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INDIUM TIN OXIDE-POLYANILINE BIOSENSOR:
FABRICATION AND PERFORMANCE ANALYSIS

presented by

Zarini Muhammad-Tahir

has been accepted towards fulfillment
of the requirements for the

Ph.D degree in Biosystems and Agricultural

 

 

 

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INDIUM TIN OXIDE-POLYANILINE BIOSENSOR:
FABRICATION AND PERFORMANCE ANALYSIS

By

Zarini Muhammad-Tahir

A DISSERTATION
Submitted to
Michigan State University

In partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Biosystems and Agricultural Engineering

2007

ABSTRACT

INDIUM TIN OXIDE-POLYANILINE BASED BIOSENSOR:
FABRICATION AND PERFORMANCE ANALYSIS

By
Zarini Muhammad-Tahir

To facilitate rapid detection of potential bioterrorist agents, development of an
inexpensive, adequately sensitive and specific, user-friendly, robust, and rapid field-
based biosensor is advantageous for preventing the spreading of infections. In this study,
fabrication and performance analysis of an indium tin oxide (ITO)-polyaniline (Pani)
biosensor are highlighted. The biosensor design is based upon the specific nature of
antibodies and electroactive properties of Pani. The ITO-Pani biosensor is comprised of
two components: immunosensor and amperometric measuring device. The immunosensor
is made of a conductive ITO substrate coated with Pani. The conductive substrate is
functionalized into a biosensor by immobilizing antibodies within the polymer matrix.
Experimental conditions such as the Pani size, antibody concentration, immobilization
method, and incubation time were assessed to find the best condition for the biosensor
fabrication. Using bovine viral diarrhea virus as a model pathogen, the sensitivity of the
biosensor was 104 cell culture infective dose per ml (CCID/ml) with a linear range up to

106 CCID/ml of virus concentration.

ACKNOWLEDGEMENTS
First, I thank my family for their unconditional love and undying support
during the toughest stage of my life. The “can do” attitude that you thought me has
helped me overcome many hurdles in my life thus far.

I would like to express my sincere gratitude and appreciation to Dr. Alocilja, for
her guidance, time, advice, encouragement and funding over the past few years of my
graduate work at Michigan State University. I am also grateful for the much needed
advice and resources provided by Dr. Grooms, Dr. Mohanty, and Dr. Steffe.

I would like to thank everyone in the Biosensor research group. The hours of help,
motivation and encouragement from Stephen Radke, Finny Mathew, Maria Rodriquez-
Lopez, Lisa Kindschy, and Sudeshna Pal can never be repaid. I would like to express my
appreciation to all undergraduate students that work with me over the past few years.

I owe many thanks to Mayk Chahine, a great friend and companion, for the
infinite patience and encouragement during the last few months. Thank you!

To my loving friends, I could have never achieved this without all of you.

iii

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TABLE OF CONTENTS

 

Table list

viii

ix

 

Figure List

CHAPTER 1: INTRODUCTION

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.1 Specific aims 1
Long-term goal 1
Short-term Goal 2

1.2 Hypothesis 2

1.3 Objectives 2

CHAPTER 2: LITERATURE REVIEW 4

2.1 Agroterrorism 4
Terminology 4
History 4
Importance of agriculture 5
Vulnerabilities of the US agriculture 6
Impact 8
Agroterrorism agents 9
Bovine Viral Diarrhea Virus 10

Acute infection 11
Persistent infection 12
Mucosal disease 12
Bovine Herpesvirus Type-1 13

iv

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.3 Detection Methods 14
2.4 Conducting Polymer 17
Mechanism for conducting polymer 2O
Doping 22
Applications of conducting polymer 24
Polyaniline 28
Self doped polyaniline 31
2.5 Biosensor 32
Transducing element- Electrochemical method 33
Conductometric biosensors 36
Amperometric biosensor 37
Potentiometric biosensors 37
Impedimetric biosensors 37
Sensing element-Antibodies 38
Assay formats 40
Difficulties in biosensor fabrication and application 43
CHAPTER 3: MATERIALS AND METHODS 46
3.1 Procedural flowchart 46
3.2 Reagents and apparatus 47
Reagents 47
Apparatus 47
Amperometric measurement 47
3.3 Pani preparation 48

 

Self doped and non self doped Pani

 

3.4 Pani characterization

 

Conductivity measurement

 

Thermogravimetric measurement (TG)

Transmission electron microscopy (TEM) and atomic force microscopy (AFM)

 

analyses

 

3.5 Fabrication of an ITO-Pani biosensor

 

3.6 Antibody immobilization

Liu et a1., 2000’s method

 

 

Tang et a1., 2004’s method

 

3.7 Confirmation of antibody immobilization

 

3.8 Detection and incubation time

3.9 Viral culturing

 

 

3.10 Viral confirmation

3.11 Sensitivity testing of the biosensor in BVDV pure culture

 

3.12 Specificity testing of the ITO-Pani biosensor

 

 

3:13 Reusability testing

CHAPTER 4: RESULTS AND DISCUSSION

 

 

Paper 1: Fabrication of indium tin oxide-Polyaniline biosensor

 

4.1 Characterization of polyaniline

 

4.2 Concept of detection

50

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50

50

51

51

51

52

52

52

53

53

53

59

52

 

4.3 Fabrication of ITO-Pani biosensor

vi

 

 

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Paper 2: Performance of the indium tin oxide —Polyaniline biosensor for bovine viral

 

 

 

 

diarrhea virus detection 70
4.4 Experimental condition analysis 70
4.5 Sensitivity and Specificity ' 76
4.6 Reusability 78
4.7 Cost analysis 79

 

CHAPTER 5: PRELIMINARY STUDY-FRACTAL ANALYSIS ON THE INDIUM

 

 

 

 

 

 

 

TIN OXIDE-PAN I BIOSENSOR 79
CHAPTER 6: CONCLUSION 86
CHAPTER 7: FUTURE MODIFICATION 89
Appendices 93
Appendix I 94
Data 94
Appendix II 99

Reference I 1 00

 

vii

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Tal
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Tat

 

TABLE LIST

 

 

 

Table 2.1: Major US animal and crop production, 2001 (USDA 2001). 6
Table 2.2 Animal and plant pathogens with potential biological agents developed for the
Defense Intelligence Agency (Christopher 1997; Atlas 1999). 10
Table 2.3: Applications of conducting polymer compounds. 25
Table 2.4: Conducting polymer based-biosensor. 27

 

Table 4.1: Experimental condition parameters evaluated for the ITO-Pani biosensor. --- 70

 

Table 4.2: Summary of data (complete data is in Appendix 1). 76

Table 5.1: Estimated binding rate coefficient and fractal dimension using a single fractal
analysis. 83

 

 

Table A4: Conductivity (S/cm) of the unused and treated ITO substrates. 98

viii

 

 

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FIGURE LIST

Figure 2.1: Conductivity range of conducting polymer compounds compared to some
metals (Friend 1993). ‘ 19

 

Figure 2.2: Conductivity of silver and polyacetylene in response to varying temperature
(Winokur, 1998). 20

 

Figure 2.3: Schematic diagram explaining the difference between an insulator, a
semiconductor, and a metal (Bott, 1986). 21

 

Figure 2.4: Pani structure (Macdiarmid, Chiang et a1. 1987; Yen Wei 1989; Genies,

 

 

 

 

 

Boyle et a1. 1990). 28
Figure 2.5: Doping of emeraldine base with 1 M hydrochloric acid (Macdiarmid, Chiang
et a1. 1987). 30
Figure 2.6: A probable mechanism for chemical polymerization of Paul doped with
camphorsulfonic acid (Rannou, Nechtschein et a1. 1999). 32
Figure 2.7: Schematic of a biosensor. 33
Figure 2.8: An electrical schematic of a three-electrode electrochemical cell (Wang
2000) 36
Figure 2.9: Antibody structure (Sadana 2002). 39

 

Figure 2.10: Potential antibody orientations after an immobilization process (Lu, Smyth
et a1. 1996). Dotted arrows correspond to antigen binding sites. 40

 

Figure 2.11: Different forms of irnmunoassays (Lukosz 1991): (a) Direct binding; (b)

 

 

 

 

 

sandwich assay; (c) displacement assay; ((1) replacement assay. 43
Figure 3.1: Apparatus for amperometric response measurement. 48
Figure 4.1: Conductivity of Pani compounds. 54
Figure 4.2: TEM images of a) 20 KDa, b) 50 KDa, and c) 65 KDa self doped Pani; and d)
non-self doped Pani. 55
Figure 4.3: Amperometric responses of ITO -self doped Pani compounds. 56
Figure 4.4: Percent of weight loss of Pani in varying temperature level. 58

 

ix

Figure 4.5: Amperometric responses of ITO substrates coated with non-self doped Pani

 

 

 

 

and 50 KDa self doped Pani in two pH controlled electrolytes. 59
Figure 4.6: Concept of detection ( Direction of electron flow). 61
Figure 4.7: Schematic of an ITO-Pani biosensor before and after antibody-antigen
binding. ' 62
Figure 4.8: ITO-Pani biosensor. 63

Figure 4.9: Atomic force microscopy images of a) untreated ITO glass and b) ITO ------ 65

 

glass treated with ammonium hydroxide (NH40H). 65

Figure 4.10: Atomic force microscopy images of a) ITO glass treated with (NH40H) and
spin coated with 65KDa, b) SOKDa, c) 20KDa Pani. 65

 

Figure 4.11: Atomic force microscopy images of a) ITO glass spin coated with Pani
(65KDa) + ab, b) Pani (50 KDa) + ab, and c) Pani (20 KDa) + ab. 66

 

Figure 4.12: Atomic force microscopy images of a) ITO-Pani biosensor (65KDa) tested
with 106CCID/ml and b) 104CCID/ml of BVDV. 66

 

Figure 4.13: Atomic force microscopy images of a) ITO-Pani biosensor (50KDa) ------- 67

 

 

 

 

tested with 106CCID/ml and h) 104CCID/ml of BVDV. 67
Figure 4.14: Atomic force microscopy images of a) ITO-Pani biosensor (20KDa) tested
with 106CCID/ml and b) 104CCID/ml of BVDV. 67
Figure 4.15: Amperometric responses of A) substrate A (ITO biosensors consisted of
self-doped Pani and B) substrate B (ITO biosensor without Pani coating). 69
Figure 4.16: Typical response of Q° and Q5 over time. 71

Figure 4.17: Response of biosensors prepared with 65 KDa Pani and varying antibody
concentrations. 74

 

Figure 4.18: Response of biosensors prepared with 50 KDa Pani and varying antibody
concentrations. 75

 

Figure 4.19: Response of biosensors prepared with 20 KDa Pani and varying antibody
concentrations. 75

 

Figure 4.20: Performance of the biosensor in varying concentrations of BVDV. --------- 77

 

 

331‘

Fla

Fig

 

Figure 4.21: Performance of the biosensor in a mixture of BVDV and BHV culture
samples. 78

 

Figure A1: Intensity profile of the FITC-labeled antibodies immobilized on an ITO
substrate based on Liu’s method. 96

 

Figure A2: Intensity profile of the FITC-labeled antibodies immobilized on an ITO
substrate based on Tang’s method. 96

 

 

Figure A3: SEM image of unused ITO substrate. 98

 

Figure A4: SEM image of treated ITO substrate. 98

xi

CHAPTER 1: INTRODUCTION

One of the lessons from the event on the September 11, 2001 is the US’s
vulnerability to unforeseen and unprecedented ways of terrorist attacks. Following this
event, a partnership between federal agencies, state and local leaders, private sectors, and
academic community is investing in several preventative measures to strengthen the
security of our nation’s agricultural industry. One such effort is in the development of a
new surveillance system to target the early detection of potential bioterrorism agents.
Development of an inexpensive, sensitive, specific, and rapid field-based systems that is
cost competitive compared with the current diagnostic methods is certainly advantageous

for controlling and preventing the spread of these bioterrorism- related diseases.

1.1 Specific aims

Long-term goal

The long-term goal of this research is to develop a rapid, cost effective, highly
specific and sensitive, self-contained, indium tin oxide (ITO)-polyaniline (Pani)
biosensor capable of identifying multiple targets of detection. A combination of highly
specific antibody molecules and excellent electronic and electrochemical properties of
conductive polymer compounds will enable the fabrication of a field-portable biosensor.
The ability to change the specificity of the antibodies will allow a redesign of the
biosensor as a multi-array detection device capable of recognizing multiple pathogens
simultaneously. Such a device can be used to enhance food safety and biosecurity of the

food supply chain, as well as to support medical diagnostics and bio-defense measures.

Short-term goal

In this study, a prototype ITO-Pani biosensor was designed, fabricated, and tested
for the detection of bovine viral diarrhea virus (BVDV), a model pathogen for
bioterrorism agents. First, the Pani properties were evaluated to assess its potential as a
transducer to translate antibody-antigen reaction into a measurable signal. Then, the ITO-
Pani biosensor platform was fabricated by 1) spin-coating Pani on the ITO glass and 2)
immobilizing antibody onto the Pani coated substrate. The experimental conditions were
evaluated based on the Pani size, antibody concentration, antibody immobilization
method, and antigen incubation time. Lastly, the ITO-Pani biosensor was tested in pure
culture samples of BVDV for its sensitivity and specificity performance. The use of spin-
coating method in this study was novel to the fabrication of the ITO-Pani biosensor.

' 1.2 Hypothesis

An ITO-Pani biosensor can be fabricated by spin-coating Pani onto an ITO glass.
Then, the resulting ITO glass can be functionalized into a biosensor by immobilizing
BVDV-specific antibodies onto the substrate. The presence of the antigen-antibody
complex can be detected by measuring the amperometric response occurring at the
surface of the biosensor.

1.3 Objectives

The following objectives led to the successful development and demonstration of

the ITO-Pani biosensor:

Objective 1: To characterize the self doped Pani.

Objective 2: To fabricate the ITO-Pani biosensor by evaluating the
experimental conditions such as Pani size, antibody concentration, antibody
immobilization method, and antigen incubation time.

Objective 3: To determine the specificity and sensitivity of the biosensor.

This manuscript is divided into the following sections:

Chapter 2 covers the motivation of the study and the background information on
the fabrication of the ITO-Pani biosensor.

Chapter 3 addresses the specific methods and materials involved in the
characterization of Pani, biosensor fabrication, and performance analysis.

Chapter 4 presents the results obtained from Objectives 1-3. The chapter is
formatted based on the manuscripts submitted for peer-reviewed journals. The first paper
addresses 1) the characteristics and roles of the self doped Pani in the biosensor design
and 2) the fabrication of the biosensor. The second paper covers the study of the
experimental conditions and the performance of the biosensor for BVDV detection in
pure culture.

Chapter 5 presents the preliminary study on fractal analysis to further understand
the antigen-antibody binding relationship.

Chapter 6 presents the novelty and the conclusion of the research.

Chapter 7 recommends several refinements for the improvement of the biosensor
performance.

The appendices provide the data generated from this study. This section also

covers preliminary studies on the effect of temperature on the biosensor performance.

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CHAPTER 2: LITERATURE REVIEW

2.1 Aggoterrorism

Terminology

The term ‘agroterrorism’ or ‘bioterrorism’ can be confusing and has never been
formally defined before. The Federal Bureau of Investigation defined terrorism as a
“deliberate act or threat committed by an individual or group for political or social
objectives” (Parker 2000). In this paper, the term bioterrorism is defined as “the threat or
use of biological agents, such as pathogenic bacteria, fungi, viruses and their toxic
products, by individuals or groups to cause disease or death in humans” (Rogers, Whitby
et al. 1999). Agroterrorism, on the other hand, is defined as “the deliberate introduction
of biological, chemical or radiological agents, either against livestock/crops or into the
food chain, for the purpose of undermining stability and/or generating fear” (Davis 2004).
Parker classified five potential targets of agroterrorism: field crops; farm animals; food
items in the processing or distribution chain; ready to eat foods; and agricultural facilities
including processing plants, storage facilities, transportation infrastructure, and research

laboratories (Parker 2000) .

Histog

Historical evidence suggests that bioterrorism or agroterrorism is not a recent
occurence. Two hundred years ago, the Romans dumped bodies into wells of enemy

drinking water supplies (Neher 1999). In the 14th century, Mongols catapulted Bubonic

4

plaque-infested bodies into the Walls of Kaffa and caused 25 million deaths (Wilson,
Logan-Henfrey et a1. 2000). During World War 1, German agents infected horses with
bacteria that caused glanders, a fatal human and equine disease (N eher 1999). The Soviet
Union military also used glanders in the early 19803 in the war with Afghanistan
(Wilson, Logan-Henfrey et a1. 2000). In 1996, chlordane, an organochlorine pesticide that
can adversely affect human health at a low concentration, was used by unidentified
perpetrators to contaminate animal feed in Wisconsin (Neher 1999). The latest case of
bioterrorism in the US is the inhalation of anthrax in 10 confirmed cases, resulting from
intentional delivery of Bacillus anthracis spores through mailed letters and packages

(Jemigan, Stephens et al. 2001).

