MIP-Si Sensor ................................................................................................... 4 7
4. 4. 3 Comparison of peak currents for IT 0 and Silicon ............................................ 49

4.5 SENSITIVITY AND SELECTIVITY TESTING MEASUREMENTS OF SUBMERGBD
WORKING ELECTRODE — CELL B .............................................................................. 51
4.5.1 Background Measurement ................................................................................ 51
4. 5. 2 Sensitivity of MIP-I T0 Sensor .......................................................................... 52
4. 5. 3 Selectivity of MIP-IT 0 Sensor .......................................................................... 61
4.6 SELECTION OF FUTURE COMPOUND FOR FOOD SAFETY ............................................ 67
CHAPTER 5: CONCLUSIONS _ - - -- - -- --70
5.1 GENERAL CONCLUSIONS ........................................................................................... 70
5.2 RECOMMENDATIONS FOR FUTURE RESEARCH ........................................................... 71
REFERENCES - _ - - 73
APPENDICES _ _ _ ..... - _ _ ............ 80
APPENDIX A: LIGHT ABSORBANCE TABLES AND RAw DATA ......................................... 81

APPENDIX B: POTENTIALS AND CURRENTS USED FOR SENSITIVITY AND

SELECTIVITY STATISTICAL ANALYSIS ....................................................... 88
APPENDIX C: PROCEDURE FOR INITIAL RESULTS FOR PATULIN ...................................... 96

vii

Table 2-1.

Table 3-1.

Table 4-1.

Table 4-2.

Table 4-3.

Table 4-4.

Table 4-5.

Table 4-6.

Table A-1.
Table A-2.
Table A-3.
Table A-4.
Table B-1.

Table B-2.

Table B-3.

LIST OF TABLES

Summary of Molecularly Imprinted Polymers in Sensor
Applications. ................................................................................................. 1 8

Procedures Used in This Research. ............................................................... 29

Ratio of Maximum Currents (ipc) of MIP and Blank on ITO
and Silicon .................................................................................................... 49

Ratio Of Minimum Currents (ipa) of MIP and Blank on ITO
and Silicon .................................................................................................... 49

Ratio of Maximum Currents (ipc) Of B-ITO and MIP-ITO
Sensors at Various Analyte Concentrations .................................................. 58

Ratio of Minimum Currents (ipa) of B-ITO and MIP-ITO
Sensors at Various Analyte Concentrations .................................................. 58

Ratio of Maximum Currents (ipc) of B-ITO and MIP-ITO
Sensor at Various Counter Analyte Concentrations ..................................... 66

Ratio of Minimum Currents (ipa) of B-ITO and MIP-ITO

Sensor at Various Counter Analyte Concentrations ..................................... 66
Light Absorbance Values for Aqueous SmM Theophylline. ........................ 81
Light Absorbance Values for Non-imprinted Polymer. ................................ 81
Light Absorbance Values for MIP. ............................................................... 81

Comparison of Light Absorbance Difference of MIP to Non-
imprinted Polymer Using t-Test for Two Samples Assuming
Unequal Variances. ....................................................................................... 82

Potentials and Currents for B-ITO Evaluated at 1 mM
Theophylline. ................................................................................................ 88

Potentials and Currents for MIP-ITO Sensor Evaluated at 1
mM Theophylline .......................................................................................... 88

Potentials and Currents for B-ITO Evaluated at 2 mM
Theophylline. ................................................................................................ 89

viii

Table 8-4.

Table B-5.

Table 8-6.

Table 8-7.

Table B-8.

Table 8-9.

Table B-lO.

Table B-1 1.

Table 8-12.

Table B-1 3.

Table B-14.

Table B-15.

Table B-16.

Potentials and Currents for MIP-ITO Sensor Evaluated at 2
mM Theophylline .......................................................................................... 89

Potentials and Currents for B-ITO Evaluated at 3 mM
Theophylline. ................................................................................................ 90

Potentials and Currents for MIP-ITO Sensor Evaluated at 3
mM Theophylline .......................................................................................... 9O

Potentials and Currents for B-ITO Evaluated at 4 mM
Theophylline. ................................................................................................ 91

Potentials and Currents for MIP-ITO Sensor Evaluated at 4
mM Theophylline .......................................................................................... 91

Potentials and Currents for B-ITO Evaluated at 1 mM
Caffeine. ........................................................................................................ 92

Potentials and Currents for MIP-ITO Sensor Evaluated at 1
mM Caffeine. ................................................................................................ 92

Potentials and Currents for B-ITO Evaluated at 2 mM
Caffeine. ........................................................................................................ 93

Potentials and Currents for MIP-ITO Sensor Evaluated at 2
mM Caffeine. ................................................................................................ 93

Potentials and Currents for B-ITO Evaluated at 3 mM
Caffeine. ........................................................................................................ 94

Potentials and Currents for MIP-ITO Sensor Evaluated at 3
mM Caffeine. ................................................................................................ 94

Potentials and Currents for B-ITO Evaluated at 4 mM
Caffeine. ........................................................................................................ 95

Potentials and Currents for MIP-ITO Sensor Evaluated at 4
mM Caffeine. ................................................................................................ 95

ix

Figure 2-1.
Figure 2-2.
Figure 2-3.
Figure 2-4.
Figure 2-5.

Figure 3-1.

Figure 3-2.

Figure 3-3.

Figure 3-4.

Figure 3-5.

Figure 4-1.

Figure 4-2.

Figure 4-3.

Figure 4-4.

Figure 4-5.

Figure 4-6.

LIST OF FIGURES

Schematic overview Of molecular imprinting process. ................................ 6
Commonly used functional monomers and cross-linkers. ......................... 11
Excitation Signal for a cyclic voltammetry experiment. ............................ 25
Typical cyclic voltammogram for a reversible system. ............................. 26
Schematic representation Of a three-electrode potentiostat. ...................... 27

Cell A — Three-electrode electrochemical cell with working
electrode at the bottom of the cell. ............................................................. 32

Cell B — Three-electrode electrochemical cell with working
electrode submerged. ................................................................................. 33

Procedure for the formation of theophylline MIP on
electrode. .................................................................................................... 35

Chemical structures of (a) methacrylic acid and
(b) ethylene glycol dimethacrylate ............................................................. 36

Chemical structures Of (a) theophylline and (b) caffeine ........................... 38

Schematic representation Of the Shrinking effect. Possible
transformation in the arrangement of imprinted polymer upon
rebinding of template molecule leading to increased electron
flow ............................................................................................................ 42

AFM image of MIP-ITO sensor after extraction of the
theophylline template for l h in 9:1 methanol/acetic acid. ........................ 43

AFM image of MIP-ITO sensor after rebinding in 5 mM
theophylline for 25 min .............................................................................. 44

Cyclic voltammograms of B-ITO in the absence Of
theophylline and in 5 mM theophylline for cell A ..................................... 46

Cyclic voltammograms of MIP-ITO in the absence of
theophylline and in 5 mM theophylline for cell A. .................................... 46

Cyclic voltammograms of B-Si in the absence of
theophylline and 5 mM theophylline for cell A. ........................................ 48

Figure 4-7.

Figure 4-8.

Figure 4-9.

Figure 4-10.

Figure 4-11.

Figure 4-12.

Figure 4-13.

Figure 4-14.

Figure 4-15.

Figure 4-16.

Figure 4-17.

Figure 4-18.

Figure 4-19.

Figure 4-20.

Figure 4-21.

Cyclic voltammograms Of MIP-Si in the absence Of
theophylline and 5 mM theophylline for cell A. ........................................ 48

Cyclic voltammogram of 5 mM potassium ferrocyanide and
0.1 M potassium nitrate with 5 mM theophylline on untreated

ITO. ............................................................................................................ 52
Cyclic voltammogram Of B-ITO sensor evaluated at 1 mM
theophylline ................................................................................................ 54
Cyclic voltammogram of MIP-ITO sensor evaluated at 1 mM
theophylline ................................................................................................ 54
Cyclic voltammogram of B- ITO sensor evaluated at 2 mM
theophylline ................................................................................................ 55
Cyclic voltammogram of MIP-ITO sensor evaluated at 2 mM
theophylline ................................................................................................ 55
Cyclic voltammogram of B-ITO sensor evaluated at 3 mM
theophylline ................................................................................................ 56
Cyclic voltammogram Of MIP-ITO sensor evaluated at 3 mM
theophylline ................................................................................................ 56
Cyclic voltammogram of B-ITO sensor evaluated at 4 mM
theophylline ................................................................................................ 57
Cyclic voltammogram of MIP-ITO sensor evaluated at 4 mM
theophylline ................................................................................................ 57
Comparison Of the ratio of maximum currents of MIP-ITO
sensor to B-ITO at various analyte concentrations. ................................... 60
Cyclic voltammogram of B-ITO sensor evaluated at 1 mM
caffeine ....................................................................................................... 62
Cyclic voltammogram of MIP-ITO sensor evaluated at 1 mM
caffeine ....................................................................................................... 62
Cyclic voltammogram of B-ITO sensor evaluated at 2 mM
caffeine ....................................................................................................... 63
Cyclic voltammogram of MIP-ITO sensor evaluated at 2 mM

caffeine ....................................................................................................... 63

xi

Figure 4-22.

Figure 4-23.

Figure 4-24.

Figure 4-25.

Figure 4-26.
Figure 4-27.

Figure A-l.

Figure A-2.

Figure A-3.

Figure A-4.

Figure A-5.

Cyclic voltammogram of B-ITO sensor evaluated at 3 mM
caffeine ....................................................................................................... 64
Cyclic voltammogram of MIP-ITO sensor evaluated at 3 mM
caffeine ....................................................................................................... 64
Cyclic voltammogram of B-ITO sensor evaluated at 4 mM
caffeine. ...................................................................................................... 65

Cyclic voltammogram of MIP-ITO sensor evaluated at 4 mM

caffeine ....................................................................................................... 65
Chemical structure of patulin (Sigma-Aldrich, 2004). .............................. 67
Initial cyclic voltammogram for patulin MIP. ........................................... 68

Light absorbance data for an aqueous solution of 5 mM
theophylline ................................................................................................ 83

Light absorbance data for non-imprinted polymer afier
extraction of template. ............................................................................... 84

Light absorbance data for non-imprinted polymer after
soaking 25min in an aqueous solution of 5 mM theophylline. .................. 85

Light absorbance data for MIP after extraction of template. ..................... 86

Light absorbance data for MIP after soaking 25min in an
aqueous solution of 5 mM theophylline ..................................................... 87

xii

2, 4-D

3-MPS

AF M
AIBN
B-ITO
B-Si
Caf
DMF
DMSO

EDGMA

Eapp
Efinal

Einital

LIST OF ABBREVIATIONS

2,4-Dichlorophenoxyacetic acid
3-(Trimethoxysilyl)propyl methacrylate
Ampere

Atomic force microscopy
2,2'-Azobisisobutyronitrile
Blank sensor on indium tin oxide
Blank sensor on Silicon

Caffeine
N,N-Dimethylformamide
Dimethyl Sulfoxide

Ethylene glycol dimethacrylate

Potential

Applied potential

Final potential

Initial potential

Peak anodic potential

Peak cathodic potential

hour(s)
High performance liquid chromatography

current

xiii

ipa Peak anodic current

ipc Peak cathodic current

ITO Indium tin oxide

MAA Methacrylic acid

min minute(S)

MIP Molecularly imprinted polymer

MIP-ITO Molecularly imprinted polymer sensor on indium tin oxide
MIP-Si Molecularly imprinted polymer sensor on Silicon
S second(S)

S Siemens

Si Silicon

Thy Theophylline

V Volts

xiv

CHAPTER 1: INTRODUCTION

1.1 Significance

Novel sensors are needed for the detection and quantification of toxins and other
harmfirl molecules. A unique group of sensors uses the principle of biomimetics, where
natural processes and components are imitated through artificial replicas. Molecularly
imprinted polymers (MIPS) use the technique of biomimetics to create artificial receptor
Sites using specific monomers, the building blocks of polymers, to create tailored
recognition Sites for the target molecule. Biomimetic sensors using MIPS Offer some
interesting advantages to typical biological-based sensors, such as those using antibodies
or enzymes. MIPS have a long Shelf life due to the inherent strength of the polymer and
are resistant to extreme conditions, such as acidic, basic, high temperature, and low
moisture environments.

Molecular imprinting is the process of forming artificial receptors for a molecule
that is also used as the template. The building blocks (monomers) are polymerized
around the template that is used as a mold. Once polymerization is complete, the
template is removed from the polymer, leaving holes that exactly match the Size and
Shape of the original template. The polymer is now considered imprinted because holes
have been created that are Specific in shape and Size to the template, and to which only it
can rebind. This thesis will explore the use of molecularly imprinted polymers for the

detection Of small molecules using a biomimetic sensor.

1.2 Hypothesis

This research will demonstrate that a biomimetic sensor can be developed through
the molecular imprinting technique. To demonstrate proof of concept, the target
molecule in this research is theophylline. To test the hypothesis, the objectives of this
research are to: (I) confirm the imprinting of the polymer; (2) attach the imprinted
polymer to an indium tin oxide (ITO) electrode; (3) test the sensitivity and selectivity of
the biomimetic sensor; and (4) initially test the formation of the MIP on a Silicon

electrode.

1.3 Specific Activities

1.3.1 C onfirmation of Imprinted Polymer

The first objective Of this research iS to verify that a molecularly imprinted
polymer can be successfully formed. This is accomplished through the comparison of

light absorption measurements of the imprinted polymer and non—imprinted (blank)

polymer.

1.3.2 Attachment to Electrode for Formation of Biomimetic Sensor

After successful formation of the MIP, the next goal of this research is to attach
the polymer onto an electrode for use as a biomimetic sensor. The successful attachment
of the MIP to the electrode is monitored using imaging by atomic force microscopy

(AFM).

1. 3.3 Sensitivity of the Biomimetic Sensor

The sensitivity of the MIP sensor on ITO is studied for varying concentrations of

theophylline.

