DEVELOPMENT OF AMPEROMETRK BIOSENSORS BASED ON DEHYDROGENASE ENZYMES

Diqing Tang

Department of Chernical and Biochernical Engineering Faculty of Engineering Science

Submitted in partial fulfillment of the requirement for the Degree of Master of Engineering Science

Faculty of Graduate Studies The University of Western Ontario

London, Ontario, Canada December 1997

O Diqing Tang i 998

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ABSTRACT

The developrnent of two amperometnc biosensors, a glucose-&phosphate (G6P)

biosensor and a nitrate biosensor have been investigated in this thesis. The biosensor

performances have been optimized for the determination of G6P in biologicd samples and

nitrate in waste water. respectively.

Both biosensors were based on dehydrogenase enzyme immobilized in carbon

paste together with NADP- or NADPH7 the electrochernical mediator TCNQ or TTF, and

a cationic polymer polyethylenimine (PEI) which served to retain the NADP- or NADPH

in the carbon paste matrix. The enzyme, glucose-6-phosphate dehydrogenase (EC

1.1.1 -49) was used for G6P sensor and nitrate redutase (EC 1-6-63) was used for nitrate

sensor. The optimal response for the G6P biosensor was obtained at pH 7.4, 0.3 V vs.

AgIAgCI and 3 0 ' ~ and for the nitrate biosensor was at pH 7.5, 0.2 V vs. .Ag/AgCI and

30'~. respectively. The stability of the G6P biosensor was found to be over two weeks

with a 50 % reduction of original response. The results from the "real sample" tests of

G6P biosensor in human blood were in excellent agreement with the measurements using

enzyrnatic assay based on spectrophotometer. A modified mathematical mode1 based on

Tatsuma and Watanabe's steady-state formulation was developed for the G6P biosensor to

predia the biosensor response.

These new biosensors may permit more econornical use for the diagnosis and

monitoring in medical and environmental applications. Future work for the G6P biosensor

should include investigations in a FIA System, and improvement of sensitivity and stability

for the nitrate biosensors.

Key words: Glucose-6-phosphate, Biosensor, Mediated Biosensors. Dehydrogenases.

Nitrate Ions. TCNQ, G6PDH, Nitrate Reductase.

The author would iike to express her sincere gratitude and appreciation to her

chief advisor, ProE Amarjeet S. Bassi, for his enthusiastic guidance, persistent

encouragement and support throughout the duration of this study and preparation of this

thesis.

The author would also like to sincerely thank her CO-supervisor. ProE Maurice A.

Bergougnou, for his excellent and timely advice, sharing of good ideas. and strong

encouragement and support during this snidy.

In addition, a deep appreciation is extended to Prof. Argyrios Margaritis, for the

use of a spectrophotometer in his laboratory.

The author also wishes to acknowledge Esther Lee, David Riveira and KeWi Long

and other graduate and undergraduate students for their support and assistance. In

addition, Dr. Keeny, and Dr. Brown at Victoria Hospital (London, Ontario) are

acknowledged for their support of their fiendship and the provision of blood samples.

Finally, the author wishes to thank her husband Chuntao and daughter Fan, and her

parents, for their suppon, understanding, and encouragement throughout the penod of this

study .