Imgortance of agriculture

Agriculture is one of the most important national infrastructures in the US. In
2000, the US was home to 2.17 million farms that encompassed 942 million acres of land
(USDA 2001). The US food and fiber system (FF S) is also a source of jobs and earnings
for millions of American workers and suppliers ranging from farms to fast food chains.
The total FF S economy contributed over $1.24 trillion to the nation’s gross domestic
product, provided employment for 23.7 (17%) million workers in 2001 (USDA 2001). Of
the $1.24 trillion, almost $339 billion came from services, $334 billion from trade, and
$73.8 billion from the farm sector (USDA 2001). Table 2.1 shows the value of major
crop and animal production in 2001.
The US agriculture industry is also heavily dependent upon exports. Based on the
2001 census, the US exported $53.7 billion of agricultural products which supported jobs

on farms, food processing facilities, manufacturing plants, and the transportation and

 

 

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trade sectors (Edmondson 2003). Thus, the agriculture sector has a vital importance to
our society and economy. Any significant disturbance and terrorism threat in the
agricultural system have the potential to increase unemployment, increase prices
domestically and internationally, reduce trade, and affect the nation’s economy as a
whole.

Table 2.1: Major US animal and crop production, 2001 (USDA 2001).

 

 

Animal/crop Production Value ($billion)
Cattle 97.3 million 70.5

Pigs 60 million 4.5

Poultry (non-broiler) 440 million 1

Sheep 7 million 0.7

Corn grain 9.5 billion Bushels 18.9

Soybeans 2.9 billion Bushels 12.6

Wheat 2 billion Bushels 5.4

 

Vulnerabilities of the US agriculture

Due to the growing size of the US agricultural sector, this industry has been
recognized as a tempting target for both domestic and foreign terrorist acts. Based on the
National Agricultural Statistical Service, the average-size farm expanded from 147 acres
in 1990 to 441 acres in 2002 (USDA 2001). The growth in the agricultural sector,
specifically in the US livestock industry, is partly due to the emerging of consolidation of
companies and farmers that control the production of “animal feeds, animal breeding,
processing, and slaughtering” (Casagrande 2002). In the traditional agricultural society, a
farm raised and slaughtered a variety of livestocks in one area. In today’s agricultural
society, farms are more geographically dispersed and specialized in a “monoculture
(single species) ‘croplands, livestock feedlots, and poultry houses” (Parker 2000). Most

livestocks are born on breeding farms, for example, pigs in Iowa, North Carolina, and

 

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Illinois; and then raised in different farms such as in Texas, Kansas, and Nebraska (Davis
2004). These animals are then transported across the states for slaughtering and
processing (Lautner 1999). The slaughtering business is dominated by three large
companies (Mac Donald, Ollinger et al. 2000) with an average of 130,000 heads of cattle
slaughtered per week (USDA 2002). This consolidation in the agricultural industry makes
the US’s agricultural industry more vulnerable to agroterrorism. Potential terrorist may
take advantage on this issue by intentionally introducing potential agroterrorism agents
into a relatively specific region of the US (e.g. in a cattle farm in Texas) and this could
result in widespread national repercussions due to the animal movement from one region
to another. For example, the movement of pigs between farms in Netherlands was shown

to partly cause the classical swine fever endemic in 1997 (Stegeman, Elbers et a1. 1999).

To date, there has not been any documented intentional introduction of
agroterrorism agent in the US. That is not to say that an attack toward the nation’s
agricultural industry will not happen or it has never been tried. The emergence of West
Nile virus in the US has been speculated by some authorities as an intentional event
although it has never been proven (Davis 2004). It is hard, however, to distinguish an
agroterrorism attack from a natural occurrence of an animal or a plant disease, thus
providing an advantage for terrorists. For example, in 2000, the first rabbit hemorrhagic
disease appeared unexpectedly on a farm in Iowa (USDA 2006). This may be the same
virus that accidentally escaped an Australian research laboratory in 1995 (Mutze, Cooke
et a1. 1998), smuggled to New Zealand in 1997 (PCE 1998), and reappeared in Cuba
(USDA 2001). Although the recent mad cow case in the US has been ruled out as

unintentional (USDA 2005), the incident certainly has caused federal regulatory agencies

to improve surveillance and monitoring systems for potential terrorism act. Another
example of a potential agroterrorism attack is an outbreak of foot and mouth disease
(FMD), a highly contagious viral disease of cloven—hoofed animals. Foot and mouth
disease, comprising over 70 different strains, is the most infectious virus known, capable
of spreading as a wind-driven aerosol over 170 miles from its source (Horn and Breeze

1999)

In summary, the US agriculture sector is exceedingly vulnerable to agroterrorism

and some of the factors leading to the aforementioned event are (Parker 2000; Davis

2004):
0 Centralized feed suppliers, breeding, and slaughtering/processing farms
0 Lack of foreign animal disease training by US veterinarians
0 Lack of education of farmers on foreign animal diseases
0 Poor farm biosecurity
o Porous national border promoting movement of infected animals
0 Limited traceability of animal movement
0 Lack of an animal identification system
Imgact

Agroterrorism has the potential to cause both the mass disruption in a society and
a substantial impact on the US’s economy. It is estimated that the 2001 outbreak of FMD
in the United Kingdom resulted in the destruction of 4 million animals and cost

approximately $9 billion (Thompson 2001). According to a study by the University of

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California, Davis, an outbreak of FMD in California alone would cost the state at least $6
billion (Ekboir 1999). Additionally, the first US case of mad cow disease, which was-
detected in a cattle in the state of Washington, was estimated to have lowered the US
farm income by $5.5 billion in 2004 (Rainford 2004). Financial losses resulting from
agroterrorism include but not limited to the direct loses of agriculture commodities, cost
related to diagnosis, surveillance system, destruction of contaminated products and
containment facilities, disposal of infected carcasses, and loses due to export and trade
restriction (Parker 2000; Davis 2004). Since the economic implication of agroterrorism is
likely to be significant, the term “econoterrorism” has been proposed as an alternative to

agroterrorism (Steele 2000).

Agroterrorism agents

There are a number of agents that could be employed in agroterrorism acts, which
include bacteria, fungi, algae, insects, weeds, and genetically modified organisms (Parker
2000). Table 2.2 presents a comprehensive listing of some potential biological agents
developed for the Defense Intelligence Agency (Christopher 1997; Atlas 1999). The list
identifies the agents that cause animal diseases recognized by the Organization
Internationale des Epizooties (OIE) as List A diseases. List A diseases are considered as
highly infectious, capable of spreading rapidly, and have the potential to inflict
catastrophic economic loses and social disruption (Christopher 1997; Atlas 1999; Wilson,
Logan-Henfrey et a1. 2000). The Organization Internationale des Epizooties strictly
requires its members to report outbreaks of List A diseases within 24 hours of laboratory

confirmation. Report of List A disease outbreaks trigger immediate trade restrictions on

the affected products (OIE 2005). Some diseases categorized under the list are African

swine fever, foot and mouth disease, bovine spongiforrn encephalopathy etc (Table 2.2).

Table 2.2 Animal and plant pathogens with potential biological agents developed by the

Defense Intelligence Agency (Christopher 1997; Atlas 1999).

 

Animal Pathoggns

Plant Pathogens

 

African swine fever”

African horse sickness*

Anthrax Avian influenza“

Foot and mouth disease*
Bluetongue“

Hog cholera/classical swine fever"
Bovine spongiforrn encephalopathy*
Ornithosis/Psittacocis

Contagious bovine pleuropneumonia*
Rinderpest“

Lumpy skin disease“
Trypanosomiasis

Newcastle disease”

Poxvirus

Paratuberculosis/Johne’s disease
Peste des petits ruminants
Pseudorabies virus

Rifi valley fever“

Sheep and goat pox“

Swine vesicular disease“
Vesicular stomatitis*

Rice blast (Magnaporthe grisea)

Wheat stem rust (Puccinia graminis)
Wheat dwarf geminivirus

Wheat smut (F usarium graminearum)
Pseudomonas fascovaginaei
Clavibacter tritic

Pseudomonas fascovaginaei
Scleropthora rayssiae
Peronoschlerospora sacchari

P. philippinensis

P. maydis (Java downy mildew)
Phakospora sachyrhizi

Pyrenochaeta glycines (Red leaf blotch)
F usarium oxysporum f. sp. Vasinfectum
Xanthomonas campestris pv.
Maloacearium

Geminivirus

 

*Office Internationale des Epizooties List A Disease

(http://www.oie.int/eng/en_index.htrn)

Bovine Viral Diarrhea Virus

Bovine viral diarrhea virus (BVDV), one of the most insidious and economically

devastating viral pathogens in cattle (Brock 2003), is chosen as a model for potential

agro/bio terrorism agents, such as pathogens indicated in Table 2.2. Though BVDV is

predominantly found in cattle, the ability of this virus to replicate in numerous wild

ruminant species such as camels, deer, elk, and bison has also been documented

10

(Nettleton 1990). Bovine viral diarrhea virus is classified in the genus Pestivirus within
the family Flaviviridae with a single stranded, enveloped RNA genome that is prone to
high mutation rates. High mutation rates lead to heterogeneity which helps BVDV and
other pestiviruses to adapt and evade host immune systems (Ridpath 2003). Bovine viral
diarrhea virus has two predominant genotypes, BVDV Type-1 and Type 2, based on their
RNA makeup, the structure of their protein capsules, and the antibodies that are made in
response to their infection (Pellerin, Van Den Hurk et al. 1994). A specific virus may also
be either cytopathic or non-cytopathic, indicating its ability to cause visible damage to
experimentally infected cells in the laboratory (Fulton, Saliki et al. 2000). Although both
biotypes can cause infections, the noncytopathic strain of BVDV is more common in the
cattle population (Dubovi 1992). Studies also show that there are two different ways that
cattle can be infected by BVDV: Acute infection of animals that have not been previously
exposed to the virus (immunocompetent cattle population) and fetal infection occurring

in the early pregnancy stage (Houe 1995).
Acute infection

Most of the BVDV infections in an immunocompetent cattle population are
subclinical, demonstrated by a small increase in body temperature and a decrease in milk
production (Baker 1995). A bovine viral diarrhea disease is manifested when the BVDV
infection becomes clinical. The virus incubation period is 5 to 7 days, and a transient
viremia occurs 4-5 days post-infection that may continue up to 15 days (Duffell and
Harkness 1985). At the time animals develop the clinical infection, they are usually
starting to make neutralizing antibodies (Brock 2003). Clinical symptoms include

depression, diarrhea, sometimes oral lesions which is characterized by ulceration, and a

11

rapid 1

 

Rebhui
is much
I985).

\Vlll'l lllx

Persistti

 

between
infectim
less that.
relilicat
0n the ‘
Breakir
E€Static
these ;
ICCID.
BVDV
dischEu
901mm

Infecn' (

.‘IIUCOS

rapid respiratory rate, which can be mistakenly diagnosed as pneumonia (Perdrizet,
Rebhun et al. 1987). The concentration of virus shed from these acutely infected animals
is much lower than that of the persistently infected (PI) animals (Duffell and Harkness
1985). In Denmark alone, an annual incidence of acute infection of 34% was estimated

with the total annual losses of $20 million per million calves (Houe 1995).
Persistent infection

Persistent infection with BVDV develops when a fetus is exposed to the virus
between 50 to 150 days of gestation (Brock 2003). The ability to induce fetal persistent
infections is a unique aspect of BVDV pathogenesis. Although PI animals may represent
less than one percent of the cattle population, they shed the virus and initiate further virus
replication and genetic variation (Brock 2003). Therefore, control programs must focus
on the prevention of persistent infections, and identification and removal of PI animals.
Breaking the cycle of exposure of pregnant animals in the first 125 to 150 days of
gestation is the key to preventing persistent infections. The concentration level of virus in
these PI animals is extremely high, up to 106 cell culture infective dose per milliliter
(CCID/ml) in serum samples (Houe 1995). Cattle that are persistently infected with
BVDV shed a high concentration of virus in their secretion and excretion, such as nasal
discharge, saliva, blood serum, tissue, semen, urine and milk (Brock 1991). Direct
contact with P1 cattle is probably the most common method of transmission of the

infection (Houe 1995).

Mucosal disease

Mucosal disease can occur is when PI animals are exposed to cytopathic strain of

BVDV that shares close homology with the non-cytopathic BVDV (Bolin 1995). Acute

12

mucosa] disease is characterized by pyrexia, depression, weakness, and anorexia (Baker
1995). Animals with chronic mucosal disease may develop high fever, continual diarrhea,
weight loss, inappetence, long-term erosive epithelial lesion, and ultimately die from
severe debilitation (Baker 1995). Generally, mucosa] disease has a high morbidity rate

and a low mortality rate (Bolin 1995).

Bovine Hemesvirus Tyge-l

In this study, the specificity of the biosensor is tested against the presence of
bovine herpesvirus type-1 (BHV). The virus causes infectious bovine rhinotracheitis, an
infectious respiratory disease of cattle (Kapil and Basaraba 1997). The virus can infect
the upper respiratory tract or the reproductive tract. The virus may remain in the latent
stage (inactive) following an infection and may become re-activated by stresses applied
to the animal (Kapil and Basaraba 1997). The primary portal of entry is the nasal cavity
where the most infections occur when an infected animal is introduced into a herd. The
main source of genital disease is venereal transmission (Kapil and Basaraba 1997).

Bovine Herpesvirus Type-1 is capable of attacking many different tissues in the
body and therefore, is capable of producing a variety of clinical disease forms according
to the infected tissues. The clinical diseases caused by this virus can be grouped as
respiratory tract infections, eye infections, abortions, genital infections, brain infections,
and a generalized infection of newborn calves (Richey 1994). Respiratory infection with
BHV varies in severity, depending on the host and the strain of the virus involved. The
symptoms can range from no signs to severe disease with approximately 10% mortality
rate (Kapil and Basaraba 1997). Clinical signs associated with BHV respiratory tract

infections are high fever (104 °F to 108 °F), inflammation of nostrils, erosion of nasal

13

mucosa, mental depression, a decrease in appetite, and conjunctivitis. The inflamed nasal
mucosa causes the nose to look very red, hence the term "red nose" is commonly used to
describe the disease (Richey 1994). In general, the respiratory form of BHV does not
cause death. The stresses associated with this disease, however, may reduce resistance to
other infections and thus cause death (Richey 1994).

2.3 Detection Methods

This section examines the current methods for microbial and viral detection. The
parameters most currently used to evaluate a method are sensitivity, specificity (Barbour
and George 1997), and detection time. These parameters will be then used to compare
some of the existing methods for the diagnosis of BVDV.

Analytical sensitivity of an assay is characterized by the assay’s ability to detect
the lowest number of the target organism and is used synonymously as “limit of
detection" and "minimal detectable concentration" (Saah and Hoover 1997). One possible
way to determine analytical sensitivity is by testing serial dilutions of samples with a
known concentration of the target organism (Annbruster, Tillman et al. 1994). Diagnostic
sensitivity, on the other hand, is defined as the ability of the assay to recognize the target
organism in a biological sample as opposed to its ability to detect the lowest
concentration of the organism. If the target organism is not present in the tested biological
samples due to inefficient sample processing method, an assay with a perfect analytical
sensitivity performance will fail to give a positive result (Saah and Hoover 1997).

Analytical specificity is the capability of the assay to “exclusively detect the
target organism rather than similar or related target groups” in a sample (e.g. detect

Escherichia coli 0157:H7 rather than the generic E. coli) (Saah and Hoover 1997). When

14

an assay is analytically specific, a negative result is produced when no target organism is
present. Diagnostic specificity, on the other hand, is “the ability of an assay to correctly
identify” if a sample is infected with the target organism (Saah and Hoover 1997).
Interestingly, several studies have shoWn that an analytically specific assay often reduces
the diagnostic specificity. For example, an analytically specific device, polymerase chain
of reaction (PCR), may cause a false-positive result in a biological “dirty” sample
(Noordhoek, van Embden et al. 1993; Zaaijer, Cuypers et al. 1993). The finding shows
that the assay is able to maintain its high analytical specificity but unable to give a
diagnostically correct result due to the presence of external contamination in the sample.

Detection time of a method is not as critical as its sensitivity and specificity
performance in an analytical laboratory setting. In farms, food processing plants,
international borders or diagnostics laboratories however, a rapid detection method is as
significant as its sensitivity and specificity performance. With a lengthy detection time,
the contaminated products could have reached the consumers or caused the spread of a
disease before contamination results are known. Current detection method ranges from
days (Saliki and Dubovi 2004) to hours (Kramps, Maanen et al. 1999), and it is definitely
a new challenge to develop a real time or near-real time detection method.

An ideal detection method or assay should be both analytically and diagnostically
sensitive and specific with rapid detection time. At the moment however, no method or
device that satisfies all these criteria is commercially available. A review of a
conventional method for BVDV detection is illustrated in the next subsequent

paragraphs.

15

Virus isolation is the most reliable and is considered the “gold standard”
diagnostic technique for BVDV detection (Dubovi 1990; Edwards 1990; Saliki and
Dubovi 2004). This method requires 4 to 5 days of virus culturing and several more hours
for virus detection (Dubovi 1990; Edwards 1990; Saliki and Dubovi 2004). Samples used
in this method include serum, whole blood, and tissue samples (Brock 1995).