1.3.4. Selectivity of the Biomimetic Sensor

The selectivity of the biomimetic sensor is evaluated by testing with caffeine, a

molecule that is structurally related to theophylline.

1.3.5. Biomimetic Sensor on Silicon

The final area of investigation is to initially test the biomimetic sensor on silicon.

CHAPTER 2: LITERATURE REVIEW
2.1 Molecularly Imprinted Polymers

2.1.] Introduction

A biosensor is an analytical device that incorporates a biological recognition
element (antibodies, enzymes, DNA probes, or cells) in close proximity to a transducer
that converts the recognition event between the biological element and the target analyte
into a quantifiable signal. A common type of biosensor is an immunosensor, which uses
the binding of an antigen to an antibody to produce a measurable Signal, allowing for the
quantification Of the antigen (Muhammad-Tahir and Alocilja, 2003; Muhammad-Tahir
and Alocilja, 2004; Radke and Alocilja, 2005). In general, antigens can be in the form of
microorganisms or pollutants that are capable of stimulating an immune response in the
host, resulting in the production of antigen-specific antibodies. Thus, the attractiveness
Of the antibody—antigen biosensor is the superior specificity that results from the antigen-
antibody binding such that structurally related molecules are rejected from the binding
Site.

Antibody-based detection is limited by the reliance on the antigen for antibody
production, the expensive extraction process of the antibodies, and the highly controlled
environment needed for antibody production. Consequently, research has led to the
development of synthetic antibody mimics that reproduce the natural sites of antibodies.
These biosensors belong to a group of devices and processes called biomimetics, which
use artificial materials to mimic biological systems found in nature. These “antibody

mimics” have similar and comparable performance when measured alongside their

antibody counterparts. One type of biomimetic receptor that has received considerable
attention in recent years is the group called molecularly imprinted polymers (MIPS).
MIPS have several attractive characteristics that make them more suitable for certain
sensor applications than their immunosensor counterparts. They are very stable and
robust, and are resistant to degradation or loss of sensor properties in a wide range of
conditions, such as extreme temperatures, as well as acidic and basic environments
(Svenson and Nicholls, 2001).

MIPS offer a viable alternative for applications where antibodies are not available
or are too expensive to obtain for a particular substrate. MIPS have been successfully
used for several applications, such as the Stationary phase in separations using high
performance liquid chromatography (HPLC) (Kempe, 1996), thin layer chromatography
(Kriz et al., 1994), catalysis (Wulff, 2002), ligand-binding assays (antibody mimics)
(Andersson, 1996; Surugiu et al., 2001a), and solid phase extraction (Rashid et al., 1997;
Stevenson, 1999). Imprinting technology is also employed in the area of sensors and
assays where MIPS are used as binding site mimics in assay systems as well as the

recognition element in biosensors.

2.1.2 Molecular Imprinting Technique

The method of molecular imprinting involves the polymerization of a functional
monomer and cross linker around a molecular template (process illustrated in Figure 2-1).
It is important to note that while the picture is a two-dimensional representation of the
process, the imprinted Site that is created is three dimensional, such as that of a lock and

key.

Functional , I
Monomers

: 1 \ . 1‘ '1':
D V [i
l; "
1. fl ‘7, >‘
A
Template

Imprinted
Polvmer

      

Figure 2-1. Schematic overview of molecular imprinting process.

Steps of non-covalent imprinting: (1a) self-assembly, (lb) polymerization, and (1c)
solvent extraction of template. Steps of covalent imprinting: (2a) synthesis of
polymerizable template, (2b) polymerization, and (2c) extraction of template

by chemical cleavage.

In the first step, the functional monomers are assembled around the template by
either covalent or non-covalent bonds. For both methods, the process must be reversible
such that the template can be removed from the polymer, but have the ability to rebind to
the site. The functional monomers are selected based upon the number of bonds they can
form with the template. For example, in a study on the agricultural pesticide atrazine

using non-covalent interactions, the functional monomer, methacrylic acid, was chosen

for its ability to form two ionic and two-three hydrogen bonds with the template, atrazine
(Sergeyeva et al., 1999). In this thesis, the term template will refer to the molecule that is
used to form the molecular imprint in the polymer, and analyte will refer to the same
molecule as it rebinds in the imprinted Site. While these are the same compound, they
differ in the purpose that each serves. The template is used as a mold around which the
polymer is assembled towards the formation of the imprinted polymer. The analyte, on
the other hand, is the target material that rebinds to the imprinted Site that was vacated by
the template and produces a measurable signal.

Following the pre-assembly step, the monomer-template complex is combined
with the cross-linker, an initiator, and, usually, a porogenic solvent. The role of the
porogenic solvent is to create pores or tiny holes in the finished polymer that will allow
the analyte to access the imprinted binding sites. Oxygen must be removed from the
reaction environment prior to polymerization either by vacuum removal or replacement
with an inert gas, such as nitrogen, due to its interference with the formation of free
radicals.

Polymerization is started either thermally (addition of heat) or photochemically
(usually exposure to UV light) with the initiator selected based upon the requirements
and restrictions of the template to either method. In a UV light initiated process,
polymerization can be performed at lower temperatures. This process is preferred for
non-covalent imprinting due to the increased strength of ionic and hydrogen bonding at
reduced temperatures. The polymerization step fixes the position of the functional

monomers around the template by creating chemical bonds between the cross linkers and

functional monomers, such that a memory of the receptor site is permanently retained in
the polymer upon removal of the template.

When polymerization is complete, the template iS removed by washing with an
organic solvent for non-covalent imprinting or by chemical cleaving from the polymer if
the covalent imprinting method is used. Subsequent processing Of the polymer is usually
required for bulk polymerization methods to obtain the beads that are used in many
procedures (Kriz et al., 1996; Baggiani et al., 1999). After removal of the template, Sites
are obtained that have a chemical “memory” of the template due tO the relative position
of the functional monomers within the imprinted polymer.

Wulff first reported work on an organic molecularly imprinted polymer in 1973
(Wulff et al., 1973). In Wulff’s research, covalent bonds were formed between the
template D-glyceric acid and two other monomers (p-amino styrene and 2,3-O-p-
vinylphenylboronic ester). The template-monomer complex was integrated with a
divinylbenzene polymer followed by chemical cleavage of the template to Obtain a
binding site.

The advance in molecular imprinting technology that allowed for several new
applications to emerge was the use of non-covalent bonding between the template and the
functional monomers. Mosbach and co-workers found that electrostatic and hydrogen-
bonding forces could be used to form a pre-assembled complex between the template
phenylalanine ethyl ester and certain monomers (styrene or acrylic based) prior to
polymerization, resulting in the ability to easily remove the template after polymerization
with an organic solvent (Andersson et al., 1984). Non-covalent bonding has been

primarily used in recent molecular imprinting procedures due to the large number Of

molecules that can be imprinted using non-covalent interactions. While non-covalent
imprinting is becoming mainstream, covalent imprinting may still find usefulness for
certain templates or when the two techniques can be used in combination. Whitcombe
combined the advantages of non-covalent and covalent bonding to produce an imprint for

cholesterol (Whitcombe et al., 1995).

2.1.3 Chemical Modification of the Polymer

Polymers can be formed by a variety Of different techniques with the most
popular method being free radical (addition) polymerization. Other techniques include
condensation polymerization, where a byproduct (usually water) is formed when the
monomers combine or the use of a catalyst to facilitate the joining Of monomers. MIPS
are mainly formed by free radical polymerization due to the variety Of functional
monomers available. In this type of polymerization, the fimctional monomers and cross
linkers generally have either a vinyl or acrylic group where monomers add onto the end
Of a growing chain. Prior to polymerization, a complex is formed between the functional
monomers and template by the self-assembly approach. In this approach, the functional
monomers bind with the template in key locations where a non-covalent bond is formed.
The interactions holding the two species together in the self-assembly complex are
usually based on hydrogen bonding or ionic interactions.

The selection of the functional monomers and cross-linking agent is critical to the
imprinting process because the reattachment of the template to the fiinctional monomers
is the basis of the signal generation. The key element for a functional monomer is to
have accessible binding Sites for the interactions to occur. Several types of functional

monomers and cross—linkers are available, the most common being methacrylic acid

(MAA) and ethylene glycol dimethacrylate (EGDMA). The structures of twenty
common functional monomers and cross-linkers are shown in Figure 2-2.

In addition to the importance of selecting the functional monomer and cross-
linker, the relative amounts of the pre-polymerization ingredients are also vital to the
formation of the polymer. Various concentrations of functional monomers and cross-
linkers can result in MIPS with noticeably different sensor properties. In a study by
Dickert and Hayden (1999), the effect of varying the concentration of a cross-linker for
both ethanol and ethanol acetate imprinting resulted in a 10% optimal cross-linker
amount with the highest sensitivities and selectivities. Increasing or decreasing the
amount of cross-linker in the MIP produced less accessible binding Sites or decreased the

imprinted effects in the sensor.

10

HM, H. H... WT“ W

ACRYLAMIDE ACROLEIN ACRYLIC ACID ACRYLONITRILE ALLYAMINE
\ N
©\\ Q\\©\\><Of)
_ O
p -DIVINYLBENZENE m-DIVINYLBENZENE STYRENE 2-(DIETHYLAMINO) ETHYL
METHACRYLATE
NH NH O o \l —
O \ OH HO
N,N-METHYLENEBISACRYLAMIDE METHACRYLIC IMIDAZOLE-4-ACRYLIC 4-VINYLPYRIDINE
ACID ACID
0 Nfi O
O
OWQJY / \ N O
\ HO
0 O
OH
ETHYLENE GLYCOL IMIDAZOLE-4-ACRYLIC METHYLENESUCCINIC
DIMETHACRYLATE ACID ETHYL ESTER ACID

OH
fin N H NH+—\ of
//——N\/l \ / fl SO3H fl
__ o O
I-VINYLIMIDAZOLE 2-VINYLPYRIDINE Z-ACRYLAMIDO-Z-METHYL - 2-HYDROXYETHYL

l-PROPANESULFONIC ACID METHACRYLATE

Figure 2-2. Commonly used functional monomers and cross-linkers.
(Subrahmanyam et al., 2001)

11

2.1.4 Methods for Pre-Screening Polymers

The large number of functional monomers and cross-linkers often makes the
selection process a difficult step. Assessing different functional monomers and cross-
linker systems as well as various concentrations of each can be time consuming due to
the tedious processing and evaluation of the resultant MIP. One technique that is used to
ensure the success of an imprinted polymer involves scaling down the MIP synthesis,
processing, and rebinding steps in an automated in situ batch process using programmed
liquid-handling equipment. In a study by Takeuchi et al. (1999), the polymerization
solution was dispensed into glass vials at varying monomer concentrations. They were
then sealed, allowed to polymerize by UV light, and washed by repeated steps Of
dispensing and removing the solvent, all occurring in an automated process. A
computational approach was demonstrated for microcystin-LR in which the template and
a computationally designed MIP were evaluated according to their affinity, specificity,
cross-reactivity, and stability and were compared to monoclonal and polyclonal
antibodies (Chianella et al., 2002). An alternative method using computationally
designed polymers entailed the screening of a virtual library of functional monomers
against the template of interest. The functional monomer that was able to form the
strongeSt complex with the template was selected, and real polymerization conditions
were compiled using commercially available software to test the polymer

(Subrahmanyam et al., 2001).

12

2.1.5 Physical Modification of the Polymer

Several procedures exist for modifying the physical characteristics of the polymer
so that it will suit the intended application. Imprinted polymers that result from bulk
polymerization in block form are relatively simple to develop, but require tedious
processing to obtain the small fragments. The processing Of the monolithic solid includes
mechanical grinding and sieving steps to obtain small particles, and the process is
inherently wasteful of chemicals (this process is commonly reported in the literature)
(Kroger et al., 1999; Tan et al., 2001; Lu et al., 2002). A slightly more complicated
method, typically used in chromatography, allows polymerization to occur with beads
where the imprinted polymer may fill in the pores or simply coat the surface Of the bead
(Tamayo et al., ; Fairhurst et al., 2004). This type of polymerization allows for
significantly better particle shape compared to grinding of a solid block, yet it also
requires careful preparation and carries the additional cost of the original beads.

More sophisticated and complex polymerization techniques involve either a
suspension in water or perfluorocarbon to produce spherical beads. The disadvantage of
using a water suspension is the incompatibility with most imprinting procedures due to
the disruption of interactions from the polar nature of water. The use of perfluorocarbon
overcomes the interference with imprinting; however, the cost of liquid perfluorocarbon
and the small literature base limit the use of this method. These suspension methods for
producing imprinted polymer beads are mainly used in packed columns for
chromatography. For a more detailed description of polymerization methods, refer to the

article by Mayes and Mosbach (1997) and the references cited within the article.

13

2.1.6 Membrane Imprinted Polymers

The other polymer form that is most suited to sensor applications is a thin
membrane. Thin films are ideal for sensor applications due to their uniform macroscopic
Shape and the variety of reproducible conditions that can be controlled during
polymerization, such as the temperature, polymerization rate, and light flux (for
photoinitiated polymerization). However, the inherent cross-linking nature Of MIPS
makes them very stiff and rigid, a property not suitable for films. Polymer films can be
made more flexible and mechanically stable by the addition of a plasticizer. In the
preparation of an imprinted film for atrazine, a common agricultural pesticide,
Oligourethane acrylate was added to the polymerization mixture containing the functional
monomer methacrylic acid and triethylene glycol dimethacrylate as the cross-linker
(Sergeyeva et al., 1999). Oligourethane acrylate served as the plasticizer in this
procedure, making the imprinted film more flexible. The Optimal ratio of cross-linker to
plasticizer was found to be 85:15 (w/w) with the specificity and flexibility of the
imprinted membrane maintained. To form the membrane, the polymerization mixture
was placed between two quartz glass slides where polymerization occurred by the
addition of UV light. The membrane thickness was between 60 and 120 pm and was
used in a conductometric sensor system (Sergeyeva et al., 1999).

