CRANFIELD UNIVERSITY MOHAMAD KAMAL ABDUL KADIR DEVELOPMENT OF ...

345 Pages·2010·3.53 MB·English

CRANFIELD UNIVERSITY

MOHAMAD KAMAL ABDUL KADIR

DEVELOPMENT OF IMMUNOSENSORS FOR
MYCOTOXINS ANALYSIS

CRANFIELD HEALTH

PhD THESIS
Academic Year: 2006 – 2009

Supervisor: Dr. Ibtisam E. Tothill
May 2010
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CRANFIELD UNIVERSITY

CRANFIELD HEALTH

PhD THESIS

Academic Year: 2006 – 2009

MOHAMAD KAMAL ABDUL KADIR

DEVELOPMENT OF IMMUNOSENSORS FOR
MYCOTOXINS ANALYSIS

Supervisor: Dr. Ibtisam E. Tothill
May 2010

This thesis is submitted in partial fulfilment of the requirements for the
degree of Doctor of Philosophy

© Cranfield University, 2010. All rights reserved. No part of this
publication may be reproduced without the written permission of the
copyright owner.
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ABSTRACT

Aflatoxin B (AFB ) and fumonisins (Fms) are mycotoxin contaminants found in
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peanuts and corn, respectively, and are known to be immunosuppressive and
carcinogenic compounds. Therefore, the development of rapid and sensitive method
for detecting these toxins especially for field analysis is required for risk assessment
and management. The work presented in this thesis reports on the construction of
sensor platforms capable of fulfilling these requirements. The use of, screen-printed
thick film electrodes, gold nano-particle application and microelectrodes on a silicone
support were investigated as suitable sensor platforms. The development of indirect
and direct competitive immunoassay formats for the electrochemical immunosensor
construction was undertaken for AFB and Fms determination.
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A spectrophotometric assay was initially developed as a first step procedure using
microtitre plates for immunoreagents concentration and conditions selection before
being transferred to the surface of the screen-printed gold electrode (SPGE) and then
the microelectrode array (MEA) sensor. Detection was performed by
chronoamperometry monitoring the reaction of tetramethylbenzidine (TMB) and
hydrogen peroxide (H O ) catalysed by horseradish peroxidase (HRP). The
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performance of screen-printed gold electrode (SPGE) immunosensor was compared to
3,3-dithiodipropionic acid (DTDPA) modified SPGE, immuno gold nano-particle
modified SPGE and amine silane modified microelectrode array (MEA). Surface
modification of MEA was successfully undertaken using silane and phenylene di-
isothiocyanate (PDITC) chemistries for the covalent binding of the recognition system
to the silicon surface area of the microelectrode arrays. The immunosensor format was
transferred to a gold microelectrode array based on a silicone support for the purpose
of signal sensitivity enhancement and miniaturisation for multiplex detection in the
prospect of field analysis.

The developed of the gold immunosensor achieved a detection limit of 5 ng L-1, 1 ng
L-1 and 1 ng L-1 for AFB using indirect assay on DTDPA thiol modified SPGE,
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immuno gold nano-particle modified SPGE and modified microelectrode array
(MEA), respectively. While, the direct competitive method for fumonisins detection
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on the SPGE sensor and modified MEA achieved a limit of detection was 0.5 µg L-1.
The sensors were also used for AFB and Fms determination in peanuts and corn
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samples, respectively, and also validated using a standard HPLC and a commercial
ELISA kit. Samples analysis involved the rapid extraction (without clean-up) and pre-
treatment using solid phase extraction (clean-up) before measuring using the
developed immunosensor platforms. The results achieved were found to be in average
82.7% (without clean-up) and 103.1% (clean-up) using gold-particle SPGE and 85.7%
(without clean-up) using MEA for AFB detection in peanut, respectively. While for
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Fms detection in corn sample was achieved in average of 73.5% (without clean-up)
and 98.6% (clean-up) using SPGE and 87.3% (without clean-up) using MEA,
respectively. The developed immunosensors (gold particle SPGE, SPGE and MEA)
had a satisfactory agreement with HPLC and ELISA kit. The performance of the
proposed sensors are highly sensitive and provide analytical system capable of
detecting very low level of toxin within the required legislative EU limit of analyses.
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ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my supervisor Dr. Ibtisam E. Tothill
for her continue guidance, support and friendship throughout the whole project. The
financial support for this research degree is gratefully acknowledged from Malaysian
Agricultural Research and Development Institute (MARDI).