An increased demand for high-throughput screening produces several
technological developments for detecting BVDV. These emerging technologies include
enzyme linked immunosorbent assay (ELISA), PCR and nucleic acid hybridization, and
immunohistochemical (IHC) staining methods (Dubovi 1990; Edwards 1990; Saliki and
Dubovi 2004). Several antigen (Mignon, Dubuisson et al. 1991; Grieser, Frey et a]. 1993;
Rossmanith, Vilcek et al. 2001) and antibody (Kramps, Maanen et al. 1999) ELISAs
have been developed for the diagnosis of BVDV infection. An antigen-capture ELISA is
much cheaper and produces a faster result, but not as sensitive as the virus isolation
method (Grieser, Frey et al. 1993). The latter method however is primarily used in
screening PI cattle and not for the diagnosis of acute BVDV infection (Saliki and Dubovi
2004). As for the antibody-capture ELISAs, a study reports that in testing one thousand
field serum samples, the ELISA test shows a sensitivity and specificity relative to virus
neutralization test of 98% and 99%, respectively (Kramps, Maanen et al. 1999). An
ELISA method however, is laborious and the test protocol requires approximately 2.5
hours of incubation time with multiple washing steps in between (Kramps, Maanen et al.
1999)

Recently, the detection of BVDV antigen in formalin-fixed paraffin-embedded

tissues by IHC staining has gained popularity for the diagnosis of BVDV infection. The

16

discovery of antigen retrieval methods (Huang, Minassian et a]. 1976) and enzyme-based
detection methods (Hsu, Raine et al. 1981) were instrumental in allowing IHC system to
be used with tissue samples for a routine diagnostic application. In screening PI animals,
the IHC method has been widely used in analyzing “ear notch” samples (Haines, Clark et
al. 1992; Bildfell, Thomson et al. 2000; Grooms and Keilen 2002). This method also has
a potential to be relatively inexpensive and accurate for screening PI animals.

During the past 10 years, reverse transcriptase polymerase chain reaction (RT-
PCR) has gained popularity as a routine diagnostic tool for BVDV detection (Saliki and
Dubovi 2004). Since RT-PCR has a high analytical sensitivity and specificity
performance, any animal specimen such as milk, urine, tissue, serum, and whole blood
can be used with this instrument. In practice, however, the efficiency of isolating RNA
from any given sample has a profound effect on the diagnostic sensitivity of RT-PCR
(Saliki and Dubovi 2004).
2.4 Conducting Polymer

This section describes the history, mechanism, and general application of
conducting polymer compounds. The employment of conducting polymer compounds in
a biosensor design is described later on in this manuscript. Historically, the research in
conducting polymer begun in the 1960’s when a Japanese scholar, Hideki Shirakawa,
discovered a silvery film of polyacetylene, by accidentally adding a thousand-fold too
much catalyst to a reaction vessel (Chiang, Fincher et al. 1977). In another part of the
world, MacDiarmid and Heeger were experimenting with a metallic-like film of the
inorganic polymer sulphur nitride (Akhtar, Kleppinger et al. 1977). The following

collaboration between MacDiarmid, Shirakawa and Alan Heeger led to the historic

l7

 

 

 

dlSt
Ch‘
1

[0w

 

 

 

discovery of polyacetylene and they became the recipients of the Nobel Prize in
Chemistry in 2000. This finding generated new interests in the scientific community
towards the discovery of new conducting polymer compounds.

Poly-paraphenylene was first discovered by Ivory and his coworkers (Ivory,
Miller et al. 1979). The discovery of poly-paraphenylene is particularly interesting due to
its processable properties which open the door for commercially viable conducting
plastics (Rabolt, Clarke et al. 1980). Polypyrrole also is extensively investigated for its
excellent conductive properties (Kanazawa, Diaz et a1. 1979). Polyaniline (Pani), on the
other hand, is probably the most rapidly growing class of conducting polymer. The
interest stems from the fact that this polymer family can be doped by a variety of
different dopants, either by chemical or electrochemical syntheses (MacDiarmid and
Epstein 1990). Many other conducting polymer compounds, such as polythiophene,
polyfuran, polycarbazole and polyindole have also been synthesized and studied
(Lagowski, Salzner et al. 1998; Ivanov, Gherman eta]. 2001). A logarithmic conductivity
ladder of some of these polymer compounds (in their undoped form) is shown below

(Heeger 1986; Friend 1993) (Figure 2.1).

18

Conductivity

(S/cm)
106 , Copper metal
5 Polyacetylene doped with
10 ASF5
Polyacetylene doped with 12
4 Liquid mercury
10
103 Poly(p-phenylene) doped with
ASF5
2 Polypyrrole doped with 12
10
1
10 Polyaniline

Figure 2.1: Conductivity range of conducting polymer compounds compared to
some metals (Friend 1993).

Unlike conventional metallic materials, conducting polymer compounds are not
inert. In fact, conducting polymer compounds are quite reactive and capable to initiate
molecular interaction. Therefore, the polymer stability in the environment and its shelf
life need to be monitored and studied. Exposure to extreme pH, temperature, or ionic
strengths also can result in a loss of conductivity and electroactivity, and a degradation of
mechanical properties (Guiseppi-Elie 1998).

One of the external factors affecting the conductivity of conducting polymer
compounds is temperature. While the conductivity of metallic materials generally
increases with decreasing temperature, the conductivity of conducting polymer however,

increases with increasing temperature (Figure 2.2). A number of studies also have been

19

done to investigate the structural response of conducting polymer to changes in
temperature (Sauvajol, Djurado et a]. 1991; Winokur 1998). The most obvious response
to thermal variations is the large fractional change in the crystal lattice repeats in the
equatorial direction. Therefore, the “free” volume within which individual chains travel
varies greatly with the temperature variation (Winokur 1998). In addition, rotational
displacements of the main chain and the chain axis due to the thermal variation have
important consequences on the polymer properties (Sauvajol, Djurado et al. 1991). The
strong rotational motion about the chain axis is reported to alter the band structure and

thus alter the material conductive properties (Sauvajol, Djurado et a]. 1991).

 

 

 

 

 

 

 

 

 

 

 

Conductivity (S/m)
10" '
10’ ‘
\ Silver
107 \
105 I
Polyacetylene
103
1 l 1 . L 1
0 100 200 300
Temperature (K)

Figure 2.2: Conductivity of silver and polyacetylene in response to varying

temperature (Winokur, 1998).
Mechanism for conductin o mer

There are several models for the mechanism of conducting polymer. The most

widely used mechanism is the electron band model (Bott 1986). This model is based on a

20

 

 

bond between two atoms over a crystalline structure. When two atoms are brought
together close enough for the orbitals to overlap, the orbitals interact with each other to
form two new orbitals: one of higher (conduction band) and one of lower (valence band)
energy levels (Figure 2.3). The difference in magnitude between the conduction and
valence bands is determined by the degree of overlapping of those two bands. In a
semiconductor or an insulator, there is a “gap” between the conduction and valence bands
(Figure 2.3). In a semiconductor, the magnitude of the band gap is small enough for the
electrons to be thermally excited from the valence band to the conduction band. An
insulator however, has a large band gap, and hence thermal excitation of carrier is not

possible.

Wide band gap — H
Increasing Narrow band gap N
- . g .. . 0 gap .

energy
level

 

Insulator Semiconductor Metal

- Energy levels in conduction band

Energy levels in valence band

Figure 2.3: Schematic diagram explaining the difference between an insulator, a
semiconductor, and a metal (Bott, 1986).
In becoming electrically conductive, a polymer has to imitate a metal, that is its
electrons are free to move. Conducting polymer compounds contain n-electron backbone

responsible for their unusual electronic properties (Macdiarmid, Chiang et al. 1987). This

21

extended n-conjugated system of the conducting polymer has single and double bonds
alternating along the polymer chain (Figure 2.4 for the structure of polyaniline). The
conjugated double bonds behave quite differently from the isolated double bonds. The
conjugated double bonds act collectively, “knowing” that the next-nearest bond is also a
double bond (Kroschwitz 1988). Hiickel’s theory and other simple theories predict that
electrons are delocalized over the entire chain and that the band gap becomes smaller for
a long chain (Shuai and Bredas 1992). This prediction takes into account the character of
a molecular orbital, including the p-orbitals of all carbon atoms along the chain of
conjugated double bonds (Shuai and Bredas 1992).

The mechanism of conductivity in conducting polymer compounds is complex
since such polymer exhibits conductivity across a range of fifteen orders of magnitude,
which involves different mechanisms within different regimes. Various studies show that
the conductive property in conducting polymer compounds is influenced by a variety of
factors including the polaron length, conjugation length, the overall chain length, and by
the charge transfer to adjacent molecules (Kroschwitz 1988). Conducting polymer
compounds also show increased conductivity by doping. The concept of solitons and
polarons is used to explain the conductivity properties in doped polymer and is discussed

later in the text.

Doping

As discussed above, a key property of conducting polymer compounds is the
presence of conjugated double bonds along the backbone of the polymer (Kroschwitz
1988). However,=conjugation is not enough to make the polymer material conductive. In

addition to the conjugated double bond, charge carriers in the form of extra electrons or

22

“holes” (a hole is an empty position where an electron is missing) have to be introduced
into the material. When a hole is filled by an electron jumping in from a neighboring
position, a new hole is created and thus allows a charge transport (Winokur 1998).

Photoexcitation, charge injection, and/or doping are employed to introduce charge
carriers necessary for electron transports (Winokur 1998). Of the three aforementioned
mechanisms, only doping yields a permanent transition to the conductive state. By
definition, doping is “an intentional introduction of a selected chemical impurity (dopant)
into the crystal structure of a semiconductor to modify its electrical properties”
(Streetrnan 1995). Doping, either by addition of electrons (reduction reaction) or the
removal of electrons (oxidation reaction) from the polymer is formed via chemical or
electrochemical methods (Salaneck and Lundstrom 1987). The conductivity of the
polymer increases as the doping level increases (Winokur 1998).

In the case of polyacetylene, a halogen (F, Cl, Br, or I) is used to perform a p-type
doping (oxidation reaction) and an alkali metal (Li, Na, K, Rb, Cs, or Fr) for an n-type

doping (reduction reaction). This is shown below (Chiang, F incher et al. 1977):
[CH ]n +3x(212) ‘7 [CH lnx+ 'I' 3513— Oxidative doping

Reductive doping
[CH in + xNa —-) [CH ]nx T + xNa +

The resulting doped polymer is a salt with the charges on the polymer being the
mobile charges. By applying an electric field perpendicular to the film, the counter ions
(13’ and Na) are made to diffuse from or into the structure, causing the doping reaction to

proceed backward or forward. In this way the conductivity is switched off or on. The

23

conductivity of polyacetylene is enhanced by several orders of magnitude from 10'9 S/cm
to 105 S/cm (Chiang, Fincher et a1. 1977).

The role of the dopant is either to remove or to add electrons to the polymer. For
example, when polyacetylene goes thrOugh oxidative doping, the iodine molecule attracts
an electron from the polyacetylene chain and becomes 13" ion (Chiang, F incher et al.
1977). The polyacetylene molecule, now positively charged, is termed a radical cation, or
polaron. The lonely electron of the double bond, fi'om which the electron has been
removed, can move easily along the polymer backbone. The positive charge, on the other
hand, is fixed by electrostatic attraction to the 13" ion and not mobile. When the
polyacetylene chain is heavily oxidized, polarons condense pair-wise into so-called
solitons. These solitons are responsible, in complicated ways, for the transport of charges
along the polymer chains, as well as from chain to chain on a macroscopic scale. This
explains why a high level of doping is required to bring the polymer to its maximum
conductive state (Heeger 1986). Additionally, an intersoliton-hopping mechanism also is
proposed to explain conductivity mechanism by charge hopping between different
polymer chain. The mechanism is based upon the idea that carriers tend to hop larger
distances to sites which lie energetically closer, rather than to their nearest neighbors
(Mott and Davis 1979). Therefore, the choice of doping agent is extremely important for

practical application of conducting polymer compounds.

Applications of conducting polymer

Conducting polymer compounds have attracted much attention largely because of
their many projected applications in solar cells, lightweight batteries, electrochromic,

chemical/bio sensors and molecular electronic devices. Generally, the two main

24

applications for conducting polymer compounds are based on their conductive and
electrochemical properties. The list illustrates the diverse applications of conducting
polymer compounds and is by no means complete (Table 2.3).

Table 2.3: Applications 0f conducting polymer compounds.

 

 

Application based on the conductive Application based on the
property electrochemical property
Electrostatic materials (Friend 1993) Electrochromic displays (Saxena and
Malhotra 2003)
Anticorrosive (Ding, J ia et al. 2002) Drug release systems (Loh, Moody et
al. 1990)
Microelectronics ((Koezuka and Electromagnetic shielding
Tsumura 1989) (Kim, Kim et al. 2003)
Chemical, biochemical and thermal Chemical, biochemical and thermal
sensors (Siturnorang, Hilbert et al. 2000) sensors (Grennan, Killard et a].
2006)
Antistatic clothing (Dall'Acqua, Tonin et Rechargeable batteries and solid
al. 2004) electrolytes (Lee, Lee et al. 1993)
Artificial muscles (Kaneto, Kaneko et al.
1995)

Nano wires (Zhu, Chang et al. 2006)

The most publicized and promising current applications of conducting polymer
compounds are the lightweight rechargeable batteries, where the polymer compounds,
such as polyacetylene, polythiophene, polypyrrole, and Pani, are used as the electrode
materials for the batteries (Lee, Lee et al. 1993; Efirnov, Belov et al. 1996; Mu 2004).
Some commercialized polymer batteries are comparable, if not better than nickel-
cadmium cells. The polymer battery, such as a polypyrrole-lithium cell, operates by the
oxidation and reduction of the polymer backbone (Tanaka and Reynolds 1995).

Organic electroluminescence is one of the most exciting developments in
conducting polymer. The first polymer based light emitting diode (LED) was discovered

by using layers of polypyrrole (Burroughes, Bradley et al. 1990). Conducting polymer

25

compounds are thought to have advantages over other materials in LEDs because of its
ease of manufacturing, greater thermal and mechanical stability, reduced tendency for
crystallization, and low operating voltage (Saxena and Malhotra 2003). The challenge in
using conducting polymer compounds however, lies in purifying the polymer to a
necessary degree (Moratti 1998).

The potential for conducting polymer compounds in the area of electronics and
photonics is also enormous. The first report of electronic devices with potentially useful
properties was the field-effect transistors based on poly (3-hexylthiophene) (Koezuka,
Tsumura et a]. 1987; Koezuka and Tsumura 1989). The scope of conducting polymer
applications in photonics area is also advancing rapidly, especially in LED (Cha and Jin
2004; Ramos, Correia et al. 2004) and capacitor fabrications (White and Slade 2003).

The use of conducting polymer also has expanded in making conducting textiles.
Since synthetic fibers are electrically insulating, they are transparent to the detrimental
electromagnetic (EM) wave (Kuhn, Kimbrell et a]. 1993; Mirmohseni and Oladegaragoze
2000). Most metals usually show a very high EM wave shielding capability but are poor
EM absorbent materials. On the other hand, conducting polymer compounds, such as
Pani, are capable of absorbing as well as reflecting EM wave, showing a significant
advantage over metallic materials (Kim, Kim et al. 2003).

Conducting polymer compounds also are used extensively in the biosensor
technology (Guiseppi-Elie 1998). Polyaniline, for example, is chosen as the electrical
transducer in biosensors due to its excellent stability in liquid form, promising electronic
properties (Syed and Dinesan 1991), and strong bio-molecular interactions (Imisides

1996). Also, Pani structure can be modified to attach to protein molecules, such as

26

 

antibodies, by binding to its polymer backbone (Situmorang, Hilbert et al. 2000).
Additionally, Pani has the ability to efficiently transfer the electric charges produced by
biochemical reactions to electronic circuits (De Taxis du Poet, Miyamoto et al. 1990).
Polyaniline also acts as an enzyme amplifier and conductivity modulating agent to
provide signal amplification in the recognition process (Sergeeva, Pilletskii et al. 1996;
Kim, Cho et al. 2000; Grennan, Strachan et a]. 2003; Morrin, Guzman et a]. 2003). The
use of Pani as an enzyme switch, which yields "on" and "off" responses, was
demonstrated by lribe and Suzuki (lribe and Suzuki 2002). Additionally, the biological
sensing elements entrapped within the polymer matrix are shown to maintain their
biological activity (Sadik and Emon 1996). A list of some conducting polymer based
biosensors is shown in Table 2.4. The next subsequent paragraph describes the structure
and mechanism of Pani in detail.

Table 2.4: Conducting polymer based-biosensor.

 

 

Biological
Detection Sensing
Biosensor Target Element Reference
Whole (Kim, Cho et al.
PANI/ whole cell cell IgG 2000)
(Killard, Smith et
PAN I/Immunoglobulin (IgG) Attrazine IgG al. 2000)
(Adeloju and
Polypyrole/Urease Urea Urease Wallace 1996)
(Deshpande and
Polypyrrole/Dopamine Dopamine Whole cell Hall 1990)
Polypyrrole/ Deoxyribose (Rodriguez and
nucleic acid (DNA) DNA DNA probe Alocilja 2005)
(Taniguchi,
Fujiyasu et al.
Polypyrole/ IgG IgG Anti- IgG 1986)
Whole (Muhammad-Tahir,
PANI/ IgG cell IgG Alocilja et al. 2005)

 

27

 

Po aniline

Polyaniline (Figure 2.4) is extensively researched for its electrical, optical,
chemical and electrochemical properties due to its simple synthesis method, stability in
air, and a myriad range of applications (Winokur 1998). Additionaly, Pani is the best-
known semiflexible rod conducting polymer system (Genies, Boyle et al. 1990). The
chemical and structural flexibility surrounding the amine nitrogen linkages in Pani
creates enormous diversity in its properties. Different forms of Pani exist based on the
oxidation levels. Polyaniline is believed to be composed of the basic chemical units

shown in Figure 2.4 (Macdiarmid, Chiang et al. 1987; Yen Wei 1989; Genies, Boyle et

  

al. 1990).
H H
N N N: N
Reduced unit ' 03:11:11le unit 14)" . .

Figure 2.4: Pani structure (Macdiarmid, Chiang et al. 1987; Yen Wei 1989; Genies,
Boyle et al. 1990).