The use of MIPS as the recognition element in a biosensor requires the
immobilization of the polymer onto the surface of the transducer. In situ polymerization
allows the polymer to be electrically synthesized on the surface of the transducer with no
after-processing requirement, excluding solvent washing for removal of the template.

The film is grown onto conducting electrodes Of virtually any Size and shape with the

14

thickness controlled by the amount of charge flowing through the electrode. The first
successful application of electropolymerization detected a glucose template in a poly(o-
phenylenediamine) polymer matrix using a quartz crystal microbalance biomimetic
sensor (Malitesta et al., 1999).

Another method for applying polymers to transducers is using a surface coating
method, either spin or spray coating. In one experiment, the pre-polymerization liquid
containing the imprint molecule, the functional monomer MAA, cross-linker EGDMA,
photoinitiator, and solvent (toluene) were mixed and sprayed onto a gold electrode where
polymerization was initiated by exposure to UV radiation (Jakoby et al., 1999). Polymer
beads of uniform size can also be used in sensors by immobilization onto an electrode
either in a gel or in a membrane. An imprinted polymer, poly(EGDMA-co-4-
vinylpyridine), specific for the agricultural herbicide 2,4-Dichlorophenoxyacetic acid
(2,4-D) was prepared by the bulk polymerization method and mechanically ground into
small particles that were suspended in acetone. The particles that remained in suspension
after 4 h of settling time were collected. The collected particles were resuspended in
methanol, pipetted onto a screen-printed electrode, and allowed to dry. The particle layer
was covered with a hot agarose solution to form a permeable membrane over the
electrode (Kroger et al., 1999).

Thin polymer layers can also be formed by adhesion to other surfaces. For the
preparation of a thin membrane for sensing of theophylline, an indium-tin oxide film was
washed and placed in a solution of MAA, EGDMA, template, and a photoinitiator,
allowed to polymerize, and ultrasonicated to remove weakly adhering copolymer

(Yoshimi et al., 2001). Another procedure developed a flow injection capillary imprinted

15

with 2,4-D in the presence of 4-vinylpyridine (functional monomer) and
trimethylolpropane trimethacrylate (cross-linker). The activated capillaries were filled
with the functional monomer and cross-linker solutions with an initiator where
polymerization was carried out in a water bath. To remove loosely adhering polymer,
this procedure utilized a sonication bath after rinsing with methanol to obtain a uniform
polymer layer (Surugiu et al., 2001b). Similar to the capillary tube, imprinted polymer
films can be polymerized between two glass slides (Duffy et al., 2002). For a more
extensive review of emerging MIP formats, refer to the article by Perez-Moral and Mayes

(2002)'

2.1. 7 Transducer Selection

In addition to the recognition element, the other component of a biosensor is the
transducer, which converts the recognition event into a measurable electric signal. The
binding of the analyte to the antigen can be verified by optical, piezoelectric, or
electrochemical devices. The two most common methods are electrochemical and optical
due to ease of measuring and the availability of instrumentation. The selection of the
transducer depends on many factors, such as the nature of the analyte, temperature range,
presence of corrosive materials, atmospheric/gaseous conditions, size or weight
constraints, and desired sensitivity (Haupt and Mosbach, 2000; Merkoci and Alegret,
2002)

Electrochemical sensor devices can be subdivided into four categories:
amperometry, potentiometry, conductometry, and impedance measuring sensors.
Amperometric sensors measure the current at a fixed voltage and are widely used due to

their simplicity and low cost. Potentiometric sensors measure the voltage at zero or near

16

zero current. The potential is measured between a working electrode and a reference
electrode. Measurements using both amperometry and potentiometry are made either in
steady-state or transient conditions since an equilibrium response is not possible.
Conductive sensors measure the change in conductivity over time as a result of the
analyte binding to the receptor. Impedance devices measure the total electrical resistance
when an alternating current is passed through a specific medium, such as a polymer
membrane (Blanco-Lopez et al., 2004).

Other transduction formats include piezoelectric, which measures changes in
mass, and optical, which monitors the emission or adsorption of electromagnetic
radiation such as spectrometry, fluorimetry, surface plasmon resonance (SPR), and
luminescence (Yano and Karube, 1999). Table 2-1 summarizes different methods of

detection in sensors and the monomer-analyte system used in each study.

17

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18

2.1.8 Food and Agricultural Applications of MIPS

2.1 .8.1 Food Applications

In recent years, people have become more health conscious, leading to
adjustments in food preparation and handling. Changes in lifestyle and attitude have led
to the consumption of raw and fresh foods that are minimally processed, with importance
given to those that are low in fat, sugar, and preservatives. These shifts in food
preparation and handling create a more suitable environment for the grth of
microorganisms. The result is an increased need for fast, cost-effective biosensors with a
high degree of sensitivity and selectivity for the detection of pathogens, microorganisms,
toxins, and pesticides in food and water. This demand has sparked the need for sensors to
rapidly identify any adulteration that may be present in food or water. By the year 2005,
the food pathogen testing market is anticipated to grow to $192 million per year with 34
million tests to be performed (Alocilja and Radke, 2003). The first study to report a MIP-
like technique for pathogen detection used a surface imprinting technique for Listeria
monocytogenes and Staphylococcus aureus to synthesize a polyamide layer around the
template bacteria, forming a microcapsule (Aheme et al., 1996). While this is not
produced like conventional imprinted polymers, the research leading in this direction will
be critical to detecting microbial food and water contaminants with MIP sensors.

The most widely imprinted templates for food applications are small molecules,
such as sugars, peptides, proteins, vitamins, oils, colorants, preservatives, and toxins. In
a study by Lai et a1. (1998), MIPS were produced for three drugs, including caffeine,
using a surface plasmon biosensor. In a later study, a bulk acoustic wave (BAW) sensor

for caffeine was developed using a spin coating method of ground MIP particles (Liang et

19

al., 1999). The detection limit of the sensor was 5.0 x 10'9 M, with a response range
between 5.0 x 10'9 M and 1.0 x 10'4 M. Several other food components have been
successfully incorporated into MIP sensors, such as cholesterol (Piletsky et al., 1999),
flavonol (Suarez-Rodriguez and Dial-Garcia, 2000), and methyl-B-glucose (Chen et al.,
1997). The variety of small compounds that can potentially be imprinted allows the
development of sensors to detect possible harmful substances in food such as toxins and
allergens. A recent review discussing MIPS for food monitoring applications including

sensors, liquid chromatography, and solid phase extraction is available (Ramstrom et al.,

2001).

2.1.8.2 Agricultural Applications — Pesticides, Toxins, and Biowarfare Agents

In the agricultural sector, the detection of pesticides that control unwanted plants,
insects, fungi, and microorganisms requires novel approaches to monitor the levels
specified by governmental agencies. MIPS constitute an emerging technology in
agricultural sensing that overcomes some drawbacks of other methods, such as the
reliance on antibodies and the irreversible binding that inhibits reuse of some sensors.
Several studies have been conducted on agricultural contaminants, many in the sensing
area. As previously described, a competitive electrochemical sensor for the herbicide
2,4-D was developed by immobilizing imprinted particles with an average diameter of 1
pm onto screen-printed electrodes. The sensor response was measured using differential
pulse voltammetry, with the time for one scan taking 15 s (30 min to l h to reach
equilibrium) (Kroger et al., 1999). Several branch studies have been performed using
2,4-D in other sensor formats, such as a thin MIP on the surface of a silicon wafer studied

with a radio ligand binding assay (Yan and Kapua, 2001). Two similar studies employed

20

chemiluminescence to detect 2,4-D in a setup similar to an enzyme-linked
immunosorbent assay using a MIP as the receptor instead of the antibody (Surugiu et al.,
2001a; Surugiu et al., 2001b). MIPS have been formed for other herbicides, such as an
imprinted polymer coating for desmetryn onto a polypropylene support film using
grafting photopolymerization (Panasyuk-Delaney et al., 2001). This sensor used
capacitive detection to test the response for desmetryn against triazine derivatives and
found the sensor to be fairly Specific to the target molecule. As mentioned in the
previous discussion on thin film MIPS, a conductometric membrane sensor for atrazine
was prepared from MAA and EGDMA with the addition of a plasticizer to increase the
flexibility of the membrane (Sergeyeva et al., 1999). The response time of the sensor
varied from 12-15 min for a 120 um membrane to 6-10 min for a 60 um membrane, with
the imprinted membranes having excellent specificity to atrazine compared to Similar
herbicides.

A thin-layer MIP for the pesticide hexachlorobenzene was attached to a quartz
crystal microbalance (Das et al., 2003). High selectivities compared to four structurally
Similar compounds and sensitivities as low as 10'12 mol/L were obtained for this MIP
with a response time of approximately 10 S. Promising results were also obtained for an
optical MIP sensor for pesticide and insecticide detection in water where the detection
limit was less than 10 parts per trillion with a response time of less than 15 min (Jenkins
et al., 2001). A recent study demonstrated the successful formation of a MIP for the
herbicide metsulfuron-methyl (Zhu et al., 2002). These studies Show that research into
the fabrication of real-time, sensitive, and selective sensors for pesticide detection can be

initiated since many of these MIPS have been proven successful.

21

Studies have also demonstrated that MIPS can be formed for toxins and biological
agents. While these molecules are not used in sensors, they are worth noting for future
sensor applications in agriculture and biosecurity. A potentially carcinogenic mycotoxin
produced by several species of fungi, ochratoxin A, was used in MIPS for solid phase
extraction (Jodlbauer et al., 2002) and liquid chromatography (Baggiani et al., 2002).
Two MIPS for the mycotoxins deoxynivalenol and zearalenone were shown to be
successful through HPLC analysis (Weiss et al., 2003). Future studies can strive to link
the above three MIPS to a transducer for use in sensing devices.

The design of a polymer Specific for microcystin-LR, a toxin produced by several
species of cyanobacteria, had a detection limit of 0.1 ug/L using an enzyme-linked
competitive assay (Chianella et al., 2002). A silane-imprinted polymer was successfully
formed for conotoxin, a neurotoxin found in snails of the Conus genus (Iqbal et al.,
2000). The results of the study indicated that silane-imprinted polymers could recognize
subtle structural differences between two closely related conotoxins. Another silane-
imprinted polymer Specific for ricin, a potent toxin from castor beans and potential
biowarfare agent, was confirmed by fluorescence binding detection and Showed a high
affinity to the imprinted Sites (Lulka et al., 2000; Piletsky et al., 2001a). The detection of
this large toxin exhibits potential for producing MIPS for other larger molecules and

biological toxins.

2. 1.9 Potential Applications

A surge in the number of groups working with MIPS (a rise from about 8 to 70
from 1990 to 1999) and the number of papers being published (a rise from about 12 to

119 from 1990 to 1999) shows that interest in imprinting technology is expanding with

22

continual opportunities for research (Piletsky et al., 2001a). Imprinting technology has
the ability to be used in separatiOn systems, such as water purification, wastewater
treatment, and in the chemical and pharmaceutical industries. The potential market for
biosensors in drug testing, drug delivery, in vitro diagnostics, military (warfare agent
detection), and electronic noses and tongues is $1.9, $1, $19, $0.64, and $4 billion,
respectively (Piletsky et al., 2001b). The use of MIPS in the solid phase extraction of
drugs and in drug separation is of growing interest as well as applications for drug
screening and in vivo monitoring. The ability of MIPS to retain receptor properties under
extreme conditions without loss of recognition may be very important in aeronautical
situations of low gravity and in situations of extreme pressures or temperatures due to the
stability of the polymer compared to other receptors such as antibodies. MIPS will also
be critical for military applications for the detection of biological warfare agents such as
toxins as well as the detection of compounds used for explosives where the stability of
the sensor and the long Shelf life (up to six months for some MIPS) will be vital to the

sensor.

2.1.10 Limitations and Challenges

The ability to incorporate MIPS with sensors combined with the stability,
resistance, and shelf life of the recognition sites allows for a new array of biosensors that
do not require controlled environments, as is the case for antibodies. The potential
applications for MIPS in the field of biosensors and assay systems are limited by the
research on MIP systems that is currently available. The number of templates that can be
successfully imprinted is constantly increasing as new functional monomer-template

combinations are assembled. Although a variety of imprinted polymers can be produced,

23

the lack of a general procedure for their preparation and confirmation of binding Sites
limits the surge of industrial interest in this technology. Often the presence of
background noise due to the remaining template in the receptor sites can interfere with
the Signal, especially in fluorescence type systems. Future research will also focus on the
development of MIPS for use in water and other polar-based solvent systems as well as
increasing the sensitivity and selectivity of current imprinted polymer systems.

Studies recently published have shown that certain templates can be successfully
imprinted, but there is a lack of feasible applications for the resulting imprinted polymers.
Most imprinting technology arises from specialized groups that seek to extend and
improve current methods and monomer systems. The challenge for future researchers
will be to find real-world applications for current polymer systems that are rapidly being
produced. The ability to link an imprinted polymer with a suitable transducer that allows
for quantitative measurements of the binding of the analyte will be critical to the
incorporation of MIP receptors into real-time sensors. In the near future, imprinting
technology has the potential to become mainstream alongside current biosensors and to

offer real alternatives in sensing systems.

2.2 Cyclic Voltammetry

Electrochemical techniques explore the interplay between electricity and
chemistry. It Specifically examines the role of charge, current, or potential, to changes in
chemical parameters, such as the concentration of a particular compound. Potentiometric
methods measure an electron transfer reaction by applying a potential and measuring the
resulting current. The purpose of a potentiometric technique is to acquire a current

response that is related to the concentration of the target analyte. Cyclic voltammetry is a

24

potentiometric method that applies a triangular potential waveform to the system under

investigation resulting in a current response (Figure 2-3).