Special thank to Faridah, which was always there sharing my Cranfield Health
experience, also to Yildiz Uludag and all friend for sharing the experience, knowledge
and for everything. Thanks.

Finally, I would like to thank my wife, Norsuriana Ahmad, for her enormous patience,
never ending care and love. Also to all my children, Atie, Rina and Fahiim for their
loving support. Without their encouragement and understanding it would have been
impossible for me to finish this work.
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LIST OF CONTENT

CONTENTS PAGE.NO
Abstract iii
Acknowledgments v
Contents vi
List of Figures xiv
List of Tables xxvi
Notation xxviii

CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW

1.1 Background 2
1.2 Mycotoxins 4
1.2.1 Aflatoxins 6
1.2.2 Fumonisins 9
1.3 Analytical methods used for aflatoxin and fumonisin analysis 11
1.3.1 Thin Layer Chromatography (TLC) method 14
1.3.2 High Performance Liquid Chromatography (HPLC) method 15
1.4 Immunochemical analysis 16
1.4.1 The antibody (Ig) molecule 17
1.4.1.1 Polyclonal antibody (PAb) 18
1.4.1.2 Monoclonal antibody (MAb) 19
1.4.2 Immunoaffinity column (IAC) 21
1.4.3 Enzyme Linked Immunosorbent Assay (ELISA) 21
1.4.3.1 Competitive immunoassay test 22
1.4.3.2 Detector labels and substrates used in immunoassay 24
1.4.3.3 Data analysis 24
1.5 Immunosensor 26
1.5.1 Background 26
1.5.2 Sensing materials (receptors) 28
1.5.3 Immobilisation (coating) 29
1.5.4 Transducers used in biosensor devices 31
1.6 Electrochemical Immunosensor 34
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1.6.1 Electrode systems used for immunosensor fabrication 35
1.6.2 Cyclic voltammetry (CV) 37
1.6.3 Chronoamperometry (CA) 39
1.6.4 Potentiometric detection 40
1.6.5 Amperometric detection 40
1.6.5.1 Hydrogen peroxide and mediators 41
1.6.5.2 A mediator of 3,5,3‘,5‘-tetrametilbenzidine (TMB) 43
1.7 Microsensors 44
1.7.1 Microelectrode arrays design 45
1.7.2 Electrochemical microelectrode arrays application 46
1.8 The functional of nano-particles for a biosensing application 48
1.8.1 Nano-particles for electrochemical immunosensor 48
1.9 Immunosensor for aflatoxin B and fumonisins analysis 50
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1.10 Aim and objectives 52
1.11 Overall activities for aflatoxin B and fumonisins analysis 55
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CHAPTER 2: DEVELOPMENT OF ELISA FOR AFLATOXIN B
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2.1 Introduction 57
2.2 Material and Methods 59
2.2.1 Chemicals and reagents 59
2.2.2 Instrumentations 60
2.2.3 Buffer solutions 60
2.2.4 Blocking solution 61
2.2.5 Standard solution 61
2.2.6 Indirect competitive ELISA 61
2.2.6.1 Optimisation of reagents 62
2.2.6.2 Effect of coating buffers and blocking agents 63
2.2.6.3 Competition assay preparation 63
2.2.6.4 Effect of concentrations of reagents 65
and incubation time for competitive assay development
2.2.7 Direct competitive ELISA 65
2.2.7.1 Optimisation of reagents 65
2.2.7.2 Competition assay preparation 66
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2.2.8 Calculation for the sensitivity of the assay 67
2.3 Result and Discussion 68
2.3.1 Indirect ELISA 68
2.3.1.1 Optimisation of anti-AFB antibody 68
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(monoclonal antibody against AFB ) and
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AFB -BSA conjugate
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2.3.1.2 Optimisation of anti-mouse IgG-Horseradish 70
peroxidase conjugates (anti-IgG-HRP)
2.3.1.3 Effect of coating buffer in indirect 72
non-competitive method
2.3.1.4 Blocking agents 73
2.3.2 Assay optimisation with free AFB 76
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2.3.2.1 Effect of the sample volume on the assay 76
2.3.2.2 Effect of incubation times in the competition steps 77
2.3.2.3 Effect of different concentration of 79
anti-aflatoxin B antibody (MAbAFB )
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2.3.2.4 Effect of different concentration of anti-IgG-HRP 81
2.3.3 Direct ELISA format 83
2.3.3.1 Optimisation of MAbAFB and AFB -HRP 83
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without free AFB
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2.3.3.2 Optimisation of anti-IgG (mouse) 85
unconjugated without free AFB
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2.3.3.3 Calibration curve of AFB 87
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2.3.4 Sensitivity of the immunoassay development 89
2.4 Conclusions 90