When 0 < y <1, they are called poly (paraphenyleneamineimines) in which the
oxidation state of the polymer increases continuously with decreasing value of y. The
firlly reduced form, also known as leucoemeraldine, has a y value equal to 1. The most
oxidized form (y equal to 0) is called pemigraniline. Finally, the intermediate form, with
y equal to 0.5, is called emeraldine. The terms leucoemeraldine, emeraldine, and

pemigraniline refer to the different oxidation states of Pani (Macdiarmid, Chiang et al.

28

1987). Between the three types of Pani, emeraldine is the most studied Pani compound

(Seegeeva 1996).

In an aqueous acidic solution, aniline exists predominantly as the aniliniurn cation

(Macdiarmid, Chiang et a]. 1987):

/ \ NHgT—A NH2+H++ e

 

(Equation 2.1)

Aniline cation may be recombined into benzidine or N—phenyl-p-phenylenediamine to

participate in the growth of Pani chains in the pemigraniline form:

NH NH;'+2 H++ 2e

(Equation 2.2)
The protonated pemigraline appears to be a blue color mixture. The acidicity of the
mixture increases during polymerization due to the release of protons. The protons

released from the oxidation process above are used in the reduction of the oxidizing agent

such as persulfate to sulfate:

29

 

 

2- 2-
8208 + 26 2 804

(Equation 2.3)
When aniline is still present in the mixture after all the oxidizing reaction is
consumed, pemigraline takes over the role of an oxidant and becomes reduced to
emeraldine. Oxidative polymerization of aniline then proceeds according to equations
(2.1) and (2.2) until all the pemigraline is converted into emeraldine. Emeraldine ranges
from fully to partially crystalline films or powders. The degree of crystallinity, however,
is quite low and never exceeds 50% (Libert, Bredas et a]. 1995). In addition to this
oxidation chemistry, the amine nitrogen can be reversibly protonated using an
electrochemical method.
Polyaniline, in the form of conductive emeraldine, is synthesized via chemical
oxidation by doping aniline in acidic media (e. g. 1 M hydrochloric acid) (Figure 2.5). The
doping mechanism is reported to increase the conductivity level of Pani from 10-10 S/cm

to 1 S/cm (Macdiarmid, Chiang et al. 1987).

iN-O—~@~H@~u—©J3

Polyaniline (eurcrnldine)
base

2 n H6019
protonation
0. is}! NH NH
3%_©—Ci 9 © Q
n

Polyaniline (emeraldine)
salt

Figure 2.5: Doping of emeraldine base with 1 M hydrochloric acid (Macdiarmid,

Chiang et al. 1987).

30

Self doped polyaniline

The electrical property of Pani mentioned previously is pH-dependent with most
studies conducted in a pH level lower than pH 4.0 (Shaolin and Jincui 1999). In a
biosensor design however, nearly neutral pH solution (pH 6-8) is used since most
biocatalyst and immunological reactions occur optimally at neutral pH. Thus, it is very
challenging to incorporate biological elements with the conventional, pH-dependent Pani
that requires acidic environment to maintain its conductive property. Recently, significant
progress has been accomplished to improve the chemical and physical properties of Pani.
One progress is by introducing sulfo-acid residues to the emeraldine base to give rise to
the so-called ‘self doped’ Pani. In contrast to the Pani discussed in the previous section,
self doped Pani has a negatively charged functional group bound to the polymer
backbone to act as an inner anion dopant. Therefore, no anion or electron exchange
between polymer and the surrounding solution is required during oxidation and reduction
process (Lukachova, Shkerin et a]. 2003). Such Pani has a conductivity of approximately
1 S/cm within a pH range of up to 7 (Lukachova, Shkerin et a]. 2003).

A self doped Pani is prepared by chemical or electrochemical synthesis. In this
study, only chemical synthesis is considered due to its myriad development and progress.
A distinct route for the preparation of self doped Pani is by chemical modification of the
emeraldine base with sulfo acidic group, such as camphorsulfonic acid (Winokur, Guo et
al. 2001). Figure 2.6 shows a probable mechanism for chemical polymerization of Pani
doped with camphorsulfonic acid as proposed by Rannou, Nechtschein et a]. (1999). The

discovery of polymer doped with camphorsulfonic acid attracts considerable attention for

31

both firndamenta] research and technological applications such as in the biosensor

development (Snejdarkova, Svobodova et al. 2004)

H H36 /
/ / / / / + 3
NH n 2n go
Emaraldine base Camphorsulfonic acid
NH
+ J"
i \ SO I \ @303 I \
3
\
NH / / / / /
OH: NH CH
Hac / /0 H36 3 /o

Polyaniline doped with camphorsulfonic acid

Figure 2.6: A probable mechanism for chemical polymerization of Pani doped with
camphorsulfonic acid (Rannou, Nechtschein et a1. 1999).

2.5 Biosensor
This section covers the terminology and some examples of existing biosensors. A
biosensor is an analytical device that integrates biological sensing elements with
electronic transducers (Turner and Newman 1998; Ivnitski, Abdel-Hamid et al. 1999)
(Figure 2.7). Biosensors make use of a variety of transducers, such as electrical,
electrochemical, optical, piezoelectric crystal, and acoustic wave. Foulds and Lowe
(1985) emphasize that a transducer should be highly sensitive for the analyte of interest
and should have a moderately rapid response time, preferably less than sixty seconds.
The transducer element also should be reliable, able to be miniaturized, and suitably

designed for practical applications (Foulds and Lowe 1985). The sensing elements may

32

be enzymes, antibodies, DNA, receptors, organelles, and microorganisms, as well as
animal and plant cells. The sensing element is placed in close proximity with a
transducing element that converts biological reactions into quantifying electrical response
(Cahn 1993). Some of the major attributes of a biosensor technology are its specificity,
sensitivity, reliability, portability, real time analysis, and simplicity of operation (D'Souza
2001). The most important features of a biosensor are its high sensitivity and specificity,
and rapid detection time (Foulds and Lowe 1985). The next few sections cover a specific

type of transducing and biological elements that are used in the biosensor fabrication in

 

 

 

    
   
  
 
 

this study.
Transducer: Biological

S Electrochemical, Sensing Element: L A
][ Electrical, Antibody, r" I:
G DNA probe, Organelle, L
1: Amperometry, DNA probe, Y
L Optical, Whole cell, _. T

Acoustic wave Enzyme E

 

 

 

 

 

 

 

 

Figure 2.7: Schematic of a biosensor.

Transducing element— Electrochemical method

In this study, an electrochemical method is employed as a transducer element in a
biosensor design. The electrochemical instruments are attractive tools, because they are
robust, relatively simple, economical to mass-produce, and is easily miniaturized to
facilitate an ultra small sample volume. Furthermore, unlike spectroscopic or optical

based techniques, electrochemical transducers are not likely to be affected by sample

33

turbidity, quenching, or interference from absorbing and fluorescing compounds in
biological samples (Wilson and Routh 2004).

Electrochemical techniques are concerned with the interplay between electricity
and chemistry, such as the measurement of current, potential or charge, and their
relationship to chemical properties (Wang 2000). The fundamental process in
electrochemical method is the transfer of electrons between the electrode surface and the
molecules in the solution (electrolyte) adjacent to the electrode. One quantity that is
particularly important in electrochemical experiments is current, which is proportional to
the electrode surface area and the concentration of the electrolyte. The differential form
of Faraday’s law (equation 2.4) shows the rate at which electrons (n) are moved across
the electrode-solution surface (Wang 2000).

f dt dt

(Equation 2.4)
(where Q is charge in coloumbs, F is faradaic constant, and N is number of moles of
molecules being processed electrochemically)
The interface between the solution and the surface electrode also creates capacitive
current which obey equation 2.5:

.=42-CdV

l ..____.

C dt dt
(Equation 2.5)
where C is the capacitance and V is the potential difference between the surface electrode

and the solution. Therefore, the total current flow at the surface electrode-solution

34

 

interface is the summation of faradaic and capacitive currents. The capacitive current
however, can be eliminated by operating the electrode at a fixed potential (i.e., dV/dt=0).
An electrochemical cell and a potentiostat are the basic electrochemical
instruments required for a controlled-potential type of measurement (Wang 2000). The
electrochemical cell is typically made up of a beaker of 5-50 ml volume and three
electrodes, namely working, reference, and auxiliary electrodes. The reference electrode
(commonly saturated calomel or a silver/silver chloride) provides a stable and
reproducible signal against which the signal of the working electrode is compared. The
auxiliary electrode, which is often a platinum wire, transfers input signal generated from
the potentiostat to the electrochemical cell. The working electrode, typically made from
carbon, gold, platinum, indium tin oxide (ITO) or Pani, is the electrode at which the
reaction of interest occurs. In this study, the working electrode consists of self-doped
Pani coated on an ITO glass. An ITO glass is chosen as part of the working electrode due
to its structural flexibility to bind directly with biological elements (Fang, Ng et a]. 2003),
stable electrical property with high density of charge carrier (Marks, Novoa et al. 2002),
and inexpensive production cost. Figure 2.8 shows a schematic of a three-electrode

electrochemical system (Wang 2000).

35

Input Amplifier

I/

Feedback

 

g
7

' Counter Electrode

   
 

 

 

’ ' ' Working

Referénce Elec ode

  

E,I (Output)
—>

 

Output

Figure 2.8: An electrical schematic of a three-electrode electrochemical cell (Wang
2000)

An electrochemical based biosensor is also known as conductometric,

amperometric, impedimetric or potentiometric biosensor depending on the type of

electrical properties being evaluated. Examples of electrochemical biosensors are

summarized below:
Conductometric biosensors

Conductometric biosensors measure the change in conductance of the biological
complex situated between electrodes (Gerard, Chaubey et al. 2002). Such biosensors have
been constructed for detection of glucose, urea, neutral lipid/lipase, and
hemoglobin/pepsin by monitoring the change in the electronic conductivity of the
polymer matrix (Contractor, Sureshkumar et al. 1994). Conductometric biosensors based
on conducting pelymer compounds are also developed for penicillin (Nishizawa, Matsue

et al. 1992), glucose, urea (Castillo-Ortega, Rodriguez et al. 2002), lipids, hemoglobin

36

(Contractor, Sureshkumar et al. 1994), bacteria and viruses (Kim, Cho et al. 2000;

Muhammad-Tahir, Alocilja et al. 2005; Tahir, Alocilja et al. 2005).

Amperometric biosensor

Amperometric biosensors measure the current or charge produced between the
electrode and the electrolyte by applying a constant potential value. The most important
factor affecting the functioning of amperometric biosensors is the electron transfer
between catalytic molecule and the electrode surface, which most often involves a
mediator or conducting polymer (Gerard, Chaubey et al. 2002). Various studies have
been done in recent years on the application of electrochemically grown conducting
polymer layers in amperometric biosensors. Enzymes or antibodies are either entrapped
within conducting polymer layers or covalently bound to functional groups (Harnmerle,

Schuhmann et al. 1992; Bartlett, Wang et a]. 1996; Rodriguez and Alocilja 2005).

Potentiometric biosensors

Little studies are done on potentiometric biosensors with enzymes or antibodies
immobilized within the polymer matrix (Gerard, Chaubey et al. 2002). This type of
biosensor normally utilizes the pH sensitivity feature of conducting polymer compounds
(Ratcliffe 1990) instead of the electrical conducting attribute. For example, polypyrrole’s
sensitivity to NH3 was used to produce a potentiometric biosensor for urea detection

(Pandey and Mishra 1988; Trojanowicz and Krawczyski 1995).
Impedimetric biosensors

Impedimetric biosensors, also known as capacitive biosensors, measure the

changes in the capacitance layer between electrodes upon binding of a biological element

37

 

 

to its receptor (J ian-Guo, Yu-Qing et al. 2004). Such biosensors are used for the detection
of whole bacterial cells (Radke and Alocilja 2005), enzyme (Myler, Collyer et al. 2005),
viruses and oligonucleotides (Davis, Nabok et al. 2005). For a whole cell detection, it is
demonstrated that an impedimetric biosensor based on a microelectrode array silicon chip
can detect as low as 104 colony forming units/ml of E. coli 0157:H7 grown in nutrient

broth (Radke and Alocilja 2005).

Sensing element-Antibodies

This section illustrates the use of antibodies as the biological elements in the
biosensor design. Antibodies are frequently used in biosensor research due to their high
specificity attributes (Sadana 2002) and are further explored in this research. The
specificity of antibodies is based on the immunological reaction involving the unique
structure recognition on the antigen surface and the antibody binding site (Barbour and
George 1997). The basic structure of all antibody or immunoglobulin molecules consists
of four polypeptides—two heavy chains and two light chains joined like a capital letter
"Y", and linked by disulphide bonds (Sadana 2002) (Figure 2.9). The Fc fragment
comprises the effector functions, such as complement activation and cell membrane
receptor interaction. The Fab fragment, on the other hand, comprises the antigen binding
sites. The amino acid sequence on the tips of the “Y” (antigen binding sites) varies
greatly among different antibodies. This antigen binding site is composed of 110-130
amino acids, giving the antibody its specificity for binding antigen (Barbour and George
1997). Typically, antibodies are prepared in the form of monoclonal or polyclona].
Polyclonal antibodies are produced by multiple clones of antibody-producing molecules.

Polyclonal antibodies recognize multiple epitopes or binding sites on the surface of the

38

 

antigen, making them more tolerant to the variability in antigen structures. Monoclonal
antibodies on the other hand, are those that are derived from a single clone antibody-
producing molecule and thus, react only to a specific epitope on the antigen
(CHEMICON International 2004). Because of their high specificity, monoclonal
antibodies are excellent to be used in immunoassay techniques. Studies also show that
result from monoclonal antibodies are highly reproducible between experiments
(CHEMICON International 2004). Monoclonal antibodies, however, are much more
vulnerable to epitope lose due to chemical treatment compared to polyclonal antibodies.
Additionally, monoclonal antibodies are more costly to produce (CHEMICON

Intemationa] 2004).

Antigen binding site

Fab

Light chain

   

Heavy chain

Figure 2.9: Antibody structure (Sadana 2002).

Antibody-based biosensors are usually fabricated by immobilizing antibodies onto
an electrode by way of adsorption (Schrieber, Feldbrugge et a1. 1997), covalent
attachment (N iwi, Y et al. 1993), electrostatic attachment (Liu, Liao et al. 2000; Grennan,
Strachan et al. 2003; Tang, Yuan et a1. 2004) or polymer entrapment (Sadik and Emon
1996). The optimization of biomolecule immobilization is a critical issue in the

performance of a biosensor. Most of the current immobilization methodologies, however,

39

are not at 100 % efficiency due to the limited immobilization capacity, partial loss of
bioactivity of the immobilized molecules (Bunde, Jarvi et al. 1998), cross-reactivity of
enzymes (Cheung, Stockton et al. 1997), nonspecific binding (analyte binding occurs at
places where it should not), and interference with the transducer elements (Scheller,
Hintscher et al. 1991). Figure 2.10 shows some possible antibody orientations after
immobilization procedures. During the immobilization process, antibody bindings may
happen on the Fc region (Figure 2.10a), on both Fe and Pb regions, or one of the antigen
binding sites (Figures 2.10b and 2.10c). Lu et al. (1996) emphasizes that if
immobilization occurs on the antigen-binding sites, the ability of antibody on the surface
to bind antigen may be lost completely or at least to a high degree, and thus affecting the

overall performance of the biosensor.

   

. Biosensor surface

£—

(a) Fully active (b) Inactive (c) Partially active
Figure 2.10: Potential antibody orientations after an immobilization process (Lu,

Smyth et al. 1996). Dotted arrows correspond to antigen binding sites.

Assay formats

Currently, there are four typical assay formats used in antibody based biosensors
(Lukosz 1991) (Figure 2.11): (a) direct binding assay, (b) sandwich assay, (c)

displacement assay, and (d) replacement assay.

40

(a) Direct binding assay: The antigen (Ag) in the solution binds directly to the
antibody (Ab) receptors immobilized on the surface of a biosensor. The Ab-Ag
complex is monitored.

(b) Sandwich assay: This system is used when low molecular weight Ag, such as
enzymes, is to be analyzed. This is because the binding between the Ag to the
primary antibody (Abl) induces only small change in signal to be measured.
After addition of the secondary antibody (Ab2), a much bigger complex of Ab]-
Ag-Ab2 is formed and detected. One of the drawbacks of this system is the need
for multiple washing steps required to remove all unspecific binding.

(c) Displacement assay: The analogue of Ag (denoted as Ag-(A)) is immobilized on
the surface of the biosensor. Then, the corresponding Ab is bound. Upon addition
of the Ag of interest, the Ab is displaced from Ag- (A) complex to bind to the
free Ag. In this system, a negative change in the signal is observed and measured.

((1) Replacement assay. In this system, Ab is immobilized on the surface of a
biosensor. An analogue of the Ag of interest, conjugated with a bigger molecule
(denoted as Ag-(AX)) is bound to the Ab. Upon addition of Ag of interest, the Ag
replaces Ag-(AX) to bind to the immobilized Ab. Similar with (c), a negative

change in the signal is measured.

41

533 6088293 A3 SEEN

EoEoomEWE 3 Sam? noguqam 3v “mac—HE 8th A3 ASE awe—nib whammeoasfifim .«o was.“ “:20wa ”:d 8sz

 

fit

 

 

g m<

 

 

 

 

 

42

Difficulties in biosensor fabrication and application

In essence, a biosensor should be highly sensitive, specific, inexpensive, user-
friendly and easily manufactured with high reproducibility. This section discusses some
of the limiting factors and difficulties in the biosensor research, especially those that are
related to conducting polymer based biosensors.