Potential

Einitial,

  
     

Reverse
Scan

 

Figure 2-3. Excitation signal for a cyclic voltammetry experiment.

Time

(Adapted from Wang, 2000)

V

Cyclic voltammetry varies the potential to obtain information about the redox

process of the system and to obtain the values of the redox potentials. A typical cyclic

voltammogram for a reversible process is Shown in Figure 2-4. The potential is plotted

on the x-axis with the measured current values plotted on the y-axis. The values of

interest on a cyclic voltammogram are the two peak currents and the corresponding

potentials. The cathodic peak current, ipc, and the anodic peak current, ipa, occur at the

reduction point and oxidation point, respectively (Bard and Faulkner, 2000).

25

 

 

 

ipc 4_ R (- O
O
‘6‘
0
§
§ ____________________________________________
t .
:3
D
0
‘6
o
3
ipa —_
R -) O
i i
E<~> Echpa E<+>
Potential

Figure 2-4. Typical cyclic voltammogram for a reversible system.

26

In cyclic voltammetry experiments, the current is measured in a three-electrode
electrochemical cell. The electrochemical cells used in this research are further discussed
in chapter 3 (Section 3.4). The schematic representation of a three-electrode potentiostat
is shown in Figure 2-5. In a potentiostat, the current is supplied through the counter
electrode and iS measured through the working electrode. The response of the working
electrode depends on the concentration and composition of the analyte being measured.
The third electrode is a reference electrode that provides a constant reference potential
against which the working electrode measures the output current. The reference
electrode draws little or no current from the system and is unresponsive to the

composition of the species under measurement (Bard and Faulkner, 2000).

Scan

WHfier
_W . . Counter
Eapp / Electrode

/l
FeedbacN

 

   
   
 

 

x. Working
Electrode

 

 

 

 

Amp lrfier Reference." E, I
Electrode Output
Current
Amplifier

Figure 2-5. Schematic representation of a three-electrode potentiostat.
(Adapted from Wang, 2000)

27

Cyclic voltammetry measures the faradaic current, or the current as a result of
electron transfer. Two factors can affect the faradaic current: the rate at which the redox
species diffuses to the electrode and the rate of electron transfer. The rate the species
diffuses to the electrode surface is discussed in Section 4.1 as it applies to this research.

The rate of electron transfer for the common redox couple Fe(CN)63'/4' is

reasonably fast. The reaction at the working electrode or cathode is
Fe(CN)6_ + e' <—> Fe(CN)6-

where one electron is added to the ferricyanide anion to reduce iron from the +3 to the +2

oxidation state. This reaction proceeds in both directions so that Species can be oxidized

to Fe(CN)63_ , then reduced back to Fe(CN)64_ (Pine Instrument Company).

28

CHAPTER 3: METHODS AND MATERIALS

3.1 Overview of Procedure for Research

The basic procedure for molecular imprinting with theophylline follows that of
Yoshimi et al. (2001) except for the modification of the template removal procedure.

Table 3-1 identifies the parts unique to this research.

Table 3-1. Procedures Used in This Research.

 

 

 

Procedure in This Research Reference
Cleaning of ITO electrode Yoshimi et al. 2001
Formation of MIP Yoshimi et al. 2001

 

Vlatakis et al., 1993; Hong et al., 1998;

Removal of template Mullett and Lai, 1998

 

 

 

 

Rebinding Yoshimi et al. 2001
Measurement Yoshimi et al. 2001
Sensitivity Original to this research
Selectivity at various concentrations Original to this research

 

MIP on Silicon measured with cyclic

voltammetry Orlginal to thls research

 

 

 

 

29

3.2 Chemicals

Methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), 2,2'-
azobisisobutyronitrile (AIBN), and 3-(trimethoxysilyl)propyl methacrylate (3-MPS) were
purchased from Sigma (St. Louis, MO). P-type silicon wafers (oxide layer 1 pm,
conductivity 1667 S/cm) were obtained from the microfabrication laboratory at Michigan
State University. Indium tin oxide (ITO) coated glass Slides were acquired from Sigma
(ITO coating 300-600 A; resistance 30-60 0). All other chemicals were of ACS grade

and used as received.

3.3 Preparation of electrodes

3.3.1 IT 0 electrode

The ITO coated glass Slides were cut with a steel wheel glasscutter into 1.3 cm
squares. Each electrode was rinsed twice with methanol followed by rinsing twice with

deionized water, and dried under nitrogen environment.

3. 3.2 Silicon electrode

The Silicon electrodes were cut with a diamond tipped pen into 1.3 cm squares.
These electrodes were soaked in hydrofluoric acid for 5 min to remove the surface oxide.
Following oxide removal, the silicon was cleaned by first immersing in a 1:1 v/v solution
of methanol and hydrochloric acid for 30 min, and then thoroughly rinsing twice with
deionized water. The silicon was next boiled in water for 30 min and rinsed twice with

deionized water. The silicon electrodes were dried under nitrogen environment.

30

3.4 Electrochemical cells

Two different electrochemical cells were used for this research. Cell A, shown in
Figure 3-1, was an inverted cone Shape where the working electrode is placed at the
bottom of the cell. The solution only contacted one Side of the working electrode that
was exposed through the opening on the bottom of the cell. The contact area between the
electrochemical solution and the working electrode in cell A was circular with an area of
0.283 cm2 (diameter of 0.6 cm). The reference and counter electrodes were secured at the
top of the cell. The amount of liquid necessary to adequately cover the counter and
reference electrodes was 25 ml for cell A.

The other electrochemical cell, cell B shown in Figure 3-2, is shaped similar to a
beaker with three openings all located on the top. The reference and counter electrodes
were secured at the top similar to the setup in cell A. In cell B, however, the working
electrode was suspended in the electrolyte solution such that the area of the working
electrode in the electrochemical solution was greater than cell A. The approximate
surface area of the working electrode exposed to the solution in cell B was 1 cm2. The
height of the working electrode relative to the cell was fixed by the rubber stopper. The

volume of liquid used in cell B was 50 ml to cover the three electrodes.

31

Reference Counter Electrode
Electrode (Untreated ITO or Si)

(Ag/AgCl)

      
      
  

Electrolyte
Solution

Working Electrode
(Treated ITO or Si)

 
    
   

Telflon O-ring

 

Copper Plate

\§ I‘“\\\\\\\\‘
F. "l‘l
. i . .'. “1.:
‘ .

Figure 3-1. Cell A - Three-electrode electrochemical cell with working electrode at
the bottom of the cell.

32

 

 

 

 

 

 

 

 

 

Rubber
Stopper
Reference
Electrode
(Ag/AgCl)
Counter Electrode
(Untreated ITO) '
\ Working Electrode
(Treated ITO)
Electrolyte
Solution

 

 

 

 

Figure 3-2. Cell B - Three-electrode electrochemical cell with working electrode
submerged.

33

3.5 Preparation of the Theophylline Imprinted Biomimetic Sensor

The process of preparing the electrode (ITO or Si) and forming the theophylline
MIP is illustrated in Figure 3-3. The formation of the MIP followed the procedure by
Yoshimi et al. (2001) with some modifications. The electrode (ITO or Si) was silanized
in a 10% solution (v/v) of 3—MPS in toluene for 6 h at 80°C under nitrogen atmosphere.
Silanization activated the surface of the electrode, which allowed the MIP to be
covalently bonded to the surface. Following Silanization, the electrode was rinsed with
methanol and dried under nitrogen environment. The polymer was prepared using
methacrylic acid (MAA) as the functional monomer and ethylene glycol dimethacrylate
(EGDMA) as the cross-linker (Figure 3-4). The initiator was 2,2'-azobisisobutyronitrile
(AIBN), the porogenic solvent was MN-dimethylformamide (DMF), and theophylline
was used as the template. Chemical inhibitors were removed from MAA and EGDMA
by passing them through an inhibitor removal column (Aldrich 30,631-2) immediately
before use. MAA (0.119 ml), EGDMA (1.20 ml), AIBN (0.036 g), and theophylline
(0.063 g) were added to 3.31 ml of DMF. The silanized electrode was immersed in 2 ml
of the above solution and placed under nitrogen atmosphere. The electrode was allowed
to polymerize for 12 h at 60°C. After this preparation, the theophylline-imprinted
polymer on ITO or Silicon would be referred to as the biomimetic MIP-ITO or MIP-Si
sensor. A reference non-imprinted polymer (blank) to be referred to as B-ITO or B-Si,

was similarly made by omitting the theophylline template.

34

a OH 0H 0“
Platform . _ I . I ,_ _ . I

 

 

 

(ITO or Si) [ _ a
CH CH CH
b) H2C\\ / 3 Hzc\\ / 3 H20\\ / 3
C C C
Platform 1 I I _ I ]
(ITO or Si) V 7

 

C)

   
  
  
  
 
 
 

(MAA-
EGDMA)

Platform
(ITO or Si)

d)

Imprinted
Site

Copolymer COO“
of MAA
and EGDMA

Platform
(ITO or Si)

 

Figure 3-3. Procedure for the formation of theophylline MIP on electrode.
(a) Clean electrode; (b) Silanize in 3-MPS/toluene (10% v/v) (c) Combine MAA,
EDGMA, AIBN, DMF, and Thy; polymerize for 12 h at 60°C; Eliminate bulk
polymer; (d) Wash with 9:1 methanol/ acetic acid for l h.

35

a) b)

ll
H H2C\ 0 CH2 /o /o
HZC=c':’C‘0H \c/ \0/ \CH2 \c/
CH3 (L C
H3 /
H20/ \CH3

Figure 3-4. Chemical structures of (a) methacrylic acid and
(b) ethylene glycol dimethacrylate.
(Sigma-Aldrich, 2004)

3.6 Extraction of Template

Both the MIP and blank sensors (ITO and Si) were washed in a 9:1 v/v solution of
methanol and acetic acid for 1 h. This washing removed the template and any excess
polymer. The MIP and blank sensors were rinsed twice with methanol followed by
rinsing twice with water and twice more with methanol. The MIP and blank sensors were

stored in water until measurement to prevent drying or cracking of the polymer.

3.7 Baseline Measurement

Two electrochemical cells were used in this research. Cell A (Figure 3-1) had a
circular area of the sensor exposed to the electrolyte solution with a diameter of 0.6 cm.

Cell B (Figure 3-2) had both sides of the sensor exposed through a dipping format.

36

Before addition of the theophylline analyte, a baseline was obtained for each
sensor (MIP and blank) using a blank electrolyte that was an aqueous solution of 0.1 M
potassium nitrate and 5 mM potassium ferrocyanide. A volume of 25 ml (or 50 ml for
cell B) was added to electrochemical cell A. Cyclic voltammetry was performed with the
blank solution using a Versastat II Potentiostat/Galvanostat (Princeton Applied Research,
Oak Ridge, TN). The working electrode was the treated (imprinted) material, the
reference electrode was the Ag/AgCl, and the counter electrode was the untreated
material (ITO or silicon). The potentiostat was run in the ramp, one vertex multi mode.
The potential was cycled between —1 V to +1 V for ITO, and between —2 V to +2 V for
silicon at a scan rate of 200 mV/S. The resulting current was measured and plotted

against the potential. After measurement, the blank solution was discarded.

3.8 Rebinding and Measurement of Theophylline Analyte

The rebinding of the analyte was performed in the same electrochemical cell by
cyclic voltammetry measurements. The analyte solution was the blank (0.1 M potassium
nitrate and 5 mM potassium ferrocyanide) with the addition of the appropriate
theophylline concentration. The analyte solution of 25 ml for cell A (or 50 ml for cell B)
was added to the electrochemical cell and cyclic voltammetry was performed as in
Section. 3.7. The sensitivity of the MIP sensor was evaluated at four theophylline
concentrations: 1, 2, 3, and 4 mM. Signal measurements were done according to Section

3.7.

37

3.9 Selectivity Evaluation

The selectivity of the MIP sensor was evaluated using caffeine, which is
structurally related to the target analyte, theophylline. AS Shown in Figure 3-5, the
structures of theophylline and caffeine differ only in the group attached to the nitrogen
atom. In theophylline, this group is a Single hydrogen atom while in caffeine this is a
CH3 group. This slight structural difference makes caffeine ideal for testing the
crossreactivity of the theophylline-imprinted polymer sensor. The selectivity testing was

performed using four concentrations of caffeine: 1, 2, 3, and 4 mM.

(a) (b)
O O
H C CH H C
3 \N N/ 3 3 \N/U\N,CH3
O O /
/ N—H /N
H3C

Figure 3-5. Chemical structures of (a) theophylline and (b) caffeine.
(Sigma-Aldrich, 2004)

38

3.10 Light Absorbance Measurements

The MIP and blank polymer were prepared according to Section 3-5. Following
template removal for 1 h in 9:1 methanol/acetic acid, 50 mg of both the MIP and non-
imprinted polymer was removed, weighed, and rebound with the analyte separately. The
rebinding concentration of 5 mM theophylline was combined with each of the polymers
and allowed to soak for 25 min. After rebinding, each polymer was immersed in fresh
water for 20 s to remove weakly adhering analyte. Each polymer was combined with 1
ml of deionized water and placed in a 1 ml cuvette. The light absorbance of only the
polymer (without the ITO or Si electrodes) was measured between a wavelength of 200

nm to 800 nm with a SmartSpec 3000 Spectrophotometer from Bio-Rad (Hercules, CA).
3.11 Statistical Analysis

3.11.1 Determination of Peak currents on Cyclic Voltammograms

The maximum (ipe) and minimum (ipa) peak currents at the oxidation-reductions

shifts (Figure 2-4) on the cyclic voltammograms were selected for statistical analysis.
The current ratio was the “peak current after addition of the analyte” to the “peak current
before addition of the analyte” and was used to compare between cyclic voltammograms.
All cyclic voltammograms were an average of three trials. Each trial was performed on a
separate day using fresh solutions and new polymers to account for slight variations in

the solutions and day-to-day error.

