Chapter I : BIOSENSORS BASED ON ENZYME INHIBITION

Index pag I

Chapter I : BIOSENSORS BASED ON ENZYME INHIBITION

Preface pag 1

GENERAL INTRODUCTION TO BIOSENSORS

1.1 Definition of biosensors pag 2 1.2 Biocomponent pag 4 1.2.1 Enzyme

1.2.1.1 Definition pag 5 1.2.2.2 Enzyme by the “Commission on Enzyme Nomenclature

of the International Union of Biochemistry” pag 6 1.2.2.3 Michaelis Menten kinetics pag 6 1.3 Immobilisation of the receptor component in biosensors pag 10 1.3.1 Adsorption pag 11 1.3.2 Physical entrapment pag 12 1.3.3 Covalent coupling pag 12 1.3.4 Cross-linking pag 12 1.4 Structure and function of transducers pag 13 1.4.1 Electrochemical transducer pag 14 1.4.1.1 Amperometric biosensors pag 14 1.4.1.2 Potentiometric biosensors pag 14 1.4.1.3 Other electrochemical biosensors pag 15 1.4.2 Optical transducers pag 15 1.4.3 Piezoelectric transducers pag 16 1.4.4 Thermal transducers pag 17 1.5 Biosensor based on enzyme inhibition pag 17

I

1.6 Cholinesterase pag 20 1.6.1The catalytic mechanism pag 22 1.6.2 Irreversible inhibition of cholinesterase pag 25 1.7 Enzyme inhibition kinetic pag 26 1.7.1 Reversible inhibitors pag 27 1.7.1.1 Competitive inhibitors pag 27 1.7.1.2 Non-competitive inhibitors pag 29 1.7.1.3 Uncompetitive inhibitors pag 31 1.7.1.4 Mixed inhibitors pag 33 1.7.2 Irreversible inhibitors pag 34 1.8 Biosensor based on cholinesterase inhibition pag 36 1.8.1 Cholinesterase inhibition pag 36 1.8.1.1 Reversible inhibition pag 37 1.8.1.2 Irreversible inhibition pag 39 1.8.2 Parameters influencing the biosensor based on cholinesterase inhibition pag 39 1.8.2.1 Effect of immobilisation pag 39 1.8.2.2 Effect of enzyme concentration pag 41 1.8.2.3 Effect of incubation time pag 41 1.8.2.4 Effect of pH pag 42 1.8.2.5 Effect of organic solvent pag 42 1.8.3 Design of cholinesterase biosensors pag 43 1.8.3.1 Three enzymes system pag 44 1.8.3.2 Bi-enzymatic system pag 44 1.8.3.3 Mono-enzymatic biosensor pag 46 1.9 Thiocholine detection pag 47 1.9.1 Methods of analysis for thiocholine detection pag 47

II

1.9.2 Chemical, physical and electrochemical properties of Prussian Blue pag 49 Concluding remarks pag 53 A1 Appendix

A1.1 Cyclic voltammetry pag 55

A1.2 Amperometric methods pag 56 A 1.2.1 Batch stirred amperometry pag 57

A. 1.3 Electrode materials pag 58 A. 1.3.1 Screen-printed electrodes pag 58

Chapter II : EXTRACTION AND DETECTION OF PESTICIDES BY CHOLINESTERASE INHIBITION IN A TWO-PHASE SYSTEM: A STRATEGY TO AVOID HEAVY METAL INTERFERENCE

2.1 Introduction pag 65 2.2 Experimental section pag 67 2.2.1 Apparatus and reagents pag 67 2.2.2 Measurement of thiocholine pag 68 2.2.3 Reaction between thiocholine and heavy metals pag 69 2.2.4 Measurement of the enzymatic activity in aqueous solution pag 69 2.2.5 Measurement of pesticide in aqueous solution pag 70 2.2.6 Measurement of the effect of organic solvents on enzyme activity pag 70 2.2.7 Measurement of inhibition by pesticide present in organic solvent pag 71 2.3 Results and discussion pag 71 2.3.1 Reaction between heavy metals and thiocholine pag 71 2.3.2 Principle of the method pag 73 2.3.3 Characterisation of enzyme activity pag 77

III

2.3.4 Effect of different organic solvents on enzyme activity pag 78 2.3.5 Measurement of inhibition by paraoxon pag 81 2.4 Conclusions pag 83

Chapter III: DETECTION OF CARBAMIC AND ORGANOPHOSPHOROUS PESTICIDES IN WATER SAMPLES USING A CHOLINESTERASE BIOSENSOR BASED ON PRUSSIAN BLUE MODIFIED SCREEN PRINTED ELECTRODE