Variation from sensor to sensor is a common obstacle in the biosensor research.
An example of the aforementioned variation can be observed in the membrane based
conductometric biosensor designed for food pathogen and viral detection (Muhammad-
Tahir and Alocilja 2003; Muhammad-Tahir and Alocilja 2003; Muhammad-Tahir,
Alocilja et al. 2005; Tahir, Alocilja et al. 2005). Although the biosensor has a promising
performance in detecting a low level of antigen concentration, the design of the
conductometric biosensor however has a disadvantage: the platform of the
conductometric biosensor can only be used once due to the non-reversible properties of
nitrocellulose and cellulose membranes employed in the biosensor design. Due to this
reason, the calibration (control sample) and the sample testing cannot be performed on
the same biosensor platform. The inability to calibrate and test samples using the same
conductometric biosensor was elucidated to contribute to the variability observed
between experiments (Muhammad-Tahir and Alocilja 2004).

Another limiting factor in the biosensor design is the stability of the sensing
elements. Antibodies, enzymes and other biological sensing elements are subjected to
denaturization either during storage or when actually used. A typical lifespan of
antibodies is about two years if stored at 2-8°C in the recommended storage buffer

(VMRD 2005) The lifespan of antibodies incorporated in the biosensor design,

43

 

however, is usually much shorter than that in storage buffer. For example, a study
indicates that the average antibody based biosensor for clinical applications has a lifetime
of about a month (Paddle 1996). The ability in finding a suitable sensing element for the
target of detection also is documented as one of the obstacles in biosensor research
(Sadana 2002). In general, if an enzyme or antibody is readily available, then the
corresponding antigen maybe detected. The ability of the biosensor to be selective to the
detection target also depends on the type of the sensing element. Therefore, the
specificity of an antibody-based biosensor is as good as the specificity of the antibodies
incorporated in the biosensor design.

Biosensors utilizing conducing polymer compounds also have several limitations.
Due to the complex process involved in polymer formation, difficulties in obtaining
uniform and reproducible polymer film have been documented (West, Jacobsen et al.
1993). Therefore, variation in biosensor performance may be observed between one batch
of experiment to another. However, the ability to control the experimental condition
during the polymerization steps may ensure minimal variations between biosensors
(Sadik and Emon 1996). Additionally, conducting polymer compounds are also subjected
to degradation. A “normal” life cycle of conducting polymer sensor was reported to be 2-
10 weeks (Sadik and Emon 1996).

Studies also show that the nonspecific-binding (N SB) phenomenon has a negative
impact on the overall performance of a biosensor (Sadana 2002). The effect of non-
specific binding becomes more critical with a decrease in concentration of the target of
interest. There are two ways NSB may occur: NSB could occur directly on the biosensor

surface area where no antibody is present and also during an immobilization process

44

which may be due to the absorption of salts and preservatives (Sadana 2002). Moreover,
a nonspecific binding is proposed as one of the reasons of the ‘hook effect’ phenomenon,
where a pseudo parabolic curve instead of linear response is observed. A ‘hook effect’
response may potentially lead to a false negative analysis as a low signal response is
typically observed at both low and high antigen concentrations (Xu, Velasco-Garcia et al.
2005)

Another problem normally encountered in fabricating a biosensor is the
uniformity and the geometry of the biological receptor immobilized on the biosensor
platform (Sadana 2002). This is a very important aspect as it is affecting the
reproducibility of the biosensor performance. Ideally, the binding site of the receptor

should be accessible to the analyte with minimum hindrances possible.

45

CHAPTER 3: MATERIALS AND METHODS
3.1 Procedural flowchart
The procedures for fabricating ITO-Pani biosensors and amperometric

measurements are summarized below: '

 

Polyaniline preparation and characterization
(Sections 3.3 and 3.4)

l

ITO glass preparation /Spin-coating procedure
(Section 3.5)

l

Antibody immobilization (Section 3.6)

l i

 

 

 

 

 

 

 

 

 

 

 

 

 

Based on Liu et al., Based on Tang et al.,
2000 2004
-Electrode charging -Treatment with
-Antibody incubation nafion
-Antibody incubation

 

 

 

 

 

 

\ /

Before antibody-antigen binding measurement (Q0)
Antigen incubation (Section 3.8)

I

After antibody-antigen binding measurement (Q5)

 

 

 

 

 

 

 

 

 

 

 

 

46

3.2 Reagents and apparatus

Reagents

The reagents included emeraldine base with molecular weight of 65, 50, and 20 K
Dalton (Da), camphorsulphonic acid, methanol, chloroform, glutaraldehyde, nafion,
phenol, phosphate buffer pH 7.4 (PB), ITO glass, silver/silver chloride electrode
(Princeton Applied Research, TN), platinum electrode (CH instruments, TX), affinity
purified swine anti-BVDV polyclonal antibody (USDA: NADL, IA), fluorescein
isothiocyanate (FITC) labeled BVDV antibody (Veterinary Medical Research &
Development, WA), chromic-sulfuric acid solution (Ricca Chemical company, IL). All
other reagents and materials were purchased from Sigma-Aldrich (MO) unless otherwise ’

noted.

Apparatus

All electrochemical measurements were carried out using a VersaStat II
potentiostat (AMETEK Princeton Applied Research, TN) in the amperometric mode. A
three-electrode electrochemical cell (MSU Scientific Glassblowing laboratory, Michigan
State University, MI), consisting of a platinum counter electrode, a silver/silver chloride
reference electrode, and a conductive substrate (e.g., ITO) as working electrode in 0.1M

PB (Figure 3.1), was used together with the potentiostat.

Amperometric measurement

The amperometric measurement was conducted by first immersing the substrate
(ITO) in 45 m] of PB. Using the three-electrode electrochemical cell set-up, a fixed

potential of 0.5 Volts (V) was applied over 25 minutes. The theoretical value of the

47

faradaic current between the working electrode and the electrolyte was obtained by

performing a linear regression analysis on the charge-versus-time experimental data.

'7

 

 

 

 

 

 

Reference
Electrode
(Ag/AgCl) —‘
Counter Electrode ,- :2; :::§::::::;:::;E§§§
(Platinum wire) E_E_§_i_f§g§g§§§§§§§§: \ Working
E:§:§:§:§:§:§:§:§:§:§:3:}: electrode
0.1 M Phosphate____ Effgifffffiéfififif: __________________________ (Biosensor)

buffer 3:3:3:32:23:3:3:323:3?:23:3:3:3:3:3:3:3:3:3:3:3:3:

 

 

 

Figure 3.1: Apparatus for amperometric response measurement.

3.3 Pani preparation

Self doped and non self doped Pani

Self doped Pani was prepared by doping emaraldine base with camphorsulfonic
acid (Cao, Smith et a1. 1993; Duic, Mandic et al. 1994; Cao, Qiu et al. 1995). Each of the
commercially available emaraldine base compound was doped with camphorsulfonic acid
in the ratio of 1.0 p-phenyleneimine (C61-14N) unit per 0.5 mol doping acid. Emeraldine
and camphorsulfonic acid were mixed using a mortar and pestle in a nitrogen
environment. The mixture was dissolved in chloroform to give a final concentration of
0.5 % by weight. To improve processability of Pani, phenol, in a ratio of 0.5 mol per p-
phenyleneimine, was added. The mixture was stirred for 15 minutes before being filtered

through a 0.45 pm filter paper. The solution was then stirred for 72 hours before further

48

used. A commercial non-self doped Pani with a molecular weight > 15 K Da was
purchased from Sigma-Aldrich (M0).

3.4 Pani characterization

Conductivity measurement

A mixture of each self doped Pani compound dissolved in chloroform was air-
dried. The dried Pani was compressed into pellets and used to measure the conductivity

of the polymer by using a four-point—probe (Signatone model 8-301, CA).

Thermogravimetric measurement (IIZG)

Thermogravimetric analysis measures weight changes in the polymer as a
function of temperature (or time) under a controlled atmosphere. The device was
operated based on the manufacturer’s instruction (TGA Q500, TA Instruments, DE) to

assess the TG analysis on Pani.

Transmission electron microsco E and atomic force microsco AF

ana 868

A transmission electron microscope (JEOL 2100FEF 200 kV field emission,
Center for Advance Microscopy, Michigan State University) and an AFM were used to
inspect the physical morphology of Pani and the biosensor. No pretreatment was
performed prior to the analysis.

3.5 Fabrication of an ITO-Pa_ni biosenfl

Indium tin oxide glass with a dimension of 1.27 cm X 2.54 cm was used as the

biosensor platform. The ITO glass was first washed with methanol/chloroform (1:1)

before being soaked in ammonium hydroxide and air-dried (Ram, Salerno et al. 1999). A

49

100- u] of Pani solution (Section 3.3) was used to spin-coat a thin layer of polymer on the
ITO platform at the speed of 500 rpm for 6 seconds. The substrate was left to air-dry and

referred to hereafter as ITO-Pani substrate.
3.6 Antibody immobilization

Liu et al., 2000’s method

Using the three-electrode cell and the potentiostat, a negative potential (-0.5 V)
was applied to the ITO-Pani substrate for 25 minutes. Forty-five ml of PB was used as
the electrolyte. After applying the potential for 25 minutes, the substrate was removed
from the cell and then immersed in 1 ml of solution mixture containing the antibody, 1%
glutaraldehyde, and PB, in a voltune ratio of 2.520.521. The substrate was left in the

antibody solution at room temperature for 30 minutes (Liu, Liao et al. 2000).

Tang et al., 2004’s method

The ITO-Pani substrate was treated with 1 m] of 5 % (v/v) nafion prepared in
ethanol for 5 minutes. The resulting substrate was thoroughly rinsed with distilled water
to remove excess nafion. Then, a 100-ul of antibody solution was applied onto the ITO-
Pani-nafion substrate. The resulting platform was incubated at 4° C for 10 hours,
followed by thorough washing with PB and air-drying (Tang, Yuan et al. 2004).

3.7 Confirmation of antibody immobilization

The presence of antibody on the ITO-Pani substrate was confirmed by repeating
the above immobilization procedures using fluorescein isothiocyanate (F ITC) labeled
antibodies. A confocal florescence microscope (a Zeiss Pascal, Center for Advance
Microscopy, Michigan State University) was used to visualize, measure the intensity of

the emitted fluorescence, and confirm the antibody immobilization.

50

 

3.8 Detection and incubation time

Detection process was conducted by 1) calculating the amperometric response
(section 3.2) after antibody immobilization, 2) incubating the biosensor from (1) with 1
ml of BVDV culture and 3) calculating the amperometric response of the biosensor from
(2). In this study, the incubation time was varied for 10, 15, and 30 minutes.
3.9 Viral culturing

A characterized strain of noncytopathic BVDV 890 from the collection of the
Department of Large Animal and Clinical Sciences (Veterinary Medical Center,
Michigan State University) was grown in bovine turbinate cells in Eagle’s minimum
essential medium (EMEM) broth containing 10% fetal equine serum for 5 days at 37 °C
and 5% C02. The culture was frozen at —80 °C for 1 hour to break apart the cells and
release the mature virus. Then, the viral culture was centrifuged at 13000 rpm for 10
minutes. The culture supernatant containing approximately 10‘5 CCID/ml of BVDV was
used as an antigen solution for the subsequent experiments.
3.10 Viral confirmation

A duplicate sample of each serial dilution was used to determine the concentration
of the virus in culture supernatant. Each dilution was inoculated quadruplicate onto the
turbinate cells in a 96-well plate. After incubating the inoculated cells for 5 days at 37°C
and 5% CO2, the cells were stained for viral infection using the immunoperoxidase assay
(Meyling 1984). Virus titer (the number of infectious units per unit volume) was

determined by the standard method (Carbrey, Brown et a1. 1971).

51

3.11 Sensitivi testin of the biosensor in BVDV ure culture

   

A series of 1:10 dilutions of culture supernatant prepared from section 3.9 was
used to determine the sensitivity of the ITO-Pani biosensor. Analysis of variance was
conducted to determine the detection limit of the biosensor at 95% confidence level.

3.12 Specificity testing of the ITO-Pani biosensor

In order to determine the specificity of the biosensor, an ITO-Pani biosensor
prepared with antibodies specific to BVDV antigen was tested in IBR virus culture. The
IBR virus was prepared as described in section 3.9 and virus titer was done by
performing a standard enzyme immunoassay procedure (V an-Oirschot 2000). The
biosensor was also used to test a mixed sample of viral cultures. The mixed culture
sample was prepared by mixing 1 ml of IBR virus culture to 1 ml of BVDV culture. The
mixture was vortexed for 30 seconds. A series of 1:10 dilutions of the mixture was
performed and used to test the biosensor. Analysis of variance also was conducted at 95%
confidence level.

3:13 Reusability testing

The used biosensors were treated with the chromic-sulfuric acid solution to
remove the coated Pani, cells, and antibodies. The conductivity of the acid-treated
substrate was measured and compared with the unused ITO substrate. In addition, a
scanning electron microscope (SEM) (JEOL 6300F, Center for Advance Microscopy,

Michigan State University) was used to evaluate the morphology of those ITO substrates.

52

 

CHAPTER 4: RESULTS AND DISCUSSION

PAPER 1: FABRICATION OF INDIUM TIN OXIDE-POLYANILINE

BIOSENSOR

4.1 Characterization of polyaniline
Sergeyeva et a1. (1996) reported that the conductivity of the non-self doped Pani

(Pani chemically polymerized with hydrochloric acid) linearly increased with increasing
molecular weight from 10 KDa up to 45 KDa. The conductivity of 100 KDa of the same
Pani, however, was reported to be lower than that of 45 KDa (Sergeyeva, Lavrik et al.
1996). Owing to this result, three commercial emeraldine base compounds were self
doped with camphorsulfonic acid: two with molecular weights larger than 45 KDa (65
and 50 KDa) and one lower than 45 KDa (20KDa). Figure 4.1 shows the conductivity of
the self and non-self doped Pani compounds using a four-point probe meter. The self
doped Pani with the 65-KDa molecular weight has the highest conductivity at 1.5
Siemens (S)/cm, followed by the 50 (0.44 S/cm) and the 20 (0.36 S/cm) KDa Pani
compounds. The conductivities of these self doped Pani are within the range of
conductivity levels found in a previous study (Lukachova, Shkerin et al. 2003) and are
also reported to be increasing with increasing molecular weight. The latter phenomenon
is further evaluated in the next couple of experiments.

Interestingly, the commercially-available non-self doped Pani has a higher
conductivity level (6.7 S/cm) even though its molecular weight (~15 KDa) is smaller than
the self doped compounds (Figure 4.1). This finding may be due to the different doping

acids used in the Pani polymerization process (Duic, Mandic et al. 1994; Stejskal, Riede

53

 

et a]. 1998). As described earlier, the self doped Pani was doped with camphorsulfonic
acid while the commercially-available non-self doped compound was doped with a
proprietary organic acid. This proposition is further observed in the subsequent

transmission electron microscope (TEM) analysis.

 

8-
7a

 
 

Conductivity (Slcm)
—- N w a OI or

 

O
1

Self doped-50 Self doped-65 Non self-doped-
KDa KDa KDa 15 KDa

 

 

 

Figure 4.1: Conductivity of Pani compounds.

A transmission electron microscope (TEM) was used to study the morphology of
Pani compounds (Figure 4.2). The TEM images show that the higher the molecular
weight of the self doped Pani, the larger is the polymer structure. For instance, the 65-
KDa self doped Pani is approximately 12pm in length (Figure 4.2c), while the 20 KDa
Pani is only 2pm (Figure 4.2a). Additionally, Figure 4.2 also shows that the self doped
Pani compounds have more globular shapes and are smaller than the non-self doped Pani.
The non-self doped Pani is shown to have a non-uniform structure with an approximate
length of 100nm. Duic et al., (1994) and Stejskal et al., (1998) also observed differences
in size and shape of Pani when using different types of doping acids in their

polymerization processes.

54

 

(C) ((0

Figure 4.2: TEM images of a) 20 KDa, b) 50 KDa, and c) 65 KDa self doped Pani;

and d) non-self doped Pani.
The conductive property of Pani was further evaluated by measuring its
amperometric response. Both the self and non-self doped Pani compounds were spin-

coated on the ITO substrate and tested for their amperometric responses using the

55

 

electrochemical set-up previously described in section 3.2. These substrates are referred
to as ITO-self doped and ITO—non self doped substrates hereafter. Figure 4.3 shows the
amperometric response of the ITO-self doped substrates in phosphate buffer (PB). The
ITO coated with the 65 -KDa of self doped Pani results in the highest amperometric signal
at 530.52 uA. This is then followed by the ITO glasses coated with 50KDa (355.78 uA)
and 20 KDa (105.13 uA) of self doped Pani (Figure 4.3). Similar observation was also
found in the earlier experiment (Figure 4.1). Therefore, it is noteworthy to conclude that
the heavier the self doped Pani, the more conductive the polymer becomes. As the
molecular weight of the polymer increases, the length of the polymer’s backbone per unit
area may potentially be increasing as well (Ryu, Chang et al. 1999), and thus enhancing
the flow of electrons and subsequently increasing the conductivity of the polymer.

Similar phenomenon was also reported by Sun Ryu et al. (1999).

 

700. 00 -
600.00 —

500.00 -

co .5
o o
.0 9
o o
o o
1

200.00 -

Amperometric response (uA)

100.00 .