39

3.11.2 Evaluation of Blank and MIP Sensors

The equality of variances was tested on the absorbance values and current ratios
using F-test prior to testing mean differences with t-test. T-test was performed on the
absorbance values and mean current ratios to determine if the difference between the MIP
and blank sensor was Significant. The difference in the mean values was considered to be

Significant when the p-value was less than 0.05, representing a 95% confidence level.

3.12 Characterization of Surface

The surface of the MIP sensor was evaluated using atomic force microscopy
(AFM). AFM experiments were performed with a NanoScope IIIa from Digital
Instruments (Santa Barbara, CA) with the tapping mode. AFM images were obtained
after template removal before rebinding and afier template removal and rebinding. The
removal of the template was performed for l h in 9:1 methanol/acetic acid. The
rebinding was performed for 25 min in a 5 mM aqueous solution of theophylline. After
rebinding, the sensor was immersed in clean water for 20 s to remove weakly adhering

analyte.

40

CHAPTER 4: RESULTS AND DISCUSSION

4.1 Theory of Detection
The rebinding of the analyte to the imprinted polymer is observed through
electrochemical measurements. The redox couple Fe(CN)63'/4‘ undergoes reasonably fast

kinetics of electron transfer and was selected for this reason. The effect the rebinding of
analyte to the MIP has upon this reversible reaction is monitored through measurement
by cyclic voltammetry.

The current in the imprinted polymer is enhanced upon binding of the target
analyte. Previous studies have Shown an increase in current through potentiometric
measurement (Yoshimi et al., 2001; Blanco-Lépez et al., 2003b, 2003a). The reason for
the increase in current upon binding of the analyte to the imprinted polymer is
hypothesized to be due to the increase in the permeability of the polymer, called the
Shrinking effect (Figure 4-1). When the analyte binds in the imprinted sites, the polymer
Shrinks around the analyte, thereby increasing the size of the surrounding pores in the
polymer. The increase in the pore Size increases the permeability of the polymer and
allows the charge to flow less restricted (Piletsky et al., 1996). The less restricted

electron flow results in an increase in the measured current.

41

 

 

 

 

 

 

 

 

 

Figure 4-1. Schematic representation of the shrinking effect. Possible
transformation in the arrangement of imprinted polymer upon rebinding of
template molecule leading to increased electron flow
(Adapted from Piletsky et al., 1996).

4.2 Confirmation of Imprinted Polymer

Theophylline at a concentration of 5 mM exhibited a maximum absorbance of
0.746 absorbance units (AU) at a wavelength of 295 nm (Data in Appendix A). Since the
maximum absorbance for theophylline occurred at 295 nm, the absorbance of the non-
imprinted polymer and MIP at this wavelength were used for further analysis.

The light absorbance values for the non-imprinted polymer and MIP at a
wavelength of 295 nm were 0.038 3: 0.025 AU for the blank and 0.203 d: 0.055 AU for

the MIP with a P-value of 0.018. The absorbance measurements of the non-imprinted

42

polymer compared to the MIP were significantly different at a 95% confidence level,

indicating that an imprinted polymer was successfully formed for theophylline.

4.3 Characterization of Surface
The surface of the MIP was evaluated after template removal and after rebinding.

The AFM image afier template removal is Shown in Figure 4—2 and the image after

rebinding is displayed in Figure 4-3.

Digital Instruments Nanoscope

Scan size 5.000 um
Scan rate 1.001 Hz
Number of samples 256
Image Data Height

    

Data scale 100.0 nm

=7 View angle

{9: light angle
0 0

Figure 4-2. AFM image of MIP-ITO sensor after extraction of the theophylline
template for 1 h in 9:1 methanol/acetic acid.

um

x 1.000 urn/div
2 100.000 nm/div

43

Digital Instruments Nanoscope

 
    

Scan size 5.000 urn
Scan rate 1.001 Hz
Number of samples 256
Image Data Height
1““ _ ‘j f ' . Data scale 100.0 nm
200 ' ‘ “ ~ ‘ '
T view angle

{:3 light angle

mm

x 1.000 urn/div 0 °
2 100.000 nm/div

Figure 4-3. AFM image of MIP-ITO sensor after rebinding in 5 mM theophylline
for 25 min.

The mean surface heights and standard deviations were 138.02 nm i 17.735 nm
for the MIP-ITO after extraction of the template and 95.522 nm :1: 9.290 nm for the MIP-
ITO after rebinding. The MIP-ITO afier template extraction (Figure 4-2) is rougher than
the MIP-ITO after rebinding (Figure 4-3). This can be observed visually from the images
as well as by comparing the standard deviations of the heights. The standard deviation of
the MIP-ITO after template extraction is almost two times greater than the standard
deviation after rebinding. The decrease in the surface roughness upon analyte rebinding

is hypothesized to be due to the Shrinking effect. Upon rebinding of the target analyte,

44

the polymer shrinks around the analyte, thereby reducing the mean surface height and

roughness of the polymer.

4.4 Initial Measurements — Cell A

4.4.1 MIP-1T0 Sensor

The first measurements of the MIP-ITO sensor and MIP-Si sensor were
performed with cell A. AS previously discussed in section 3.4, the exposure area was
circular with a diameter of 0.6 cm. The cyclic voltammograms of the B-ITO and MIP-
ITO in the presence and absence of theophylline are Shown in Figures 4-4 and 4-5,
respectively. The addition of theophylline in the MIP-ITO resulted in an increase in the
peak current of 0.036 mA. Addition of theophylline in the B-ITO resulted in a Slight
decrease in the peak current of 0.006. The ratio of the change in peak current after
addition of theophylline for the MIP-ITO increased by a factor of six compared to the B-

ITO.

45

 

0.05 — 1 No Thy
0.04 — 2 5 mM Thy

003 ~
0.02 -
0.01 -
0.00 ~ '\
-0.01 -
002 ~
-0.03T
-004 ~
-0.05 . . . . . .
-1.2 -0.8 -0.4 0 0.4 0.8 1.2

Potential (V)

Current (mA)

 

 

 

 

 

Figure 4-4. Cyclic voltammograms of B-ITO in the absence of theophylline and in 5
mM theophylline for cell A.

 

0-05 ‘ 1 No Thy
0.04 - 2 2 5 mM Thy

0.03 - /

0.02 a
0.01 -
0.00 -
-0.01 ~
-0.02 ~ 1
-0.03 -
-0.04 ~
-0.05 I i T . . 1
-1.2 -0.8 -0.4 0 0.4 0.8 1.2

Potential (V)

 

Current (mA)

 

 

 

 

 

Figure 4-5. Cyclic voltammograms of MIP-ITO in the absence of theophylline and
in 5 mM theophylline for cell A.

46

In a previous study by Yoshimi et al. (2001), ultraviolet radiation was used to
induce polymerization. In this research, the MIP was polymerized thermally by the
addition of heat. The bulk polymer was formed on the electrode and removed by
breaking and subsequent washing steps. For removal of the template, Yoshimi et al.
utilized sonication in water, while this research employed rinsing in a solution of
methanol and acetic acid. Early trials in these experiments found that sonication removed
the thin film polymer, which was confirmed through cyclic voltammetry. For this reason,

sonication was rejected, and washing with an alcohol-acid solution was adapted.

4. 4.2 MIP-Si Sensor

This research also conducted some initial tests of a thin MIP layer directly
attached to Silicon as a sensor. The initial measurements were performed in cell A,
similar to the MIP-ITO sensor. The cyclic voltammograms for the B-Si and MIP-Si are
shown in Figures 4-6 and 4-7, respectively. The change in the peak current before and
after the addition of theophylline was 0.058 mA for the B-Si and 0.078 mA for the MIP-
Si. The ratio of the changes in the peak current of the MIP-Si and the B-Si was only a
factor of 1.3 and was not as marked as the six fold increase of the MIP-ITO to the B-ITO

The Shapes of the curves for the Silicon platform in Figures 4-6 and 4-7 are
comparable to those on ITO shown in Figures 4-4 and 4-5. As in Figure 4-4 (B-ITO), the
current response curve of B-Si before addition of theophylline is not flat compared to that
of the MIP-Si. However, the response curve after addition of theophylline follows the
‘curve before addition. This indicates that the response from B-Si is non-positive, because

the difference between B-Si before and after addition of the analyte is small.

47

 

Current (mA)

 

1

Figure 4-6. Cyclic voltammograms of B-Si in the absence of theophylline and 5 mM

0.15 -
0.10 -
0.05 -
0.00 —
-0.05 ~
-0.10 ~
-0.15 —

 

-0.20 -—-—

-2.25

 

2 1 No Thy
/ 2 5 mM Thy
1
-1.5 -0.75 O 0.75 1.5 2.25

Potential (V)

 

 

 

 

 

theophylline for cell A.
0-15 ‘ 1 No Thy
0_10 _ /2 2 5mMThy
g 0.05 H \
v 0.00 —
‘E
g -0.05 - l \
3 1
O —0.10 —
-0.15 —
'0-20 I I T l l I
-2.25 -1.5 -0.75 0 0.75 1.5 2.25
Voltage (V)

 

Figure 4-7. Cyclic voltammograms of MIP-Si in the absence of theophylline and 5

mM theophylline for cell A.

48

 

 

4. 4. 3 Comparison of peak currents for IT 0 and Silicon

The ratios of the peak currents of the MIP and non-imprinted polymer for ITO

and Silicon at the maximum and minimum potentials are shown in Tables 4-1 and 4-2,

respectively. The standard deviations of 3 trials are included in the table as well as the P-

value from performing a t-test on the B—ITO compared to the MIP-ITO on each material.

Table 4-1. Ratio of Maximum Currents (ipc) of MIP and Blank on ITO and Silicon

 

 

 

 

 

 

 

Platform Blank MIP P-Value
ITO 14.114 :1: 23.177 74.408 :t 28.970 0.037
Silicon 1.169 :1: 0.377 8.868 :h 7.027 0.008

 

Means + standard deviations (n=3). Data was tested within a single row.

Table 4-2. Ratio of Minimum Currents (ipa) of MIP and Blank on ITO and Silicon

 

 

 

 

 

 

 

Platform Blank MIP P-Value
ITO 0.192 d: 1.043 62.364 i 30.336 0.048
Silicon 2.131 d: 1.296 12.818 i 3.507 0.131

 

Means :1: standard deviations (n=3). Data was tested within a single row.

49

 

 

The increase in the ratio of the peak currents of the MIP-ITO compared to the B-
ITO at a given potential showed that the MIP on ITO was successful. The ratios at the
maximum potential were 74.4 and 14.1 for the MIP-ITO and B-ITO, respectively. The
factor of increase between the MIP-ITO and B-ITO is 5.3 when only considering the
maximum current. Yoshimi et al. (2001) found these ratios to be 2.94 for the MIP-ITO
and 0.99 for the B-ITO with a factor of increase between the MIP and blank of three.

The increase in the ratio of the MIP to blank for this research compared to
previous results might be attributed to the modification of the template removal
procedure. The removal of the template by Yoshimi et al. was performed in water, while
this research used a mixture of methanol and acetic acid. The alcohol-acid mixture was
used in this research because of its prevalence for the removal of theophylline from a
MAA-EGDMA copolymer (Vlatakis et al., 1993; Hong et al., 1998; Mullett and Lai,
1998). The template removal procedure will be investigated in a continuation of this
research for its effectiveness as well as other factors that may contribute to the MIP
Signal, such as the thickness and uniformity of the MIP layer.

The ratios of the peak currents for Silicon are Shown for the MIP-Si and B-Si in
Tables 4-1 and 4-2. These initial results demonstrated the potential for MIPS to be
produced on a Silicon electrode. The ratio of the peak currents increased by a factor of
7.5 for the MIP-Si compared to the B-Si at the maximum potential. The minimum
currents were not considered because the means were not statistically different. The
variation between different batches of MIPS on silicon merits further investigation to
obtain a consistent response. Additional studies will be being conducted to increase the

success of the theophylline MIP on Silicon.

50

MIPS immobilized on silicon Show potential for research. Silicon is a relatively
inexpensive material whose properties can be uniquely and precisely controlled (Zhang,
2001). Previous literature has shown difficulty forming a thin MIP layer on silicon
(Hedborg et al., 1993). The potential for research into MIPS attached to silicon will likely
increase because few studies have been conducted, and many possibilities for MIP
sensors still exist.

The initial results of this research were conducted with cell A, where the working
electrode was at the bottom of the electrochemical cell. The inconsistent results obtained
with cell A were hypothesized to be due to contact area between the working electrode
and the copper plate where the alligator clip was attached (refer to Figure 3-1). Because
the ITO layer was only on one Side of the glass slide, there was no direct connection from
the ITO layer to the potentiostat. The glass Slide acted as a barrier and interfered with the
signal from the sensor. Due to the this issue, electrochemical cell B was used for all
further testing because the sensor (working electrode) was directly connected to the
potentiostat through the contact between the ITO layer and the alligator clip. In addition
to the improved connectivity, cell B was also used because the measurements were

occurring over a greater surface area.

4.5 Sensitivity and Selectivity Testing Measurements of Submerged Working

Electrode - Cell B

4. 5.1 Background Measurement

The cyclic voltammogram of the background measurement of 5 mM potassium
ferrocyanide and 0.1 M potassium nitrate with 5 mM theophylline is shown in Figure 4-8.

The working electrode was cleaned, untreated ITO (no Silanization or polymer). The

51

presence of theophylline does not alter the Shape of the cyclic voltammogram compared

to a typical voltammogram in Figure 2-4.

 

2.5 4
1.75 ~

0.254
-05—
-125 -
-2-
-275 . 4 . . . .
-1.2 -0.8 .04 0 0.4 0.8 1.2

Potential (V)

 

Current (mA)

 

 

 

 

 

Figure 4-8. Cyclic voltammogram of 5 mM potassium ferrocyanide and 0.1 M
potassium nitrate with 5 mM theophylline on untreated ITO.