CHAPTER 3: DEVELOPMENT OF ELECTROCHEMICAL
IMMUNOSENSOR USING GOLD WORKING ELECTRODE FOR AFB
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DETECTION

3.1 Introduction 92
3.1.1 Electrochemical immunosensor 92
3.1.2 Immuno gold nano-particle application 93
3.2 Materials and Methods 93
3.2.1 Chemicals and reagents 93
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3.2.2 Buffer and solution 94
3.2.3 Apparatus 95
3.2.4 Screen-Printed Gold Electrode (SPGE) 95
3.2.4.1 Preparation of SPGE fabricated in house 95
3.2.5 Electrochemical Measurement 97
3.2.5.1 Voltammetric studies for characterisation of SPGE 98
3.2.5.2 Surface characterisation of screen-printed gold electrode 99
with SEM
3.2.5.3 Electrochemical study of TMB/ H O / HRP 100
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3.2.6 Electrochemical immunosensor development for competitive 100
assay of AFB
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3.2.6.1 Optimisation of antibody and HRP conjugate 101

3.2.6.2 Physical adsorption procedure for competitive assay 101
3.2.6.3 Thiol self assembled monolayer (SAM) modification 102
of the gold surface
3.2.7 Electrochemical immuno gold nano-particle sensor detection 103
3.2.7.1 Self Assembled Monolayer (SAM) on gold working 103
electrode (SPGE)
3.2.7.2 Optimisation and preparation of immuno gold 103
nano-particle conjugated HRP
3.2.7.3 Indirect competitive assay using immuno gold 104
nano-particles-HRP
3.2.8 Sample analysis 105
3.2.8.1 Sample extraction for immunoassay analysis 105
3.2.8.2 Sample extraction for immunoassay and HPLC 106
3.3 Results and Discussion 107
3.3.1 Characterisation of screen-printed gold electrode by 107
cyclic voltammetry
3.3.2 Surface characterisation of SPGE using SEM 110
3.3.3 Cyclic voltammetry of 3,3‘5,5‘-tetramethylbenzidine 110
(TMB) as an electron shuttle
3.3.4 Chronoamperometry study of enzyme activity using TMB/H O 114
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3.3.5 Potential determination 115
3.3.6 Optimisation of TMB and hydrogen peroxide concentrations 117
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3.3.7 Optimisation of reagents concentrations 117

3.3.7.1 Optimisation of monoclonal antibody against AFB 119
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3.3.7.2 Optimisation of HRP conjugate 120
3.3.8 Development of competitive assay on SPGE (ERCON) 121
3.3.8.1 Passive adsorption immobilisation 121
3.3.9 Development of Competitive assay on SPGE DuPont 124
3.3.9.1 Characterisation of the formation of thiols monolayer 124
3.3.9.2 Passive adsorption and covalent immobilisation 127
calibration curve
3.3.10 Application of gold nano-particle to enhance the 131
sensor response
3.3.10.1 Formation of immuno gold nano-particle for 131
signal amplification
3.3.10.2 Electrochemical characterisation of the electrode 132
with nano-particle
3.3.10.3 Optimisation of immuno gold nano-particle sensor 135
3.3.10.4 Competitive assay using immuno gold nano-particle 136
3.3.11 AFB detection in peanuts 141
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3.3.11.1 Competitive method of detection used with sample matrix 141
3.3.11.2 Sample analysis 143
3.4 Conclusions 146

CHAPTER 4: DEVELOPMENT OF ELECTROCHEMICAL
IMMUNOSENSOR FOR FUMONISINS ANALYSIS

4.1 Introduction 149
4.2 Materials and Methods 150
4.2.1 Chemicals and reagents 150
4.2.2 Buffer Solutions 151
4.2.3 Apparatus 151
4.2.4 Preparation of stock solutions 151
4.2.5 Preparation of Standard solution 152
4.2.6 Spectrophotometric ELISA for fumonisins analysis 152
4.2.6.1 Optimisation by ELISA Procedure (Checkerboard method) 152
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