   

 

0.00 4
Self doped- 20 KDa Self doped- 50 KDa Self doped- 65 KDa

 

 

 

Figure 4.3: Amperometric responses of ITO -self doped Pani compounds.
Thermogravimetric (TG) analysis was conducted to study the effect of
temperature on the Pani weight. Figure 4.4 shows the changes in the weight loss of the

self doped Pani (65 KDa) after exposing the compound to temperatures ranging from

56

22°C to 300°C. Similar trend was also observed when testing the 50 and 20 KDa of Pani
compounds. Figure 4.4 shows a steep rate of weight loss change in regions A (22°C to
75°C) and B (210°C to 300°C), and a slight increase rate of change in region C (75°C to
210°C). From this finding, it is optimal to use the Pani between the temperature levels of
75°C and 210°C (region C) since a temperature fluctuation within this region leads to
only a small change in Pani weight loss (Figure 4.4). Since it was concluded earlier that
the weight of Pani affects the conductive property of the polymer, the use of Paul in
region C ensures minimal changes in the polymer conductive property. Temperature
levels at this region, however, are too high for any biological elements in a biosensor
design, such as antibodies, to be functional. An antibody thermal stability study
concluded that a heat treatment at 60°C resulted in the cleavage of the antibody heavy and
light chains and promoted the denaturation of the protein (Alexander and Hughes 1995).
For this reason, most biosensor operations are conducted in a room temperature (~ 25°C)
(Radke and Alocilja 2005; Rodriguez and Alocilja 2005). Since the Pani weight is
sensitive to temperature changes between the temperature levels of 22 °C and 75°C
(region A), a temperature-controlled mechanism needs to be introduced to the biosensor

design to minimize temperature-dependent variations in the polymer properties.

57

 

 

(a)
O

l-I-I-l-l-o-c--I-o-l-

 

 

%weightloes
.A _|. N N
o 01 o 01 o O:
l l 1 l l

 

 

200 250 300
Temperature (“0)

o
s
s
s

 

 

 

Figure 4.4: Percent of weight loss of Pani in varying temperature level.

The pH-dependency of Pani compounds was also conducted in this study by
testing the self and non-self doped Pani compounds in electrolytes with different pH
levels: PB at pH 7.4 and 1 M hydrochloric acid at pH 1.0 (Figure 4.5). Results show no
statistical difference in responses between the ITO-self doped Pani (50 KDa) tested in
PB, and that in 1 M hydrochloric acid. This finding suggests no changes in the
conductivity level of the self doped Pani from a highly acidic to a near neutral pH
environment. A significant difference in responses however, was observed between the
ITO-non self doped Pani in PB and that in 1 M hydrochloric acid. Though the above
substrate resulted in a high amperometric response (3400.16 uA) in 1M hydrochloric
acid, the ITO-non self doped Pani was observed to be not conductive in PB (Figure 4.5).
Additionally, this ITO-non self doped Pani substrate was observed to be at least 10 times
higher than that of the 50 KDa self doped Pani (355.78 pA) in 1 M hydrochloric acid.
Though the reason for this is not yet stipulated, the difference in types of acids used in the

doping process could be a factor for the difference in the conductivity level of the two

58

tested Pani compounds. A study reported that different anions present in a doping acid
influenced the conductivity, solubility, and other chemical characteristics of Pani (Plesu,
Iliescu et al. 2005; Gok, Sari et al. 2006). Nevertheless, this finding confirms that the
conductivity of the non-self doped Pani depends highly on pH levels and therefore, is not
suitable to be used in a neutral pH environment where most immunological reaction
occurs optimally (Garjonyte and Malinaiuskas 2000). Due to this reason, the self doped

Pani compounds were chosen to be incorporated in the subsequent biosensor fabrication.

 

 

4500.00

... 4000.00 - a pH 7.4
3500.00 - ' PH 1-0
3000.00 -
2500.00 -
2000.00 ~
1500.00 ~
1000.00 ~
500.00 «
0.00 -

 

 

 

 

 

Amperometric response (uA

   

 

 

 

 

Self doped- 50 KDa Non self-doped Pani

 

 

 

Figure 4.5: Amperometric responses of ITO substrates coated with non-self doped
Pani and 50 KDa self doped Pani in two pH controlled electrolytes.
4.2 Concept of detection
The ITO-Pani biosensor uses a direct antibody-antigen binding format with the
self doped Pani as the transducer. By using the three-electrode electrochemical setup
(section 3.2), the input signal is transferred from the auxiliary electrode to the working
electrode by the ionic charges forming in the electrolyte solution. When a fixed potential

is applied, electrons are allowed to flow freely from the auxiliary electrode to the ITO-

59

 

Pani substrate (working electrode) due to the conductive property of Pani and ITO
substrate (Figure 4.6). When proteins (e.g., antibodies with a molecular weight of 150
KDa), are immobilized within the polymer backbones, electron flows are restricted. This
phenomenon could be caused by the insulating protein membrane, interfering the transfer
of electrons within the polymer rt-backbone (Kim, Cho et al. 2000). The electron flow is
restricted even more when a bigger antigen-antibody complex (molecular weight of
BVDV is at least 4 MDa) present within the Pani backbone. It is here hypothesized that
the bigger the protein complex present in the Pani backbone, the more restricted is the
flow of electron.

A potential of between 0.2 V and 0.8 V was demonstrated to be a sufficient input
signal, especially when dealing with whole cells or biological elements (Cattaneo, Luong
et al. 1992; Darain, Park et al. 2003; Tsiafoulis, Prodromidis et al. 2004). Therefore, in
this study, a constant potential of 0.5 V was chosen arbitrarily as the input signal.

The biosensor detection concept is based on the difference between the signal
before (1°) and the signal after (13) antibody-antigen binding (Figure 4.7). This current
drop (AI) is expressed mathematically as follows:

A I= 1° —18
Theoretically, the higher the current drop between 1° and Is, the more antibody-antigen
complexes are formed on the biosensor surface, blocking the transfer of electrons.
Therefore, the value of current drop (AI) should be increasing with increasing antigen

concentration.

60

 

 

Non-restricted electron flow

A
y

 

   

Ant' gen-antibody

 

Restricted electron flow

 

Semi-restricted electron flow_

 

Figure 4.6: Concept of detection ( —> Direction of electron flow).

61

Pani

       

       
      
     
 

  
     

   

      

a??? Antrbody g??? 2 3553?
Electrode g??? Aft 'b d . 22321 (31153.:
substrat—Te 3??? er anti 0. y-antrgen a??? km.
”a ' blndlng ””1 ..;.;._
(”I ”M ’2-
’,” ”I. /.-.-.
a” ~ ~ —> 'Il « riff.‘
”” p’ ,
a» a”.
559$ as" :-:-:-
”I! u»: ‘2'
as: are -:-:-
III! fill: v I .
”M
”I ”It. . <
a ”M »
”M ’4-
3”; ..4.-:-:-
(III:

   

Figure 4.7: Schematic of an ITO-Pani biosensor before and after antibody-antigen
binding.

4.3 Fabrication of ITO-Pani biosensor

The ITO-Pani biosensor consisted of two components: immunosensor and an
amperometric measuring device (Figure 4.8). The immunosensor was constructed from
an ITO glass, and layered with Pani and antibodies. The amperometric measuring device
is described in the previous section. Indium tin oxide glass is a common substrate used in
an amperometric biosensor due to its structural flexibility to bind directly with biological
elements (Fang, Ng et al. 2003), stable electrical property with high density of charge
carrier (Marks, Novoa et al. 2002), and inexpensive production cost. In this study, an
ITO glass substrate with a dimension of 1.27 X 2.54 X 0.1 cm3 and a resistance value

ranging fiom 15 to 25 ohms were prepared for biosensor fabrication.

62

Platinum \x'trc ; "
(Counter ,,_ ,f
clcctrudcl\T\:f \ym-kinu

: A/L‘ICCII’UtIL‘

Ag Agt'l
(Reference
clcclmtlc)

 

Figure 4.8: ITO-Pani biosensor.

To fabricate the biosensor, the ITO substrate was first cleaned and treated with a
strong oxidizing agent, ammonium hydroxide, to enable the adherence of Pani to the ITO
substrate (Ram, Salerno et al. 1999). Then, the self doped Pani was layered onto the ITO
substrate using a spin coating method. Spin coating method was chosen because of its
ease of use, rapid processing time, reproducibility and is inexpensive compared to other
types of polymer coating mechanism, such as Langmuir-Bloggert technique (Rubner
1991) or layer by layer molecular deposition (Ferreira and Rubner 1995). With a speed
of 500 rpm for 6 seconds, a thickness of 324.62, 134.2, and 91.05 nm were observed on
the ITO substrates coated with 65, 50 and 20 KDa Pani, respectively (Figure 4.9). After
the coating procedure, the substrate was then functionalized into a biosensor by
immobilizing antibodies onto the surface. A three-electrode electrochemical cell was

used to charge the substrate by applying a small (0.5 V) negative potential. This step was

63

essential to promote electrostatic bonding between the negatively charged substrate and
the NIT site of the antibodies (Liu, Liao et a1. 2000). The use of a divalent crosslinker,
glutaraldehyde, also helped facilitate the antibody binding mechanism (Irina, Sanchez et
al. 1983; Diao, Ren et al. 2005).

The successful fabrication of the biosensor was evaluated by using an atomic
force microscope (AF M). Figures 4.9-4.14 show AFM images of an ITO-Pani biosensor
prepared with 20, 50, and 65 KDa Pani, functionalized with antibodies, and incubated
with 104 and 10° CCID/ ml of BVDV. An increase in height in the z direction (thickness)
between plain ITO (Figure 4.9) and ITO-Pani substrates (Figure 4.10) indicates
successful polymer coating. Figure 4.10 also shows that the higher molecular weight Pani
formed a thicker layer on the ITO substrate. Similarly, an increase in thickness was
observed between Figure 4.10 and Figure 4.11 where each of the ITO-Pani substrate was
immobilized with antibodies, suggesting successful antibody immobilization process.
More importantly, Figures 4.12-4.14 show the AF M images of the biosensor surface after
incubating them in the BVDV culture. A thicker substrate was observed when incubating
the biosensors with the higher concentration of BVDV supporting the logical
phenomenon that the higher the antigen concentration, the more antibody-anti gen
complex occurs. However, when the biosensors were tested with the same level of
BVDV concentration (e.g., 10° CCID/ml), biosensors coated with the higher molecular
weight Pani (e.g., 65 KDa) were observed to be thicker than that of the Paul with a lower
molecular weight (e.g., 20 KDa) (Figures 4.12a and 4.14a). This finding shows that the
higher the molecular weight of Pani, the more antibody binding sites is available. This

proposition is further evaluated in the next subsequent experiment.

64

 

 

 

~ 100.0 nm

50.0 nm

  

0.0 nm

 

Average thickness in z direction (a) 22.86 nm (b) 63.34 mm

 

 

Figure 4.9: Atomic force microscopy images of a) untreated ITO glass and b) ITO

glass treated with ammonium hydroxide (NH40H).

 

 

.500.0 nm

~i'250.0 nm

  

0.0 nm

 

Average thickness in z direction a) 324.62 nm, b) 234.2 mm, c) 91.05 nm

 

 

Figure 4.10: Atomic force microscopy images of a) ITO glass treated with (NI-14011)

and spin coated with 65KDa, b) 50KDa, c) 20KDa Pani.

65

 

 

Average thickness in z direction a) 344.62 nm, b) 228.1 mm, c) 122.05 nm

 

 

 

Figure 4.11: Atomic force microscopy images of a) ITO glass spin coated with Pani

(65KDa) + ab, b) Pani (50 KDa) + ab, and c) Pani (20 KDa) + ab.

 

, 1000. 0 nm

 

0.0 nm

 

Average thickness in z direction a) 524.62 nm, b) 345.1 nm

 

 

Figure 4.12: Atomic force microscopy images of a) ITO-Pani biosensor (65KDa)

tested with 10°CCID/ml and b) 10°CCID/ml of BVDV.

66

 

 

,, 1000.0 nm

500.0 nm

 

0.0 nm

 

Average thickness in z direction a) 364.22 nm b) 278.1 nm

 

Figure 4.13: Atomic force microscopy images of a) ITO-Pani biosensor (50KDa)

tested with 10°CCID/ml and b) 104CCID/ml of BVDV.

 

 

.500.0 nm

  

10.0 nm

 

Average thickness in z direction a) 267.11 11111 b) 145.1 nm

 

Figure 4.14: Atomic force microscopy images of a) ITO-Pani biosensor (20KDa)

tested with 10°CCID/ml and b) 104CCID/ml of BVDV.

67

 

 

To elucidate the use of Pani as a mediator or a “glue” for the binding of antibody
on the biosensor platform, ITO glasses undergoing A) the fabrication process from
sections 3.5 and 3.6 and B), the same fabrication process without the Pani spin-coating
step, were tested for their amperometric responses (Figure 4.15). Depending on the
fabrication process, the substrate is referred to as substrate A or substrate B thereafter.
Figure 4.15 (A) shows a significant difference between (1°) and (IS) for substrate A. The
(1‘5 and (1‘) responses for substrate B, on the other hand, are statistically insignificant
between each other. The latter finding is proposed to be caused by the absence of Pani on
the ITO substrate which may contribute to the lack of antibody binding, and subsequently
to the insignificant responses between (1°) and (Is). To investigate this theory further, the
presence of antibodies on both substrates was confirmed by repeating the above
experiment with flourescent-tagged antibodies. Results show that a much higher
fluorescence emission level (250 out of 256-bit color mode) was observed from substrate
A than that from substrate B (20 out of 256-bit color mode). This finding implies that
more antibodies are present from substrate A than substrate B. Therefore, in this study, it
can be concluded that Pani is not only required as the biosensor transducing system but
also as a mediator for the antibody binding. Kim et a]. (2000) also demonstrated the use
of Pani as a mediator between the antibodies and gold electrodes in his Pani—based
biosensor.

The finding shown in Figure 4.15 (A) also supports the theory of detection
described earlier in section 4.2. An increase in current response was observed after an
ITO substrate was coated with Pani. Then a decrease in current response was observed

after antibody immobilization, suggesting the reduction in the electron flow that could be

68

due to the insulating property of antibodies. A much higher drop in current response was
observed after the antibody-antigen binding. This result supports the proposition made
earlier that the bigger the protein molecules present on the surface of the biosensor, the

lower the flow of electrons and thus the smaller the signal response.

 

 

 

I—6—Substrate B +Substrate A I
600 —
500 e
400 - A
2
5 300 ~
_
200 -
100 —« / B

 

 

 

 

0 Pretreatment Polyaniline Before Ag After Ag
Coating Binding (|°) Binding (IS)

Different fabrication stages
Figure 4.15: Amperometric responses of A) substrate A (ITO biosensors consisted

of self-doped Pani and B) substrate B (ITO biosensor without Pani coating).

69

 

PAPER 2: PERFORMANCE OF THE INDIUM TIN OXIDE —POLYANILINE

BIOSENSOR FOR BOVINE VIRAL DIARRHEA VIRUS DETECTION

4.4 Experimental condition analysis

Before assessing the biosensor performance, experimental conditions, such as
Pani size, antibody concentration, antibody immobilization method, and antigen
incubation time were evaluated. The varying parameters of the experimental conditions
are shown in Table 4.1. Temperature setting and measurement time were set at room
temperature (~25°C) and 25 minutes, respectively. Biosensors prepared with the
following parameters were tested for the detection of 10° CCID/ml of BVDV culture
using the three-electrochemical setup.

Table 4.1: Experimental condition parameters evaluated for the ITO-Pani biosensor.

 

 

Molecular weight of Paul compounds (K Da) 65, 50 and 20
Antibody concentration (mg/ml) 1, 0.5, 0.25, and 0.15
Method of antibody immobilization Based on Liu et a1.,
2000 and Tang et al.,
2004
Incubation time (minutes) 10, 15, and 30

 

A set of 72 experiments was conducted representing a combinatorial of all
parameters shown in Table 4.1. A representative response of the biosensor is shown in
Figure 4.16, where the charges before (Q°) and after (Q5) the antibody-antigen binding
measured over time was recorded. A linear regression was performed on all the
experimental data with the following equation:

Y (Charge) = slope*X (time) + Y-intercept

(Equation 4.1)

70

Only the scenarios with a regression coefficient (r-squared) and Shapiro-Wilk statistic
(W) greater than 0.98 were chosen for further analysis. Since current is the rate of charge
flowing over time, the theoretical values of 1° and I5 were obtained by calculating the
slope of the fitted curve. The duration of time at which the charge is measured is not
critical as the slope can be evaluated by using any three points on the curve. With the
ability of the potentiostat to record 10 data points in a second, the minimum Q° and Q5
values required to obtain 1° and IS can be done in a matter of seconds. In this study,

however, the charge was allowed to accumulate for 25 minutes (1500 seconds).