4.5.2 Sensitivity of MIP-1T0 Sensor

The sensitivity of the MIP-ITO sensor was evaluated using the fully submerged
cell (cell B). This electrochemical cell was used to improve the sensitivity. The
submersion of the working electrode in cell B was hypothesized to give a higher Signal
than cell A due to the increased surface area of the ITO electrode and the MIP. Cell B
was selected for the sensitivity measurements due to the increased surface area. The
surface area of cell B was approximately 1 cm2 while the surface area of cell A was only

0.28 cm2 (diameter of 0.6 cm). Furthermore, cell B was used for the selectivity testing

52

because the Shape of the background measurements in cell B resembled those of a typical
cyclic voltammogram (Figure 4-8 compared to Figure 2-4).

The sensitivity of the MIP-ITO sensor was evaluated by testing the response at 4
analyte concentrations: 1, 2, 3, and 4 mM. The cyclic voltammograms of the B-ITO and
MIP-ITO sensor in the presence and absence of various analyte concentrations are shown
in Figures 4-9 to 4-16. The response before addition of the analyte served as a baseline
for evaluating the response of each sensor. For this reason, the response of each sensor to
the analyte was compared to the baseline measurement. This allowed the sensors to be
compared and evaluated, and accounted for any Slight differences in conductivity that
may have been present between samples.

The results obtained with cell B did not have as large an increase in the signal as
was the case for cell A. Measurements with cell B were still explored due to the overall
increase in current and because the shape of the cyclic voltammogram resembled that of a

typical curve in Figure 2-4.

53

 

 

Current (mM)

0.4 1
0.3 -
0.2 ~
0.1 -
0.0 _
-0.1 4
-O.2 -
-0.3 —
-O.4 —
-0.5 ~
-0.6 -
-0.7

 

    
  

\

1 No Thy
2 1mM Thy

2...”?

 

-1.2

I

-0.8

I I I I I

-0.4 O 0.4 0.8 1.2
Potential (V)

 

Figure 4-9.

Cyclic voltammogram of B-ITO sensor evaluated at 1 mM theophylline.

 

 

Current (mA)

0.4 -
0.3 -
0.2 ~
0.1 4
0.0 -
-0.1 -
-0.2 -
-0.3 -
-0.4 ~
-0.5 -
-O.6 1
-O.7

 

 

1 No Thy
2 1 mM Thy

 

-1.2

I

-0.8

-0.4 0 0.4
Potential (V)

 

Figure 4-10. Cyclic voltammogram of MIP-ITO sensor evaluated at 1 mM

theophylline.

54

 

 

 

0.15 — 1 No Thy
0,10 _ 2 2 mM Thy
0.05 -
0.00 -
I -0.05 - 1
-0.10 a
-0.15 -
-0.20 -
-0.25 —
-0.30 1 I . . . 1
-1.2 -0.8 -0.4 0 0.4 0.8 1.2

Potential (V)

mA)

Curren

 

 

 

 

 

Figure 4-11. Cyclic voltammogram of B-ITO sensor evaluated at 2 mM
theophylline.

 

0-15 _ 1 No Thy
o_1o — 2 2 mM Thy
0.05 —
0.00 —
I: -0.05 ~
—0.10 ~
-0.15 —
-0.20 -
-0.25 — .
-0.30 . . w I I .
—1.2 -0.8 -0.4 0 0.4 0.8 1.2

Potential (V)

  

mA)

Curren

 

 

 

 

 

Figure 4-12. Cyclic voltammogram of MIP-ITO sensor evaluated at 2 mM
theophylline.

55

 

0.4 4 1 No Thy
0,3 4 2 3 mM Thy
0.2 4

0.1 1
0.0 ~
-0.1 ~
-0.2 -
-0.3 —
-O.4 ~
-0.5 a
-0.6 r w I r I

-1.2 -0.8 -0.4 0 0.4. 0.8 1.2

Potential (V)

  

 

Current (mA)

 

 

 

 

 

Figure 4-13. Cyclic voltammogram of B-ITO sensor evaluated at 3 mM
theophylline.

 

0.4 - 1 No Thy

0.3 — 2 3 mM Thy
0.2 4

0.1 ~
0.0 .
-0.1 .
-0.2 —
-0.3 ~
-0.4 ~
-0.5 ~
-0.6 . . T . . .
-1.2 -0.8 -0.4 0 0.4 0.8 1.2

Potential (V)

 

Current (mA)

 

 

 

 

 

Figure 4-14. Cyclic voltammogram of MIP-ITO sensor evaluated at 3 mM
theophylline.

56

 

0.15 — 1 NoThy
0.10 _ 2 4mMThy

0.05 ~ K
0.00 -

-0.05 1 I
-0.10 4

-0.15 ~

'0.20 I I I I I I
-1.2 -O.8 -0.4 O 0.4 0.8 1.2

Potential (V)

    
 

Current (mA)

 

 

 

 

 

Figure 4-15. Cyclic voltammogram of B-ITO sensor evaluated at 4 mM

 

 

 

 

theophylline.
0.15 — 1 No Thy
2 4mM Thy
0.10 4
2 0.05 — .
g 0.00 r \‘t
c 1
2 -0.05 -
s /
0 -0.10 —
-O.15 ~ 2/
‘0.20 - I I I I T ‘1
-1.2 -0.8 -0.4 O 0.4 0.8 1.2
Potential (V)

 

 

 

Figure 4-16. Cyclic voltammogram of MIP-ITO sensor evaluated at 4 mM
theophylline

57

The ratio of the maximum (ipe) and minimum (ipa) peak currents for 1, 2, 3, and
4 mM the0phylline are Shown in Tables 4-3 and 4-4, respectively. The ratio refers to the
peak current on the curve of the selected concentration (1, 2, 3, or 4 mM Thy) divided by
the peak current on the baseline curve without theophylline (no Thy). The resulting p-

values using t-test are shown in the tables for each concentration that was tested.

Table 4-3. Ratio of Maximum Currents (ipc) of B-ITO and MIP-ITO Sensors at

Various Analyte Concentrations

 

 

 

 

 

 

 

 

 

Co [22:32:01] B-ITO MIP-ITO P-Value
1 mM Thy 1.022 d: 0.058 1.418 3 0.347 0.123
2 mM Thy 1.073 :1: 0.075 1.223 :I: 0.054 0.049
3mM Thy 113930.142 1.659i0.169 0.015
4 mM Thy 1.024 :t 0.026 1.188 d: 0.115 0.074

 

Means i standard deviations (n=3). Data was tested within a row (one concentration).

Table 4-4. Ratio of Minimum Currents (ipa) of B-ITO and MIP-ITO Sensors at

Various Analyte Concentrations

 

 

 

 

 

 

 

 

 

Co 1:233:20“ B-ITO MIP-ITO P-Value
1 mM Thy 1.055 a 0.152 1.413 :1: 0.368 0.217
2 mM Thy 1.093 :1: 0.136 1.196 :1: 0.129 0.393
3mM Thy 1.126i0.181 151510.203 0.068
4 mM Thy 0.926 3 0.152 1.327 i 0.575 0.363

 

Means i standard deviations (n=3). Data was tested within a row (one concentration).

 

 

The ratio of the maximum currents was compared for the B-ITO and the MIP-ITO
sensors at each analyte concentration. The currents ratios (max or min) were not
significantly different for the 1 mM theophylline concentration. The ratio of the
maximum currents was significantly different (P<0.05) at a theophylline concentration of
2 mM, but not Significant at the minimum current. When testing was performed with 3
mM theophylline, the ratio of the maximum currents was significantly different (P<0.05),
and the ratio of the minimum currents tended to significance (P-value of 0.068). The
ratio of maximum currents at a concentration of 4 mM tended to Significance (P-value of
0.074).

The MIP-ITO sensor could detect the theophylline analyte in the range of 2 to 4
mM. The optimum concentration of theophylline for detection was 3 mM, which
resulted in the highest P-value between the B-ITO and MIP-ITO.

The maximum currents were used for evaluating the sensitivity of the MIP—ITO
sensor. This was the region where the most current would flow based upon the increase
in the permeability and highest number of free electrons that would be present. In this
research, the current at a Single potential was used to evaluate the success or failure of
each sensor. Future researchers may consider other analysis techniques such as principal
component analysis or some type of modeling method to determine what region of data
on the cyclic voltammogram should be compared for increased examination of the

results.

59

A graphical comparison of the current ratios between the MIP-ITO and B-ITO is
presented in Figure 4-17. The MIP-ITO sensor was able to Significantly detect the
analyte at concentrations between 2 to 4 mM theophylline. The ratio of currents at 3 mM
theophylline yielded the lowest P-value. Therefore, the optimum analyte concentration

found in this study was 3 mM theophylline.

 

 

l MIP-ITO
I B-ITO

Current Ratio (Analyte/Blank)

 

 

 

Concentration (mM)

 

 

 

Figure 4-17. Comparison of the ratio of maximum currents of MIP-ITO sensor to B-
ITO at various analyte concentrations.

Theophylline is a small bronchodilator drug that is used in the treatment of
asthma, bronchitis, emphysema, and other airways diseases. The amount needed in the
blood to relieve airway constriction is between 5 to 15 ug/ml (National Jewish Medical
and Research Center, 2005). The sensitivity of the MIP-ITO sensor was between 2 to 4
mM theophylline, which was 360 to 900 ug/ml when converted into units used for the

60

monitoring of theophylline. The current sensitivity target would be to make the
biomimetic sensor able to detect theophylline in the 5 to 15 ug/ml range needed for
delivery and monitoring.

The hook effect has been well established in immunoassay sensors (Rodbard et
al., 1978; Amarasiri Fernando and Wilson, 1992) and states that at higher analyte
concentrations, the over abundance of analyte may interfere rather than enhance the
signal. The hook effect may be present in the MIP-ITO sensor and would account for the
decline in sensitivity around the optimum concentration of analyte. At the optimum
concentration, most of the analyte in the solution rebinds to the MIP surface. As the
concentration of analyte increases past this concentration, the remaining analyte in the
solution hinders the movement of the charge carriers in the electrolyte solution.

The maximum current, ipe, yields a higher current and a better Signal because as
the redox reaction proceeds, more analyte binds to the surface of the MIP. As previously
described, the permeability of the polymer is thought to increase as more analyte binds to
the surface. The increased permeability results in more electron transfer, thus yielding a

higher Signal.

4.5.3 Selectivity of MIP-1T0 Sensor

The selectivity of the sensor was tested using the structurally related molecule
caffeine. The cyclic voltammograms for the MIP-ITO sensor tested at concentrations of

1, 2, 3, and 4 mM caffeine are Shown in Figures 4-18 to 4-25.

61

 

 

 

 

0.4 - 1 No Get
2 1 mM Caf
0.2 _ /
25‘ 0.0 — \ 1
.. /
E '0.2 " \
5 -0.4 ~ /
-O.6 4 2
“0.8 I I I I I *1
-1.2 -0.8 -0.4 O 0.4 0.8 1.2
Potential (V)

 

 

 

Figure 4-18. Cyclic voltammogram of B-ITO sensor evaluated at 1 mM caffeine.

 

04— 1 NoCd
2 1mMCfl
02—

 

0.0 3

 

 

-0.2 -

Current (mA)

-0.4 ~
-0.6 -«

 

-O.8 . j
-1.2 -0.8 -O.4 0 0.4 0.8 1.2
Potential (V)

 

 

 

Figure 4-19. Cyclic voltammogram of MIP-ITO sensor evaluated at 1 mM caffeine.

62

 

J

1 NoCd
2 2mMCfl

 

I

 

vvvv

1

Current (mA)
.<'> .c'> .<'> .6 .o .0 .o
4:- 00 N A O A N

 

 

-1.2 -0.8 -0.4 O 0.4 0.8 1.2
Potential (V)

 

 

 

Figure 4-20. Cyclic voltammogram of B-ITO sensor evaluated at 2 mM caffeine.

 

 

 

 

 

 

0.2 — 1 No Caf
2 2mM Caf
0.1 — g
E
E; -0.1 ~ 2
5 -0.2 -
o
-0.3 ~ 1 /
'0.4 I I I I I I
-1.2 -0.8 -0.4 0 0.4 0.8 1.2
Potential (V)

 

 

 

Figure 4-21. Cyclic voltammogram of MIP-ITO sensor evaluated at 2 mM caffeine.

63

 

0.6 — 1 No Caf
0.4 _ 2 3 mM Caf
0.2 ~
0.0 ~
-0.2 ~
-0.4 —
-0.6 —
-0.8 -
-1.0 T .
-1.2 -0.7 -0.2 0.3 0.8
Potential (V)

    
    

x.“

Current (mA)

 

2/

 

 

 

 

Figure 4-22. Cyclic voltammogram of B-ITO sensor evaluated at 3 mM caffeine.

 

1 No Caf
2 3 mM Caf

95.09.09
NON-ho}
1111]

Current (mA)
.6
.5

b
a:
l

 

9'3
CD
1

 

Lt
.
O
—4
_.

_4

—1

—.

._l

 

_'.
N
l
.0
co

-O.4 0 0.4 0.8 1 .2
Potential (V)

 

 

 

Figure 4-23. Cyclic voltammogram of MIP-ITO sensor evaluated at 3 mM caffeine.

64

 

015— 2 1 Need
010- p/ 2 4mM0m

005-
000—
005-
010—
045—
0204
025— ,
030 I . . . . .

-12 -08 -04 0 04 08 12

Potential (V)

 

 

Current (mA)

 

 

 

 

 

 

Figure 4-24. Cyclic voltammogram of B-ITO sensor evaluated at 4 mM caffeine.

 

 

 

 

0.15 ~ 1 No Caf
A 0.05 -
<
E 000— ‘\‘+-
E 005~ 2
" -0.10 1
3 /
-0.15 a
-O.20 3 1 /
'0.25 I I I I I I
-1.2 -0.8 -0.4 O 0.4 0.8 1.2
Potential (V)

 

 

 

Figure 4-25. Cyclic voltammogram of MIP-ITO sensor evaluated at 4 mM caffeine.