3.1 Introduction pag 88 3.2 Experimental section pag 90 3.2.1 Apparatus pag 90 3.2.2 Electrodes pag 90 3.2.3 Reagents pag 91 3.2.4 Thiocholine sensors pag 92 3.2.4.1 Preparation of Prussian Blue modified SPE pag 92 2.2.4.2 Preparation of Cobalt Phthalocyanine bulk pag 92

modified screen printed electrodes

3.2.4.3 Preparation of the SPEs with gold film pag 92 3.2.5 Thiocholine measurements pag 93 3.2.6 Cholinesterase biosensor based on Prussian Blue modified SPE pag 94 3.2.7 Acetylthiocholine and butyrylthiocholine measurement pag 95 with AChE and BChE biosensors

3.2.8 Inhibition measurement using AChE and BChE biosensors pag 95 3.2.9 Sample collection pag 96 3.3 Result and discussion

3.3.1 Thiocholine sensors pag 97 3.3.2 Cholinesterase enzyme biosensor based on Prussian Blue modified SPE pag 102

IV

3.3.2.1 AChE biosensor pag 102 3.3.2.2 BChE biosensor pag 104 3.3.2.3 Pesticide determination using AChE and BChE biosensors pag 106 3.3.4 Waste and river water measurements pag 110 3.4 Conclusions pag 111

Chapter IV: TOWARDS A NERVE GAS SENSOR FOR SECURITY PURPOSES: A SOLUTION PROVIDED BY ANALYTICAL CHEMISTRY FOR FAST DETECTION OF NERVE AGENTS USING COST EFFECTIVE, FAST FIELD INSTRUMENTS AND SENSITIVE PROBES

4.1 Introduction pag 116 4.2 Experimental section pag 119 4.2.1 Reagents pag 119 4.2.2 Apparatus pag 119 4.2.3 Preparation of Prussian Blue (PB) modified SPE pag 120 4.2.4 Preparation of a biosensor based on PB-SPE pag 120 4.2.5 Preparation of Sarin and VX solutions pag 121 4.2.6 Preparation of Sarin gas pag 121 4.2.7 Biosensor enzymatic activity measurement pag 122 4.2.8 Stability of BChE immobilized onto PB-SPE pag 123 4.2.9 Paraoxon standard solution measurements pag 123 4.2.10 Sarin and VX solution measurements pag 123 4.2.11 Sarin gas measurements pag 124 4.2.12 Safety considerations pag 124 4.3 Results and discussion pag 124 4.3.1 Biosensor based on PB-SPE pag 124

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4.3.2 The BChE biosensor pag 126 4.3.3 Preliminary studies: paraoxon determination using BChE biosensors pag 127 4.3.4 Sarin and VX determination using the BChE biosensor pag 129 4.3.5 Sarin gas measurements pag 130 4.4 Conclusions pag 134

Chapter IV: A RAPID METHOD FOR AFLATOXIN B DETECTION BASED ON ACETYLCHOLINESTERASE INHIBITION

5.1 Introduction pag 139 5.2 Experimental section pag 141 5.2.1 Apparatus and reagents pag 141 5.2.2 Measurement of cholinesterase activity pag 142 5.2.3 Measurement of the effect of methanol on Ellman’s reaction pag 142 5.2.4 Measurement of the effect of methanol on AChE activity pag 143 5.2.5 Measurement of the effect of methanol on AChE inhibition pag 143 5.2.6 Measurement of aflatoxin stock solution pag 144 5.2.7 Spiked barley sample preparation and extraction pag 144 5.2.8 Measurement of AFB

in barley pag 145 5.3 Result and discussion

5.3.1 Aflatoxin inhibition on different ChEs pag 145 5.3.2 Mechanism of the AChE inhibition by AFB

pag 146

5.3.2.1 Measurement of AChE activity pag 146 5.3.2.2 The reversibility of the AChE inhibition by AFB

pag 146 5.3.2.3 Study of the type of the AChE inhibition by AFB

pag 147 5.3.3 Optimization of the AFB

assay based on AChE inhibition pag 149 5.3.4 Effect of pH pag 150

VI

5.3.5 Effect of temperature on enzyme activity in storage condition pag 151 5.3.6 Study of methanol effect pag 152 5.3.7 Calibration curve pag 156 5.3.8 Cross-reactivity pag 156 5.3.9 AFB

measurement in fortified barley samples pag 157 5.4 Conclusions pag 158 General conclusions pag 163

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