 

0.5 4
0.45 . Before antibody-antigen binding Q°

0.4 - \

y = 0.0003x - 0.0045

0.35 « ,
R=0ww

.9
DJ
1

0.25 —
After antibody-anti gen binding

QS
y=3EOSx+0.0007 /

O

' .0
N
1

Charge (Columbs)

0-1 ‘ R2 =0.9998
0.05 i
0 I I I I I I

-0.05 9 200 400 600 800 1000 1200 1400
Tirm (seconds)

 

 

 

 

 

Figure 4.16: Typical response of Q° and QS over time.
For each set of combination, the difference between 1° and I5 was analyzed statistically to
evaluate which parameters resulted in the best response at 95% confidence level.
Statistical analysis shows that only biosensors prepared using Liu‘s method produced I5
that is significantly different than the 1°. The 1° and Is for the biosensors prepared with
Tang’s method 'were not significantly different. The presence of antibodies on the ITO

glass was then confirmed by repeating the immobilization process with fluorescent-

71

tagged antibodies. A fluorescent intensity level of 256 out of 260 bit color mode were
observed with Liu‘s method, while less than 1% of the total emitted fluorescence was
found on the biosensors treated with Tang’s method. Evidently, this finding reveals that
more antibodies were successfully immobilized on the ITO substrate via Liu‘s method.
While Liu’s method consists of both the electrostatic (negatively charged substrate and
NH+ site of the antibodies) and covalent (from glutaraldehyde) attachments, Tang’s
method only employs the electrostatic binding (SO3'l from nafion and NH+ site of the
antibodies). As demonstrated in this study, the incorporation of glutaraldehyde as a
secondary binding method certainly helps to promote more antibody binding on the ITO
glass. Although nafion, a perfluorinated sulfonated cation exchanger, was used to bind
antibodies in the study by Tang et al., (2000), this method resulted in a poor antibody
binding in this study. The use of nafion to immobilize antibodies was successfully done
only on metal electrodes, such as gold (Tang, Yuan et al. 2005), glass carbon (Dong,
Wang et al. 1992), and platinum (de Mattos, Gorton et al. 2003) and not on the polymer
substrate. The application of nafion on a Pani coated substrate, such as the one used in
this study, posses a much more complex chemical structure than that of metal, and thus
certainly need to be further investigated. It is also noteworthy to realize that there is no
blocking reagent involve in both antibody immobilization methods. Though the use of
blocking reagent, such as casein, helps in minimizing the occurrence of non-specific
binding (Sergeyeva, Lavrik et al. 1996; Kim, Cho et a]. 2000), the use of this blocking
reagent may potentially affect the conductivite property of Pani.

Different molecular weight of self doped Pani compounds were tested in this

study. Based on the data found in sections 4.1 and 4.3, Pani with 65 KDa molecular

72

weight resulted in the highest conductivity. When testing biosensors prepared with this
type of Pani with varying antibody concentrations, biosensors prepared with 0.5 mg/ml of
antibody concentration resulted in the highest signal response at 247.9 uA. This then was
followed by the biosensors prepared with 0.25 (113.3 uA) and 0.15 (61.3 uA) mg/ml of
antibody concentrations (Figure 4.17). The linear relationship between antibody
concentration and molecular weight of Pani in this region is expected since the fewer the
concentration of antibody immobilized on the substrate, the lower antigen binding is to be
found. Therefore, the lower the amperometric response is to be generated. Interestingly,
biosensors prepared with 1 mg/ml of antibody concentration were observed to have the
lowest A] response (26.3 uA) than all the tested antibody concentrations (Figure 4.17). A
high concentration of antibody (e.g., 1 mg/ml) could saturate the binding sites on the Pani
backbone. The presence of these “extra” antibodies could inhibit the electron flow and
therefore resulted in a low AI response.

Figure 4.18 shows the response of the biosensor prepared with 50 KDa of Pani
with an increasing antibody concentration. A linear relationship between the antibody
concentration and the molecular weight of Pani was observed (Figure 4.18) as the most
A] response was generated by the biosensors with 1 mg/ml (55.4 uA), and then followed
by the biosensors with 0.5 (13.9 uA), 0.25 (9.4 uA) and 0.15 (7.0 uA) mg/ml of antibody
concentration. Biosensors prepared with 20 KDa of the self doped Pani, however, show a
significant response between 1° and IS when using only the 1 mg/ml of antibodies. The
rest of the tested antibody concentrations resulted in insignificant responses (Figure
4.19). Since the self doped Pani is speculated in the previous section to act as “glue” for

the binding of antibody on the biosensor surface, it can be concluded here that there is an

73

 

interesting relationship between antibody concentration and the molecular weight of Pani.
Evidently, a more thorough study needs to be conducted to better understand the
mechanism behind this relationship in order to fully optimize the performance of the

biosensor.

 

300

 

250 ~

M)
N
8

Mean of AI (
5 3.
O O

 

 

 

1 0.5 0.25 0.15
Antibody concentrations (mg/ml)

 

 

 

Figure 4.17: Response of biosensors prepared with 65 KDa Pani and varying

antibody concentrations.

74

 

 

Mean of AI (pA)
8

 

 

 

1 0.5 0.25 0.15
Antibody concentrations (mg/ml)

 

 

 

Figure 4.18: Response of biosensors prepared with 50 KDa Pani and varying

antibody concentrations.

 

L»
O

 

Mean of AI (M)
I — -‘ N N
LII O U! 0 UI 0 Ln
1 1 LL 1 I 1 l

 

 

 

 

I
.—
O

 

 

Antibody concentration (mg/ml)

Figure 4.19: Response of biosensors prepared with 20 KDa Pani and varying
antibody concentrations.
The detection time of the biosensor depends on the time required to incubate the

biosensor and the time to measure the Q° and Q5. Statistical analysis shows that only

75

biosensors incubated for 30 minutes resulted in the I5 value that is statistically different
than the 1°. The 10 and 15 minutes of incubation times were inadequate to produce
significant responses (Appendix 1). The time for measuring Q° and Q, on the other hand,
varies from a few seconds to infinity. In this study, the detection time of the ITO-Pani
biosensor was 55 minutes: 30 minutes for incubation time and 25 minutes for measuring
the Q° and QS responses. The detection time, however, can be shortened to only 30
minutes by minimizing the time to measure the amperometric responses.

A smnmary of the data with mean AI is shown in Table 4.2. Among all the
parameter sets evaluated, parameter set 2, which is the biosensors prepared with 65 KDa
of Pani, 0.5 mg/ml of antibody concentration immobilized using Liu’s method, and
antigen incubation time of 30 minutes yielded the highest A1 of 248 uA and thus, chosen
as the standard parameter in the subsequent sensitivity and specificity analyses.

Table 4.2: Summary of data (complete data is in Appendix 1).

 

 

Parameter Molecular Antibody Mean of A1 (pA) StdErr (uA) P value
set Weight of concentration
Pani (KDa) (mgml)
1 65 1 26.2966 1.7349076 0.0001
2 65 0.5 247.9368 7.6728816 <.0001
3 65 0.25 113.25 5.2932384 <.0001
4 65 0.15 61.3221 3.9170177 <.0001
5 50 1 55.3735 0.31311395 <.0001
6 50 0.5 13.9633 4.1088955 0.0273
7 50 0.25 9.384639 2.8577997 0.0304
8 50 0.15 7.0056652 2.4784959 0.0475
9 20 1 13.9633 421088955 0.0273
10 20 0.5 8.5714881 14.7883 0.5933

 

4.5 Sensitivity and Specificity

Biosensors prepared with parameter set 2 were tested with a varying
concentration of BVDV in pure culture. Figure 4.20 shows that the biosensor response

was proportional to the virus concentration between concentrations 104 to 10° CCID/ml.

76

Within this region of virus concentration, the biosensor response was significantly
different than that of the blank and also between virus concentration levels (e. g., between
104 and 10° CCID/ml) (Figure 4.20). The detection limit of the biosensor was found to be
10‘ CCID/ml. All virus concentrations below 104 CCID/ml resulted in insignificant

difference between 1° and IS.

 

 

Mean of AI (uA)

 

 

 

 

Virus concentration (log CCID/ml)

 

 

 

*Mean with the same letter is not significantly different at 95% confidence level.

Figure 4.20: Performance of the biosensor in varying concentrations of BVDV.

An insignificant difference between 1° and I8 was also found when testing the
biosensor in varying concentrations of IBR virus, indicating the biosensor
irresponsiveness to the presence of the virus. The biosensor also was tested with a mixed
sample of 1BR and BVDV cultures (Figure 4.21). Statistical analysis shows that the
biosensor is able to detect BVDV even in the presence of 1BR virus in the sample. The
magnitude of AI measured in this study, however, was a lot smaller that the one obtained
from the sensitivity study. For instance, the A1 of the biosensor tested in a mixture of
culture sample containing 10" CCID/ml of BVDV and 106 CCID/ml of IBR virus (100 of

dilution factor) is 20 times smaller that of the signal generated in the 10° CCID/ml of

77

BVDV culture alone. It is speculated that the presence of [BR virus in the culture sample

may potentially hinder the attachment of BVDV to the biosensor surface.

 

16
14—
12~
1o—
8-
6~
4-
24
0-

 

Mean of A1 (11A)

 

 

     

 

10'2 10'1 100
Dilution factor for the sample mixture

 

 

*100 dilution factor means that the virus concentration is 10° CCID/ml of BVDV and 10°
CCID/ml BHV. 10'2 and 10'1 mean 1:10 and 1:100 of dilution factors.

Figure 4.21: Performance of the biosensor in a mixture of BVDV and BHV culture
samples.

4.6 Reusability
The only part of the biosensor that can be reused is the ITO glass since the
detachment of antigen from the antibody and Pani cannot be done without affecting the
property of the protein and polymer. The reusability study of the ITO glass was
conducted by comparing the unused substrate with the used ITO glass treated with
chromic-sulfuric acid solution. Results show that the conductivity of the two substrates
was not significantly different between each other (Appendix 1). This finding shows that
the ITO glass is able to retain its conductive property even after the treatment of the acid
solution. The ability of the chromic-sulfuric acid solution to remove Pani, antibody, and

antigen from the ITO surface was confirmed by using a scanning electron microscope

78

 

(SEM). The SEM images show no protein or polymer structure on the treated substrate.
Moreover, this treated substrate was observed to have the same morphological structure
as the unused ITO substrate (Appendix I). As for the conclusion, the study show that the
ITO glass is reusable and the chromic-sulfirric acid solution can be used to remove the
polymer and protein molecules from the substrate.
4.7 Cost analysis

The cost of the raw materials for fabricating each of the ITO-Pani biosensor is
estimated to be $4.00. This cost, however, excludes the cost of the ITO substrates since
they are reusable, and also the cost of the amperometric measurement system. The major
component contributing to the cost is the antibodies. An effort to reduce the price of the

biosensor needs to be focused in finding ways for more efficient antibody usage.

79

CHAPTER 5: PRELIMINARY STUDY-FRACTAL ANALYSIS ON THE

INDIUM TIN OXIDE-PAN] BIOSENSOR
5.1 Introduction

Antibody-antigen complexes have long served as models to study the general
principle of protein-protein interaction in heterogeneous reaction both experimentally and
computationally. Heterogeneous chemical reactions occur at interfaces of different phases
such as liquid-solid interface (Kopelman 1988). Typically, a reaction system can be made
homogenous by vigorous stirring. Convective stirring, however, is not always possible in
heterogeneous reactions, where reactions occur in/on media that are solid, viscous or
structured. Under these dimensional or topological constraints, self-stirring or diffusion-
limited reactions occur and has been used to explain the mechanism occurring at the
heterogeneous system (Kopelman 1988)

The heterogeneous antibody-antigen reaction that is taking place at the electrode-
solution interface of a biosensor is:

Ag + Ab <—> [Ag.Ab]
where Ag is the antigen and Ab is the antibody. Generally, the binding kinetics of
antigens and antibodies are quite complex with two or more steps to adequately explain
its mechanism. A study has proposed a two-step binding kinetics: encounter and docking
steps (Sinha, Mohan et a1. 2002). The encounter step involves diffusion—limited reaction
of the two molecules driven by non-specific electrostatic forces. The second docking step
occurs when a stronger, non-covalent bond occms between the two molecules (Sinha,
Mohan et a1. 2002). An affinity value, which is the strength of antibodies to bind to an
epitope, is also important in the antigen-antibody binding reaction. Sinha et al. (2002)

indicates that a stronger electrostatic interaction leads to a higher affinity value.

80

 

Considering numerous factors playing significant roles on the antibody-antigen
binding reaction, the application of fiactal analysis has been investigated to study the
dynamic mechanism of the reaction, and how it can be utilized to enhance the biosensor
performance (Charcosset 1998; Ramakrishnan and Sadana 2000; Ramakrishnan and
Sadana 2002). Fractals are disordered systems which are described by non-integral
dimensions (Pfeifer and Obert 1989). As long as surface irregularities show invariance,
they can be characterized by the fractal dimension. An increase in the disorder on the
surface leads to a higher value of fractal dimensions (Sadana 2002). A study indicates
that heterogeneous diffusion-limited reactions, such as in antigen-antibody binding
reaction, are expected to exhibit fractal-like kinetics (Kopelman 1988). The inclusion of
the nonspecific binding and the heterogeneity of polyclonal antibodies also has been
shown to increase the fractal dimension of the biosensor (Sadana 2002).

Theory
Single fractal analysis
Kopelman (1988) indicates that the reaction rate for a heterogeneous reaction is in

the form of :
k1 =k't'b O<b<1 (t>1)
(Equation 5.1)
Generally, k1 depends on time whereas k]: k' (b=1) does not. The author indicates that in
a three-dimensional space (homogenous space), b=0, and k1 is independent of time

(Kopelman 1988). However, when a diffusion-limited reaction occurs in fractal spaces,
Such as in antigen-antibody binding reaction system, b is greater than zero, and this yields

k, as a time-dependent reaction. A study also indicates that the diffusion of a particle

81

(e.g., Ag) from a homogenous solution to a solid surface (e.g., Ab coated on a biosensor
surface) on which it reacts to form a product (e. g., Ag—Ab) is in the form of (Havlin and
Ben-Avraham 1987):

(Ab.Ag)~tp, p=(—3:22fl t<tc

(Ab.Ag) ~ 11/2, I > tc

(Equation 5.2)

where Df is the fractal dimension of the surface, which reflects the extent of
heterogeneity that exists on the biosensor. For t > to, the reaction is considered

homogenous. The value of to also is arbitrarily chosen.

Dual fractal analysis

In a dual fractal analysis, the above single model is extended (Equation 5.3).

.. p1 =_(°’Df1) =
(Ab.Ag) t , pl 2 t t1
(3-Df)
(Ab.Ag)~Ip2, p2=——2——2— t1 <I<I2

(Ab.Ag)~t1/2, t>tc

(Equation 5.3)
The time at which the first fractal dimension changes to the second one (t1 = t2) is
arbitrary and empirical (Sadana 1999).
5.2 Preliminary result and discussion

A preliminary testing was conducted to investigate the feasibility of using fractal
analysis. Table 5.1 shows the values of the binding rate coefficient, k, and fractal
dimension, Df, obtained from a regression analysis of the experimental data (charge-

Versus-time response after antibody-antigen binding) using the following equation 5.4:

82

<3 — Dfl)
(Ab.Ag) = kt 2
(Equation 5.4)
In this study, only a single fractal analysis is evaluated. In all cases presented in Table
5.1, the regression coefficient is greater than or equal to 0.96. Theoretically, a dual fractal
analysis will provide better fitting but the ability to choose the transition time between the
first and second fractal dimension will be challenging.

As the analyte concentration increases, the binding rate coefficient, k, also
increases by a factor of 13.3 from a value of 7.770 to 103.7 (Figure 5.1a). Similarly, the
fractal dimension, Df, also increases by 26% from 1.055 to 1.328 (Figure 5.1b).
Therefore, an increase in fractal dimension leads to an increase in the binding rate
coefficient (Figure 5.1c). A predictive relationship for all the cases presented is also
evaluated (Figure 5.1). The curves fit the data from Table 5.1 relatively well with an r-
squared greater than 0.95. A better fit for these predictive equations, however, may be

achieved by analyzing the experimental data using the dual fractal analysis.

83

 

Table 5.1: Estimated binding rate coefficient and fractal dimension using a single fractal

 

 

Analyte Binding rate coefficient, k (10°) Fractal dimension, Df
concentration Mean 2: SE (n=3) Mean :1: SE (n=3)
10‘ 7.770 t 2.317 1.055 i 0.043
102 9.983 i 5.052 1.134 i 0.090
103 29.28 i 7.032 1.216 i- 0.076
104 37.29 1- 16.47 1.254 r: 0.078
105 50.59 i 10.63 1.293 i 0.032
106 103.7 i 6.581 1.328 i 0.035

 

 

 

 

 

 

 

 

 

 

 

 

(b)

84

.§ 120
E 100 ~ y=4.5355e0‘5]°2x
g _.2 80 4 R2=0.96O6
3 ”T 60 4
on c
:5 5 40 5
.E
.1: 20 .
t:
g 0 . . . . .
0 2 3 4 5 6
Virus concentration ( log CCID/ml)
(a)
g“ 1-4 - y=1.0491x°"302
g R2 = 0.9944
.E 1.3 -
.5 Q—
3 a 1.2 4
O
a
~’= 1.1 -
1:
i
1 . r . . .
0 2 3 4 5 6
Virus concentration (log CCID/ml)

 

 

 

 

 

120

 

 

E
OE 100 I 93944 I
L3 y=0.0003e' "
8 80 — R2=0.9562
a ‘-’ .
a A
I- V? 60 .4
50
.5 E
.5 4O ‘
.D
S 20 .
0
2
0 I I I I I l

 

 

1 1.05 1.1 1.15 1.2 1.25 1.3 1.35

Mean fractal dimension, Df

 

 

 

(C)

Figure 5.1: Influence of virus concentration (log CCID/ml) on a) the binding
coefficient and (b) fractal dimension. (0) Influence of fractal dimension to the binding
coefficient.

In general, an increase in the binding rate coefficient value should lead to an
enhanced sensitivity and a faster response time. The predictive relationship for the
binding rate coefficient as a function of an analyte concentration will provide an avenue
in which one can manipulate the parameter for a better biosensor performance. More
thorough analyses on different experimental conditions, using both single and double
fractal analyses, will also give a better insight on how those external parameters affect the
binding rate coefficient.

Finally, more studies are required to determine whether the binding rate
coefficient is sensitive to the fractal dimension or the heterogeneity of the surface. If this
is indeed so, more attention should be given on how to manipulate the surface chemistry

of a biosensor platform.