65

The MIP-ITO sensor and B-ITO did not Show a marked response at any
concentration of caffeine. The current ratios of the B-ITO and MIP-ITO at the maximum
and minimum currents are Shown in Tables 4-5 and 4-6, respectively. The mean cm'rent
ratios and standard deviations are presented in the tables. The maximum current ratios of
the B-ITO were compared to the MIP-ITO to determine the selectivity of the biomimetic

SCIISOI'.

Table 4-5. Ratio of Maximum Currents (ipc) of B-ITO and MIP-ITO Sensor at
Various Counter Analyte Concentrations

 

 

 

 

 

 

 

 

 

ngzzzzfigly: B-ITO MIP-ITO P-Value
1 mM Caf 1.403 i 0.350 1.130 :t 0.265 0.343
2 mM Caf 1.172 3 0.217 1.065 2 0.236 0.595
3 mM Caf 1.250 .4; 0.452 1.473 3: 0.721 0.681
4 mM Caf 1.169 :1: 0.111 0.970 :1: 0.092 0.075

 

 

Means 3: standard deviations (n=3). Data was tested within a row (one concentration).

Table 4-6. Ratio of Minimum Currents (ipa) of B-ITO and MIP-ITO Sensor at
Various Counter Analyte Concentrations

 

 

 

 

 

 

 

 

 

C3223; 3:331“ B-ITO MIP-ITO P-Value
1 mM Caf 1.516 3: 0.530 1.068 3: 0.134 0.229
2 mM Caf 1.139 :t 0.190 1.028 :1: 0.098 0.419
3 mM Caf 1.525 :1: 0.861 2.020 :1: 1.62 0.672
4 mM Caf 1.329 :1: 0.502 1.025 :1: 0.575 0.419

 

Means + standard deviations (n=3). Data was tested within a row (one concentration).

66

 

The differences in the mean currents ratios (maximum and minimum) for the B-
ITO and MIP-ITO were not Significant at a 95% confidence level when tested for
selectivity. The p-values of the biomimetic sensor compared to the blank sensor were all
greater than 0.05 when the sensor was tested with caffeine. The biomimetic sensor did
not Show any response to caffeine at the tested concentrations; therefore, the sensor was

selective to the target analyte, theophylline.

4.6 Selection of Future Compound for Food Safety

Patulin is a mycotoxin that may be found in a variety of foods such as grain, fruit,
and cheese (chemical structure found in Figure 4-26). It is produced by certain species of
the Penicillium, Aspergillus, and Byssochylamys molds and is primarily of concern in
apples and apples products. The current action level for patulin set by the Food and Drug
Administration is 50 ug/kg (FDA, 2000). Patulin is a good candidate for molecular
imprinting due to its small chemical structure and the large number of polar groups able

to interact with a functional monomer.

HO

./\___
/ 0

Figure 4-26. Chemical structure of patulin (Sigma-Aldrich, 2004).

67

An initial experiment for the mycotoxin patulin is Shown in Figure 4-27. The
procedure was modified due to the high cost of the toxin (Appendix C-3). Briefly, the
rebinding and measurement procedures for patulin were modified by submerging the
sample into a known concentration of analyte followed by measurement in
electrochemical cell A. Due to the change in procedure, the graph only shows two
curves, one for the patulin-imprinted electrode and the non-imprinted polymer. The

following results Show only one replicate.

 

0.015 — 1 Non-imprinted
1 2 Patulin-MIP

 

Current (mA)

 

 

 

'0.015 I I I 7 7 I
-1.2 -0.8 -0.4 0 0.4 0.8 1.2
Potential (V)

 

 

 

Figure 4-27. Initial cyclic voltammogram for patulin MIP.

68

The current for the non-imprinted curve (1) is greater than the patulin MIP (2).
This trend was not observed with the theophylline MIP, where the current was
consistently larger for the MIP than the blank polymer. This may have been due to the
change in the procedure or to the interaction between patulin and the MIP upon
rebinding. Additional experimentation will be necessary to investigate these results and
determine their consistency.

Patulin will continue to be of interest in the area of food safety. Current detection
methods use a liquid-liquid extraction of patulin with ethyl acetate to remove it from the
sample followed by detection and quantification, usually by HPLC (Dombrink-
Kurtzman, 2005). The identification of patulin through antibody-based detection
methods is being investigated due to the speed and sensitivity of immunological
procedures. However, the low molecular weight of patulin (154 g/mol) makes it difficult
to generate antibodies without conjugation to a protein (McElroy and Weiss, 1993; Sheu
et al., 1999). Investigation into rapid MIP-based sensors for patulin will be essential to
food safety because of the difficulty of producing antibodies and the processing for

current detection methods.

69

CHAPTER 5: CONCLUSIONS

5.1 General Conclusions

This research demonstrated the successful formation of a MIP and its attachment
to an electrode. The successful formation of the MIP and attachment to indium tin oxide
(ITO) was monitored by AFM imaging. The initial results of the theophylline MIP on
ITO supported the findings of a previous study (Yoshimi et al.) using the same MIP and
template. The presence of theophylline in the initial study increased the maximum peak
current by a factor of 5.3 for the MIP-ITO sensor compared to the blank or non-imprinted
polymer (omission of theophylline). The increase in the peak cmrent in the presence of
the analyte (a rise of 0.006 mA for B-ITO and 0.036 mA for MIP-ITO) allowed the
system to act as a qualitative sensor to the presence of theophylline.

The sensitivity and selectivity of the MIP-ITO sensor was successfully evaluated.
The biomimetic sensor was able to detect the theophylline analyte between
concentrations of 2 mM to 4 mM. The optimum analyte concentration was 3 mM
theophylline. The MIP-ITO sensor Showed no cross reactivity to caffeine at
concentrations of 2 mM to 4 mM and, therefore, was selective to theophylline. Initial
studies of the MIP on silicon found the ratio of maximum currents to increase by a factor
of 7.5 when comparing the MIP-Si to the B-Si. The MIP on silicon was able to detect the

analyte at the 5 mM concentration.

70

5.2 Recommendations for Future Research

The ability to control the thickness of the MIP may be important to obtaining a
uniform signal. Additional research should be performed to control the thickness and
uniformity of the polymer, such as a Spin coating method. Reducing the thicknesses of
the polymer may increase the sensitivity of the sensor. Future investigation into a
monolayer of polymer may be useful to evaluate the maximum sensitivity of the sensor.

Additional testing would also help to evaluate the effect of other stressing
conditions on the response of the MIP sensor, such as high-temperatures, acidic and basic
conditions, and long storage times. It may be useful to determine or measure the density
of the analyte in the polymer or MIP pockets. This may be critical for determining the
amount of residual analyte trapped in the polymer matrix, which could be affecting the
response of the biomimetic sensor.

In addition to the improvements that can be made to the MIP on ITO, the response
of the MIP on Silicon may be further improved by modifying the surface characteristics
of silicon. One test that may be tried is to increase the conductivity by using Silicon that
is more highly doped with charges. The higher current may promote enhanced rebinding
of the analyte. Various surface profiles, such as porous structures, could be investigated
for their effect upon the resulting Signal. The method of detection may also affect the
Signal. Other detection techniques may be investigated to obtain an optimal signal, such
as various pulse voltammetry techniques (normal, differential, square wave, or staircase).

The biomimetic sensor for theophylline Should be improved to achieve the
sensitivities necessary to compete with current detection methods. The preceding

recommendations for improvement to the sensor will allow the detection range to reach 5

71

to 15 ug/ml needed by the medical industry. Once the sensitivity is increased to the
needs of industry, the stability and simplicity of the sensor will allow it to compete with
other detection methods for theophylline.

The final aim of this research was to assess and recommend a toxin for study in
the area of food safety. The mycotoxin patulin was suggested for future research. This
research may lead to a sensor that would detect patulin and other toxins, improving food
safety. Future studies Should target small toxins where natural receptors are difficult to

obtain or are unavailable.

72

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79

Appendix A:

Appendix B:

Appendix C:
C. 1

C2

APPENDICES

Light Absorbance Tables and Raw Data

Potentials and Currents Used for Sensitivity and Selectivity
Statistical Analysis

Procedure for Initial Results for Patulin
Preparation of Patulin MIP

Rebinding and Measurement of Patulin MIP

80

 

 

Appendix A: Light Absorbance Tables and Raw Data

Table A-1. Light Absorbance Values for Aqueous 5mM Theophylline.

 

 

 

 

Polymer Absorbance (AU)
Sample

1 0.731

2 0.753

3 0.753

 

 

 

Table A-2. Light Absorbance Values for Non-imprinted Polymer.

 

 

 

 

 

 

 

 

 

Absorbance Before Absorbance After
Sample Rebinding (AU) Rebinding (AU) Difference
1 0.12 0.175 0.055
2 0.15 0.2 0.05
3 0.04 0.05 0.01

Table A-3. Light Absorbance Values for MIP.

 

 

 

 

 

’ Absorbance Before Absorbance After
Sample Rebinding (AU) Rebinding 0w) Difference
1 0.08 0.245 0.165
2 0.142 0.321 0.179
3 0.179 0.445 0.266

 

 

 

 

81

 

Table A-4. Comparison of Light Absorbance Difference of MIP to Non-imprinted
Polymer Using t-Test for Two Samples Assuming Unequal Variances.

 

 

Variable 1 Variable 2
Mean 0.038333333 0.203333333
Variance 0.000608333 0.002994333
Observations 3 3
Hypothesized Mean Difference 0
(If 3
t Stat -4.761376574
P(T<=t) one-tail 0.008795191
t Critical one-tail 2.353363016
P(T<=t) two-tail 0.017590382
t Critical two-tail 3.182449291

 

82

l) 2) 3)

3183914012.". ' mgum mamas
. 'me 0.8 'fi‘m mm 1.010 0.9011

 

 

 

I

I.

 

 

 

 

 

 

 

 

 

 

I
I' L
— ’ h
. I I.
} I-
j 1
ti. .....
no x axis! 50 tin/division x axis! 50 tin/division
x axis: 50 III/division ‘

Figure A-l. Light absorbance data for an aqueous solution of 5 mM theophylline.

83

1)

:11: 31 1 1:113 3
.030 0.20:}

h

I-
I-
b
b

. I'

F

 

I.

%

I

4

x axis: 53 nI/dtviston

2)

next 445 z .897 NJ

run: 291 t .062 NJ

—P.1100 1.38%
W

 

 

vi

 

 

)-

are no
x axis: 58 ran/division

3)
21°31 33% 1 3:11

- .208 085%

2m
6w

1

3mm

x axis! 50 Ira/division

 

Figure A-2. Light absorbance data for non-imprinted polymer after extraction of

template.

84

1)
:11: 3? l 4:313 51
19 W1

[43

I

—'I

_:e-rwa.Aaaqr-Ja~vag_gh_3wx2___i‘FJ,.2\..JncJ"-\‘r\{h__

 

x axis! 30 na/division

Figure A-3. Light absorbance data for non-imprinted polymer after soaking 25min

2)

313313313211

 

 

800 no
,2 axis! 50 waivision

 

—
—
h
0
h
b
-
b
_
~

3)
:12: $1 3:811 211’
_P

 

~92? 0.103

 

800 n-
x axis: 50 na/diuision

in an aqueous solution of 5 mM theophylline.

85

1) 2) 3)

 

 

 

 

 

 

max: 419 = 6.12? AU , taxi : .179 RU
min: 388 : 4.831 AU 3:; 23359: 36% $ mm 3% = 3.885 nu
. .1“ 8"

16'“? 3'23? .180 1 0.239J ? 1 79%
F- P [-
b— '- r-

I

l
I- I I-
b r
P L
'- >-

’—
~ +-
" _ F
r . l
889 no ” l 880 n-
x axis! 58 nI/division 880 n. x axis: SB nI/division

x axis: 50 no/division

Figure A-4. Light absorbance data for MIP after extraction of template.

86

1) 2) 3)

m: i 2 38:; i zusnu
sac mm am: am 233 gm
1 13.10? mag

. 0.309 a one 9,433]
k

f ;

 

7

 

 

 

l- __ -

r - .

8W no~ 3“ ml sea In

x axis: 50 tun/division x axis: 59 Wdivision x axis: 50 waivision

Figure A-S. Light absorbance data for MIP after soaking 25min in an aqueous
solution of 5 mM theophylline.