85

CHAPTER 6: CONCLUSION

The ITO- Pani biosensor described in this research represents a novel technique
for viral detection. The combination of highly specific antibody molecules and excellent
electronic and electrochemical properties of Paul compounds enables the fabrication of a
field-portable biosensor that is highly sensitive, specific and rapid for the detection of
disease causing agents. The novelty of this biosensor is in the use of spin coating method
to deposit Pani on the substrate for an antibody attachment. Spin coating method was
chosen because of its ease of use, rapid processing time, reproducible, and is inexpensive
compared to other types of polymer coating mechanisms, such as Langmuir-Bloggert
technique and layer-by-layer molecular deposition. Moreover, the ability to change the
specificity of the antibodies will allow a quick redesign of the biosensor for other
detection targets and a multi-array detection device. The ability to use the same biosensor
for both the baseline and sample testing is also an advantageous feature to minimize
variability which is faced by the Pani-based membrane strip immunosensor (Muhammad-
Tahir and Alocilja 2003; Muhammad-Tahir and Alocilja 2003; Muhammad-Tahir,
Alocilja et al. 2005; Tahir, Alocilja et a]. 2005).

This study had three major objectives. The first objective was to synthesize and
characterize the self doped Pani to determine its feasibility to be incorporated into the
biosensor. Conductivity testing, TEM analysis, pH-stability, and TG analysis were
performed to characterize the self doped Pani. A commercially-available non-self doped
Pani was used to compare these characteristics. The TEM analysis showed that the self
doped Pani compounds had more globular shapes and were smaller than the non—self

doped Pani. The conductivity of the self doped Pani was also shown to be lower than the

86

non-self doped compound. The conductivity of the non-self doped Pani, however,
depended highly on pH levels and thus may not be efficient to be used in neutral pH
environment that is required for an optimal biological reaction. Due to this reason, a self
doped Pani was chosen as the transducing element for the biosensor.

The second objective was to further evaluate the possibility of fabricating the
ITO-Pani biosensor. The fabrication of an ITO-Pani biosensor, which included several
processes, such as surface treatment, Pani coating, and functionalization with antibodies,
was shown to be successful by using the AF M analysis. The role of Pani as “glue” in the
antibody binding to the biosensor surface was also demonstrated by comparing the
amperometric response of a plain ITO and an ITO coated with Pani. A significant A] was
observed from Pani-coated ITO substrate and not from no-Pani ITO. This finding
revealed the need of the self doped Pani for the antibody binding on the ITO glass
substrate. The next part of the second objective was to evaluate the experimental
conditions, such as Paul size, antibody concentration, antibody immobilization method,
and incubation time. Among all the parameters evaluated, biosensors coated with 65 KDa
Pani, 0.5 mg/ml of antibody concentration, immobilized using Liu et al. ’5 method, and
incubated with antigen for 30 minutes resulted the most significant signal. These
parameters were further used to evaluate the performance of the biosensor.

The last objective of this research was to evaluate the performance of the
biosensor in terms of sensitivity and specificity. The sensitivity of the biosensor was 104
CCID/ml with a linear range of up to 10° CCID/ml of BVDV in pure culture. The
biosensor also was shown to be not responding to the presence of the IBR viruses,

indicating the specificity of the BVDV antibody. While the detection time of the

87

biosensor was found to be 55 minutes in this study, the time can be cut down to 30
minutes by minimizing the time for measuring amperometric responses. The study also
concluded that the ITO glass was reusable by using the chromic-sulfuric acid solution to

remove the polymer and protein molecules from the substrate.

88

CHAPTER 7: FUTURE MODIFICATION

The ITO-Pani biosensor faces several issues that need to be addressed before it
can be adopted as a field-portable biosensor. It is important to have a better
understanding of the mode of the Operation of the biosensor in order to improve its
performance (sensitivity and specificity) and speed of detection. Below is the summary of
some areas of the biosensor that need more research.

The detection time of the biosensor may be decreased by minimizing the antigen
incubation time. An introduction of a stirring mechanism in the biosensor architecture
may expedite the antigen and antibody binding kinetic. Increasing the temperature during
the antigen incubation period may also speed up the attachment of antigen to the
antibodies immobilized on the substrate. The technique however, needs to be further
evaluated as temperature has a profound effect on the Pani property (Chapter 4) and
protein stability (Appendix II).

A better understanding of an antigen—antibody binding reaction is essential to
improve the biosensor sensitivity, as it is the primary parameter in the biosensor design.
A preliminary study using a fractal analysis is conducted as one of the efforts to better
understand the antibody-antigen mechanism occurring on the biosensor platform
(Chapter 5). This study also shows the need for the Pani compound to mediate the
antibody attachment on the biosensor surface (Chapter 4). The relationship between the
antibody concentration and molecular weight of Pani needs to be investigated to further
understand the mechanism occurring on the surface of the ITO glass.

Several studies have shown that a non-specific binding (N SB) phenomenon plays

a critical role in understanding and improving the design of a biosensor. Since not much

89

is currently known about the science of NSB phenomenon, and how it influences the
specific binding, several attempts may be possible to minimize the occurrence of NSB.
One possible way to do this is by using only the Fab fragment of antibodies that contain
only the antigen-binding site. This is because the whole immunoglobulin antibody
molecule contains the Fc fragment to which non-antigen components may stick to and
cause NSB (Sadana 2002). Antibody molecules containing only a single binding site,
such as a single chain antibody fragment, also may minimize NSB by preventing the
formation of antigen—bridge complexes. Also, the use of monoclonal antibodies
consisting only a single epitope may prevent NSB and increases sensitivity and
specificity of the biosensor. One of the possible drawbacks of using genetically
engineered antibodies over polyclonal antibodies is their expensive manufacturing cost.

Although sensitivity result (Chapter 4) shows that the biosensor has a promising
ability to do a quantitative analysis, there is still a tremendous need to identify some
potential factors contributing to the formation of NSB. The knowledge of NSB and its
effect to the biosensor performance is critical especially when testing field samples
containing contaminants that may contribute to an erroneous detection.

The shelf life study of the ITO-Patti biosensor is critical for it to be
commercialized and used for an on-fleld testing. Since the shelf life of the biosensor
depends on the stability of the antibody and the Pani, more thorough studies on the effect
of external (e.g., temperature) parameters to the biosensor performance need to be done.
A preliminary study on the effect of temperature to the biosensor performance is
presented in Appendix 11. Although in this research, the ITO substrate is shown to be

reusable, the shelf life of the ITO substrate still needs to be evaluated as the chemical and

90

electrical properties of the ITO may be diminished gradually from a repetitive acid
treatment.

The current biosensor design has an active area of 1.27 cm X 2.54 cm. By
reducing the size of the active area, it may be possible to increase the sensitivity of the
biosensor, as less antigen concentration is required to interfere the charge transport
between the working electrode (ITO-Pani substrate) and the auxiliary electrode.
Therefore, a more sensitive biosensor may be redesigned. One possible obstacle that may
be happening with a smaller size biosensor is the occurrence of a NSB or a ‘hook effect’
phenomenon which may lead to a false negative response. Additionally, it may be harder
for a smaller biosensor to find the detection target in a larger volume of sample.

Sample processing is an area that has significant effects on the performance of a
field-based biosensor, especially for it to be used in farms, food processing and
slaughtering plants. Even the most analytically specific and sensitive biosensor may not
be able to detect the presence of the target organism from field samples if it is not being
integrated with a sample processing mechanism. It was evident from the specificity
testing (section 4.5) that the presence of non-target virus interfered with the binding of
the target virus to its biological receptor on the biosensor. The presence of much bigger
protein molecules, for example in blood sample, could completely block the antigen-
antibody binding, and thus generate a false negative result. Therefore, it is imperative to
incorporate a filtration device onto the ITO-Pani biosensor in order to fully enhance its
performance in naturally ‘dirty’ samples. The filtration mechanism may utilize a simple
membrane filter, which has been accepted worldwide as an effective microbial and viral

filtration unit.

91

The area of conducting polymer is evolving rapidly as more and more polymer
compounds with better conductive property and higher compatibility with biological
elements are discovered. Therefore, a comparative study using different types of
conducting polymer compounds such as polypyrrole, polythiophene, polyfuran, and
polycarbazole need to be assessed.

Finally, the current biosensor design lacks computer system integration and user
interfacing. The biosensor concept needs to be taken one step further by building a
prototype model that is automated and easily operated by untrained users. A field-ready
ITO- Pani biosensor may include a filtration system, a battery-powered potentiostat with
built-in mathematical and statistical softwares for rapid data analysis, and miniaturized
three—electrode electrochemical cells capable of testing multiple targets simultaneously in

a highly contaminated field samples.

92

APPENDICES

93

APPENDIX I

DATA

Table A1: Summary of data.

 

 

Molecular Antibody Incubation Incubation Mean of AI StdErr (uA) P
Weight of concentration time method (11A) value
Pani (KDa) (mfll) (minutes)

65 1 30 Liu's method 26.2966 1.73E-06 0.0001
65 0,5 30 Liu's method 247.9368 7.67E-06 <.0001
65 0,25 30 Liu's method 113.25 5.29E-O6 <.0001
65 0,15 30 Liu's method 61.3221 3.92E-O6 <.0001
65 1 15 Liu's method 20.472 0.00002427 0.4464
65 0, 5 15 Liu's method 10.796 1.65243E-05 0.5492
65 0,5 15 Liu's method 2.03E+00 2.43E-06 0.4511
65 0,25 15 Liu's method 10.5032 8.55E-06 0.2868
65 0,25 15 Liu's method 9.38E+00 2.86E-06 0.0304
65 0,15 15 Liu's method 18.749 1.83578E-05 0.3649
65 1 10 Liu's method 4.84E+00 1.14614E-05 0.6947
65 0,15 10 Liu's method 8.53E+00 6.80E-06 0.2783
50 1 30 Liu's method 55.3735 3.13E-07 <.0001
50 0_ 5 30 Liu's method 13.9633 4.11E-06 0.0273
50 0.25 30 Liu's method 9.38E+00 2.86E-06 0.0304
50 0.15 30 Liu's method 7.01E+00 2.48E-06 0.0475
50 0.5 15 Liu's method 7.41E+00 2.07199E-05 0.7386
50 0.25 15 Liu's method 3.09E+00 9.85E-06 0.7693
50 0.15 15 Liu's method 17.262 2.13466E-05 0.4641
50 1 10 Liu's method 10.93 6.79E-06 0.1828
50 0.5 10 Liu's method 8.67E-01 1.17731E-05 0.9448
50 0.25 10 Liu's method 23.365 7.54E-07 <.0001
50 0.15 10 Liu's method 4.25E+00 1.46244E-05 0.7857
20 1 30 Liu's method 13.9633 4.11E-06 0.0273
20 0.5 30 Liu's method 8.57E+00 1.47883E-05 0.5933
20 0.25 30 Liu's method 10.103 1.19361E-05 0.445
20 0.15 30 Liu's method 2.73E+00 1.22445E-05 0.8346
20 1 15 Liu's method 9.28E+00 9.16E-06 0.3683
20 0.5 15 Liu's method 2.92E+00 2.11261E-05 0.8967
20 0.25 15 Liu's method 9.42E+00 1.46352E-05 0.555
20 0.15 15 Liu's method 9.42E+00 1.46352E-05 0.555
20 1 10 Liu's method 1.12E+00 2.59E-06 0.6886
20 0.5 10 Liu's method 6.84E-01 3.60E-06 0.8585
20 0.25 10 Liu's method 3.87E+00 1.87E-06 0.1079
20 0.15 10 Liu's method 1.17E-01 4.76E-06 0.9817

 

94

 

 

Molecular Antibody Incubation Incubation Mean of AI StdErr (uA) P
Weight of concentration time method (uA) value
Pani (KDa) (mglml) (minutes)

65 0.5 30 Tang's method 7.47E-01 3.59E-06 0.8453
65 0.25 30 Tang's method 12.109 1.05639E-05 0.3156
65 0.15 30 Tang's method 26.163 2.06843E-05 0.2746
50 0.5 30 Tang's method 1.84E+00 9.98E-06 0.8625
50 0.25 30 Tang's method 1.86E+00 2.59E-06 0.5121
50 0.15 30 Tang's method 10.4668 1.46129E-05 0.5134
20 0.5 30 Tang's method 16.944 1.63206E-05 0.3578
20 0.25 30 Tang's method 1.21E+00 4.04E-06 0.7792
20 0.15 30 Tang's method 2.16E+00 4.17E-06 0.632
65 1 30 Tang's method 3.68E+00 4.07E-06 0.4172
50 1 30 Tang's method 19.435 1.05245E-05 0.1385
20 1 30 Tang's method 2.90E+00 4.53E—06 0.5564
65 0.5 15 Tang's method 7.33E+00 1.35707E-05 0.6177
65 0.25 15 Tang's method 12.3524 1.51689E-05 0.4612
65 0.15 15 Tang's method 2.96E+00 2.57E-06 0.3138
50 0.5 15 Tang's method 6.10E+00 1.99E-06 0.0374
50 0.25 15 Tang's method 2.83E+00 8.01E-06 0.7415
50 0.15 15 Tang's method 9.67E+00 7.06E-06 0.2429
20 0.5 15 Tang's method 1.44E+00 1.13248E-05 0.9051
20 0.25 15 Tang's method 6686.9564 0.006689187 0.374
20 0.15 15 Tang's method 4.39E+00 6.75E-06 0.5507
65 1 15 Tang's method 13.7344 1.36881E-05 0.3725
50 l 15 Tang's method 7.76E+00 1.23546E-05 0.5638
20 1 15 Tang's method 16.8455 2.43762E-05 0.5275
65 0.5 15 Tang's method 4.42E+00 5.47E-06 0.4648
65 0.25 15 Tang's method 7.01E+00 2.48E-06 0.0475
65 0.15 10 Tang's method 7.79E+00 7.74E-06 0.3713
50 0.5 10 Tang's method 1 1.2888 1.05485E-05 0.3448
50 0.25 10 Tang's method 6.99E+00 0.0000112590.5684
50 0.15 10 Tang's method 8.76E+00 1.58638E-05 0.6103
20 0.5 10 Tang's method 9.47E+00 9.07E-06 0.3552
20 0.25 10 Tang's method 12.2105 1.26671E-05 0.3897
20 0.15 10 Tang's method 8.70E+00 3.40E-06 0.0627
65 1 10 Tang's method 5.41E+00 8.91E—06 0.5768
50 1 10 Tang's method 4.25E+00 1.41239E-05 0.7786
20 1 10 Tang's method 4.55E-01 6.82E-06 0.95

 

95

Confocal Microscopy Analysis

Intensity

250

III I I. II, IIi
.l 'l‘ i l

"I'I: ‘
1 . l .

| ' I'. l' ' i ‘
l l I I I :i' I i it] i! i !!

I'lii.‘

I

ll __ , II ,,
I
I

 

l l I I I l I

0 20 40 80 80 100 1 20 1 40 130 1 80 200 220
Distance [um]

Figure A]: Intensity profile of the FITC-labeled antibodies immobilized on an ITO

substrate based on Liu’s method.

Intensity

200-

150 -

100 '

50-1

 

0

2...,

11.11 1Ill .111 . .1... I 11. 111.1111. 1 ll .1

I I I

20 40 ED 80 100 120 140 1 50 1 BO 200 220
Distance [urn]

Figure A2: Intensity profile of the FITC-labeled antibodies immobilized on an ITO

substrate based on Tang’s method.

96

Specificity data

Table A2: Specificity data.

 

Concentration Mean of AI (uA) StdErr P value

 

of IBR virus (uA)
(CCID/ml)
10I 29.74 30.49 0.3845
104 14.97 14.96 0.3734
106 36.94 16.12 0.0838

 

Table A3: Specificity data for mixed- culture sample.

 

Dilution factor for the Mean of AI (uA) StdErr (uA)tValue P
sample mixture

 

value
10‘2 6.6697299 1.051~:+00 6.36 0.0031
10‘1 9.3119871 9.43E-Ol 9.88 0.0006
100 11.6801 2.58E+OO 4.53 0.0106

 

97

Reusability data

  
    

 

2um
Figure A3: SEM image of unused ITO substrate.

2um

Figure A4: SEM image of treated ITO substrate.

Table A4: Conductivity (Slcm) of the unused and treated ITO substrates.

 

Unused ITO substrate 0.84 :1: 0.003
Treated ITO substrate 0.83 :1: 0.0078
*The means are not significantly different (p=0.182184)

 

 

98

APPENDIX II

Temperature-Dependent Analysis

A preliminary study is conducted to study the effect of temperature to the
biosensor performance. The ITO-biosensor prepared with the standard parameters was
used to test 106 CCID/ml of BVDV virus at two different temperatures: 10°C and 80°C.
This is accomplished by heating up or cooling down the electrolytes in the
electrochemical cells to the desired temperatures. The biosensor response of Al was
found to be statistical different for biosensors tested at 10°C and not at 80°C (Table 5). In
addition, the mean A1 of the biosensors tested at 10°C is observed to be smaller than the
response produced by the biosensor operated at the room temperature indicating poor
antibody-antigen binding or inefficient charge transfer. Although higher electrolyte
temperature may increase the diffusion and mobility of charges (Sadana 2002), the
temperature is not optimal for antibody and antigen binding as it may denature the
antibodies (Barbour and George 1997).

Table A5: Data at different temperatures.

 

Temperature Mean of AI (pA) StdErr (pA)P value

 

10°C 16.5875 1.82E-06 0.0008
Room temp 247.9368 7.6728816 <.0001
(~25°C)

80°C 8.55 4.52E-06 0.1315

 

99

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