87

Appendix B: Potentials and Currents Used for Sensitivity and Selectivity Statistical
Analysis

Table B-1. Potentials and Currents for B-ITO Evaluated at 1 mM Theophylline.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Maximum
No Thy lmM Thy
Sensor Potential (V) Current (A) Potential (V) Current (A) Current Ratio
1 -0.635 2.99E-05 -0.635 3 2413-05 1.08
2 -0.290 2.53E-05 -0.290 2.56E-05 1.01
3 0.063 3.57E-04 0.063 3.46E-04 0.97
Average 1.02
Minimum .
No Thy lmM Thy
Sensor Potential (V) Current (A) Potential (10 Current (A) Current Ratio
1 0.349 -7.13E-05 0.349 -8.76E-05 1.23
2 0.600 -9.16E-05 0.600 -9.06E-05 0.99
3 0.428 -5.96E-04 0.428 -5.65E-O4 0.95
Average 1.06

 

Table B-2. Potentials and Currents for MIP-ITO Sensor Evaluated at 1 mM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Theophylline.
Maximum
No Thy 1 mM Thy
Sensor Potential (10 Current (A) Potential (10 Current (A) Current Ratio
1 0.094 2.79E-05 0.094 3 .01 E-OS 1.08
2 -0.012 1.46E-04 -0.012 2.05E-04 1.40
3 -0.204 3 .26E-04 -0.204 5 .78E-04 1.77
Average Minimum 1 .42
No Thy 1 mM Thy
Sensor Potential ([0 Current (A) Potential (10 Current (A) Current Ratio
1 0.396 -1.06E-04 0.396 -1.16B-04 1.09
2 0.565 -3.31E-04 0.565 —4.40E-04 1.33
3 0.702 -6.23E-04 0.702 -1.13E-03 1.82
Average 1.41

 

 

 

88

 

 

 

 

 

Table B-3. Potentials and Currents for B-ITO Evaluated at 2 mM Theophylline.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Maximum
No Thy 2 mM Thy
Sensor Potential (10 Current (A) Potential (10 Current (A) Current Ratio
1 -0.404 2.02E-05 -0.404 2.08E-05 1.03
2 -0.533 4.50E-05 -0.533 5.22E-05 1.16
3 -0.475 1.86E-04 -0.475 1.91E-04 1.03
Average Minimum 1 .07
No Thy 2 mM Thy
Sensor Potential (V) Current (A) Potential (10 Current (A) Current Ratio
1 0.875 -7.01E-05 0.875 -8.76E-05 1.25
2 0.879 -1.90E-04 0.879 -1.92E-04 1.01
3 0.788 -2.80E-04 0.788 -2.85E-O4 1.02
Average 1.09

 

 

Table B-4. Potentials and Currents for MIP-ITO Sensor Evaluated at 2 mM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Theophylline.
Maximum
No Thy 2 mM Thy
Sensor Potential (V) Current (A) Potential (10 Current (A) Current Ratio
1 -0.341 1.01E-04 -O.34l 1.21E-04 1.21
2 -0.341 9.65E-05 -0.341 1.14E-04 1.18
3 -0.435 2.51E-05 -0.435 3 .22E-05 1.28
Average Minimum 1 .22
No Thy 2 mM Thy
Sensor Potential [19 Current (A) Potentiam Current (A) Current Ratio
1 0.898 -2.84E-04 0.898 -3.21E-04 1.13
2 0.965 -2.65E-04 0.965 -2.95E-04 1.1 1
3 1.000 -1.15E—04 1.000 -1.55E-04 1.35
Average 1.20

 

89

 

 

 

 

 

Table B—5. Potentials and Currents for B-ITO Evaluated at 3 mM Theophylline.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Maximum
No Thy 3 mM Thy
Sensor Potential (10 Current (A) Potential (10 Current (A) Current Ratio
1 -0.063 8.01 E-06 -0.063 7.90E-06 0.99
2 -0.655 7.40E-05 -O.655 8.59E-05 1.16
3 0.059 6.85E-06 0.059 8.69E-06 1.27
Average Minimum 1 .14
No Thy 3 mM Thy
Sensor Potential (10 Current (A) Potential (V) Current (A) Current Ratio
1 0.651 -3.89E-05 0.651 -3.78E-05 0.97
2 1 .000 -2.47E-04 1.000 -2.67E-04 1 .08
3 0.879 -5.92E-05 0.879 1-7.84E-05 1.32
Average 1.12

 

Table B-6. Potentials and Currents for MIP-ITO Sensor Evaluated at 3 mM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Theophylline.
Maximum
No Thy 3 mM Thy
Sensor Potential (10 Current (A) Potential (V) Current (A) Current Ratio
1 0.071 6.04E-05 0.071 9.21E-05 1.52
2 -0.302 2.62E-05 -0.3 02 4.20E-05 1.60
3 -0.408 2.57E-05 -O.408 4.75E-05 1.85
Average Minimum 1 .66
No Thy 3 mM Thy
Sensor Potential (10 Current (A) Potential (10 Current (A) Current Ratio
1 0.592 -1.82E-04 0.592 -2.37E-04 1.30
2 1.000 -1 .16E-04 1.000 -1.79E-O4 1.54
3 1.000 -1.16E-04 1.000 -1.98E-04 1.70
Average 1.51

 

 

90

 

 

 

 

 

Table B-7. Potentials and Currents for B-ITO Evaluated at 4 mM Theophylline.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Maximum
No Thy 4 mM Thy
Sensor Potential (10 Current (A) Potential (V) Current (A) Current Ratio
1 -0.381 5.57E-05 -0.381 5.58E-05 1.00
2 -0.820 4.53E-05 -0.820 4.77E-05 1.05
3 -0.353 6.00E-05 -0.353 6.10E-05 1.02
Average Minimum 1 .02
No Thy 4 mM Thy
Sensor Potential (10 Current (A) Potential (V) Current (A) Current Ratio
1 0.788 -1.71E-04 0.788 -1.73E-04 1.01
2 0.879 -1.42E-04 0.879 -1.44E-04 1.02
3 0.408 -1.11E-04 0.408 -8.33E-05 0.75
Average 0.93

 

Table B-8. Potentials and Currents for MIP-ITO Sensor Evaluated at 4 mM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Theophylline.
Maximum
No Thy 4 mM Thy
Sensor Potential (10 Current (A) Potential ([0 Current (A) Current Ratio
1 -0.569 7.60E-07 -0.569 1 .00E—06 1.32
2 -0.506 1.87E-05 -0.506 2.12E-05 1.13
3 -0.831 6.85E-05 -0.831 7.60E-05 1.11
Average Minimum 1.19
No Thy 4 mM Thy
Sensor Potential (V) Current (A) Potential ([0 Current (A) Current Ratio
1 1.000 -1.98E-06 1.000 -3.93 E-06 1.98
2 0.910 -8.08E-05 0.910 -8.72E-05 1.08
3 1 .000 -2.42E-O4 1 .000 -2.22E-04 0.92
Average 1.33

 

 

91

 

 

 

 

 

 

Table B-9. Potentials and Currents for B-ITO Evaluated at 1 mM Caffeine.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Maximum
No Thy 1 mM Caf
Sensor Potential (10 Current (A) Potential (10 Current (A) Current Ratio
1 -0.384 2.25E-05 -0.384 3.31E-05 1.47
2 -0.526 2.13E-05 -0.526 3.65E-05 1.71
3 0.020 6.77E-04 0.020 6.93E-04 1.02
Average Minimum 1 .40
No Thy 1 mM Caf
Sensor Potential (10 Current (A) Potential (V) Current (A) Current Ratio
1 1.000 -8.82E-05 1.000 -1.22E-04 1.38
2 1.000 -6.89E-05 1.000 -1.45E-04 2.10
3 0.443 -9.72E-04 0.443 -1.04E-03 1 .07
Average 1.52

 

 

Table B-10. Potentials and Currents for MIP-ITO Sensor Evaluated at 1 mM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Caffeine.
Maximum
No Thy 1 mM Caf
Sensor Potential (10 Current (A) Potential (10 Current (A) Current Ratio
1 -0.400 2.48E-05 -0.400 3 .47E-05 1.40
2 -0.028 2.61E-04 -0.028 2.27E-04 0.87
3 -0.028 7.33E-04 -0.028 8.22E-04 1.12
Average . Minimum 1 .1 3
No Thy 1 mM Caf
Sensor Potential (10 Current (A) Potential (IO Current (A) Current Ratio
1 1.000 -1.06E-04 1.000 -1.21E-04 1.15
2 0.482 -4.61E-04 0.482 -4.21E-04 0.91
3 0.526 -1.20E-03 0.526 -1.38E-03 1.15
Average 1.07

 

 

 

 

 

 

 

 

 

Table B-ll. Potentials and Currents for B-ITO Evaluated at 2 mM Caffeine.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Maximum
No Thy 2 mM Caf
Sensor Potential (10 Current (A) Potential (10 Current (A) Current Ratio
1 -0.486 2.50E-05 -0.486 3.52E-05 1.41
2 -0.581 6.92E-05 -0.581 7.79E-05 1.13
3 -O.286 2.66E-04 -0.286 2.61E-04 0.98
Average Minimum 1 .1 7
No Thy 2 mM Caf
Sensor Potential (V) Current (A) Potential (V) Current (A) Current Ratio
1 1 .000 -1 .09E-O4 1.000 -1 4213-04 1 .30
2 1 .000 -2.45E-04 1 .000 -2.92E-04 1 .19
3 1.000 -5.68E-04 1.000 -5.27E-04 0.93
Average 1.14

 

 

 

 

Table B-12. Potentials and Currents for MIP-ITO Sensor Evaluated at 2 mM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Caffeine.
Maximum
No Thy 2 mM Caf
Sensor Potential (V) Current (A) Potential (10 Current (A) Current Ratio
1 -0.3 73 1.17E-04 -0.373 9.49E-05 0.81
2 -0.302 1.12E-04 -0.302 1.24E-04 1.10
3 -0.467 2.54E-05 -0.467 3 .25E-05 1.28
Average Minimum 1 .06
No Thy 2 mM Caf
Sensor Potential (10 Current (A) Potential (10 Current (A) Current Ratio
1 1.000 -3.1 1E-04 1.000 -2.93E-04 0.94
2 1.000 -3.23E-04 1.000 -3.25E-04 1.01
3 1.000 -1.14E-04 1.000 -1.30E-04 1.13
Average 1.03

 

93

 

 

 

 

 

 

Table B-l3. Potentials and Currents for B-ITO Evaluated at 3 mM Caffeine.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Maximum
No Thy 3 mM Caf
Sensor Potential (10 Current (A) Potential (10 Current (A) Current Ratio
1 -0.067 3.24E-04 -0.067 3.21E-04 0.99
2 -0.377 1.35E-04 -0.377 2.39E-04 1.77
3 -0.051 7.63E-04 -0.051 7.53E-04 0.99
Average Minimum 1 .25
No Thy 3 mM Caf
Sensor Potential (10 Current (A) Potential (V) Current (A) Current Ratio
1 0.616 -5.46E-04 0.616 -5.66E-04 1.04
2 1.000 -3.12E-04 1.000 -7.86E-04 2.52
3 0.541 -1.22E-03 0.541 -1.24E-03 1.02
Average 1.53

 

 

Table B-14. Potentials and Currents for MIP-ITO Sensor Evaluated at 3 mM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Caffeine.
Maximum
No Thy 3 mM Caf
Sensor Potential (10 Current (A) Potential (10 Current (A) Current Ratio
1 -0.392 1.03E-04 -0.392 2.38E-04 2.30
2 -0.455 5.91E-05 -0.455 6.50E-05 1.10
3 -0. 102 5 .79E—04 -0.102 5.87E-04 1.01
Average Minimum 1 .47
No Thy 3 mM Caf
Sensor Potential (10 Current (A) Potential (10 Current (A) Current Ratio
1 1.000 -2.74E-04 1.000 -1.07E-03 3.89
2 1.000 -2.17E-04 1 .000 -2.46E-04 1.13
3 0.604 -9.80E-04 0.604 -1 .02E-03 1.04
Average 2.02

94

 

 

 

 

Table B-15. Potentials and Currents for B-ITO Evaluated at 4 mM Caffeine.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Maximum
No Thy 4 mM Caf
Sensor Potential (10 Current (A) Potential (10 Current (A) Current Ratio
1 -0.361 7.14E-05 -O.361 7.88E-05 1.10
2 -0.600 4.17E-05 -0.600 5.41 E-05 1.30
3 —0.506 8.75E-05 -0.506 9.68E-05 1.11
Average Minimum 1 . 1 7
No Thy 4 mM Caf
Sensor Potential (V) Current (A) Potential (10 Current (A) Current Ratio
1 1 .000 -2.67E-04 1 .000 -2.64E-04 0.99
2 1.000 -1.59E-04 1.000 -1.39E-04 0.87
3 1.000 -2.14E-04 1.000 -2.25E-04 1.05
Average 0.97

 

Table B-l6. Potentials and Currents for MIP-ITO Sensor Evaluated at 4 mM

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Caffeine.
Maximum
No Thy 4 mM Caf
Sensor Potential (V) Current (A) Potential (10 Current (A) Current Ratio
1 -0.357 8.05E-05 -0.357 8.63E-05 1.07
2 -0.530 1.82E-05 -0.530 3 .48E-05 1.91
3 -0.341 9.66E-05 -0.341 9.74E-05 1.01
Average Minimum 1 .33
No Thy 4 mM Caf
Sensor Potential (V) Current (A) Potential (IQ Current (A) Current Ratio
1 1.000 -2.88E-04 1.000 -2.53E-04 0.88
2 1.000 -7.68E-05 1.000 -1.05E-04 1.37
3 1.000 -2.56E-04 1.000 -2.1 lE-04 0.82
Average 1.02

 

 

95

 

 

 

 

 

Appendix C: Procedure for Initial Results for Patulin

C. 1 Preparation of Patulin MIP

1.

2.

Wash and rinse ITO electrodes according to Section 3.3.

Silanize ITO electrode in 10% solution (v/v) of 3—MPS in toluene for 6 h at 80°C
under nitrogen atmosphere. Rinse electrode with twice with methanol followed
by deionized water and dry under nitrogen environment.

Combine 0.125 ml MAA, 1.1 ml EDGMA, and 3.3 ml dimethyl sulfoxide
(DMSO). Distill by passing solution through a column of inhibitor removal
material.

Measure 0.5 ml of the above-distilled solution and add 0.01 g of AIBN.

Add 200 pl of patulin in DMSO (5 mg patulin in lml DMSO)

Polymerize in 60°C water bath for 12 h.

Break bulk polymer and rinse in 9:1 methanol in acetic acid for 1 hr. Rinse twice
with methanol followed by rinsing twice with water.

Store sensors in water prior to rebinding and measurement

C.2 Rebinding and Measurement of Patulin MIP

The rebinding and measurement follows the basic procedure of (Blanco-Lépez et al.,
2003b) with some modifications:

1.

2.

Place sensors in a solution of 1mg patulin in 2ml DMSO for 30 min.

Immerse sensors in fresh DMSO for l min to remove weakly adhering analyte
from surface of sensor.

Set up electrochemical cell A with treated sensor at bottom as the working
electrode. Place 25 ml of 10% DMSO in deionized water (pH 3) into the
electrochemical cell.

4. Perform cyclic voltammetry from —1 V to 1 V at a scan rate of 50 mV/s.

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