TABLE OF CONTENTS

CERTIFIC ATE OF EXAMINATION

ABSTRACT

ACKNO WLEDGMENTS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

NOMENCLATURE

AE3BREVIATION

CHGPTER 1 INTRODUCTION

1 . 1 Scope of this thesis

1 2 Objectives

CHAPTER 2 LITERATURE REVIEW

2.1 Historical Background of Amperometnc Biosensor Development

2.2 P ~ c i p l e of Amperometnc Biosensors

2.3 The Biological Sensing Component

2 -4 Electrochernical Aspects

2.4.1 Three-electrode ce11

2.4.2 Redox reaction

2.4.3 Cyclic voltammetry

Page

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xi

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XX

1

1

4

5

5

7

9

13

14

15

18

2.5 Construction of Amperometric Enzyme Electrodes

2.5.1 Irnmobilktion of enzymes on the biosensor surface

2.5 2 Electron tramferring mediators

2.5 -3 Techniques of chemicai modification based cofactor

dependent enzyme biosensors

2.5 -4 Bienzyrne based biosensors

2.6 Applications of Biosensors

2.6.1 Medical application

3.6.2 Environmental monitoring

2.6.3 Bioprocess control

2.7 Principles of an Arnperornetric G6P Biosensor

2.8 Principles of an Amperometric nitrate Biosensor

2.9 Summary

C~APTER 3 MODELLING OF STEADY STATE RESPONSE

OF G6P BIOSENSOR

3.1 Model Description

3.2 Model Equations

3 -3 Model Formulation

3.4 Determination of Model Parameters

CHAPTER 4 MATERIALS AND METHODS

4.1 Chernicals and Apparatus

4.1.1 Chernicals

4.1 .2 Electrochernical apparatus and accessories

4.2 Experimental Techniques

4.2.1 Preparation of enzyme and chernicai solutions

4 2 - 1.1 Phosphate buffer

4-2-12 Enzyme solutions

4.2.1.3 Standard solutions

4.2.1.4 Solution of PEI -+ NADPc and

PEI + NADPH

4.2.2 Electrochernical apparatus description

4.3 The G6P Biosensor

4.3.1 Construction of the G6P electrodes

4.3 2 Immobilization of the electrochemicai polymer film

4.3.3 Experimental procedure for the characterization

of the G6P biosensor and the nitrate biosensor

4.3.4 Cyclic vokammetry of the G6P biosensor

4.3.5 St~i.age of the G6P biosensors

4.3.6 Assays for the examination of interferences

4.3.7 Determination of the G6P in human blood using

the G6P biosensor

4.3.8 Determination of the G6P in human blood

using a spectrophotorneter

4.4 The Nitrate Biosensors

4-4.1 Construction of the nitrate electrodes

4.4.2 Optimization of the biosensor for nitrate response

CELAPTER 5 RESULTS AND DISCUSSIONS

5.1 Development of the G6P Biosensor

5.1.1 Optirnization of the G6P biosensor response

5.1.1.1 Effect of the construction techniques

5.1.1.2 Effect of pH

5.1.1 -3 EEect of temperature

5.1.1.4 Effect of operating potential

5.1.1.5 EfFect of activator Mg- ions on the response

of the G6P biosensor

5.1.1 .6 Effect of interferences

5.1.2 Charactekation of the response of G6P electrodes

5.1.3 Measurement of G6P in "real samples"

5.2 Determination of Mode1 Parameters and Modei Simulations

5.3 The Development of Nitrate Biosensors

5.3.1 Optimization of the response of nitrate biosensors

5.3.1.1 Effect of pH

5 3 1 . 2 Effea of temperature

5.3.1 -3 Effecf of operating potential

5 -3.1 -4 Effect of enzyme loading

5 -3.1 -5 Stabiiity of the nitrate biosensor

5.3.1 .6 Performance of nitrate electrodes based on

different designs

5 -3 2 Calibration curve for nitrate biosensors

5.3 .3 Determination of nitrate in waste water

CHAPTER 6 CONCLUSIONS AND RECOMMENDATION

6.1 Development of the Glucose-6-Phosphate Biosensor

6.2 Development of the Nitrate Biosensor

6.3 Recornmendations for Future Research

REFERENCES

APPENDIX

VITA

LIST OF TABLES

Table Title Page

Table 2.1 Biological components in the construction of arnperometric biosensors. 10

Table 2.2 Enzyme classification 1 1

Table 2.3 Amperornetric biosensors in the medical analysis 3 i

Table 2.4 Amperometnc biosensors in the environmental monitoring 33

Table 2.5 Amperometric biosensors in the bioprocess control 34

Table 4.1 Surnrnary of protocols for the construction of nitrate biosensor 63

Table 5.1 Response of G6P electrode based on dEeerent construction methods 65

Table 5.2 Characteristic parameters of G6P biosensor in this study 80

Table 5.3 Cornparison of G6P concentration in human blood measured by biosensor

and spectrophotometer using Standard Calibration Methos (SCM) and

Standard Addition Method (SAM)

Table 5 -4 Determination of mode1 parameters

Table 5.5 Sumrnary of important findings in the development of G6P and nitrate

biosensors

LIST OF FIGURES

Figure Tiîie

Figure 2.1 Principle of an amperometric biosensor

Figure 2.2 The Lineweaver Burk Plot

Figure 2.3 A simple three electrode ceii circuit

Figure 2.4 Scheme of redox reaction on the surface of the working electrode

Figure 2.5 Typical Cyclic Voltammogram showing method of extrapolating base

lines and determinkg peak currents

Figure 2.6 Various approaches in the construction of biosensors

Figure 2.7 Methods of enzyme immobilization.

Figure 2.8 Schematic diagram of charge transfer process of the determination

of glucose at a ferrocene-modified electrode

Figure 2.9 Tentative description of the ADH-NAD--PEI compiex within

the CMCPEs

Figure 2.10 (a) Oxidation of NADH by the SAM PQQ electrode. @) Electron

transfer communication of a SAM of PQQ and enzyme by

a dfisional NAD(P) cofactor

Figure 2.1 1 Reaction scheme for a mediated amperometric glucose-6-phosphate

biosensor

Figure 2.12 Reaction scheme for a mediated amperometric nitrate biosensor

Figure 3.1 Model of biosensor mechanism

Figure 4.1 Expenmental set up for amperometric biosensor studies

Page

8

13

15

16

Figure 4.2 Electrochernical ce11 and a Teflon electrode

Figure 4.3 (a) Construction of G6P electrode based on carbon paste (CP)

and chernical modified carbon paste (CMCP). (b) Conceptual view

of entrapped biosensing material

Figure 5.1 The reproduction of the response of G6P biosensor fonn three

electrodes (a, b, c) constructed using CP + CMCP technique.

Steady state currents were measured at pH 7.4, 0.3 V vs. AgIAgC1,

and room temperature (ca. 2 2 ' ~ ) respectively.

Figure 5.2 EEect of the enzyme (G6P-DH) loading on the response of G6P

biosensor. Steady state currents was measured at pH 7.4, G6P 0.8 mM,

0.3 V vs. AgiAgCl, and ca. 22'~ . 69

Figure 5.3 Effect of pH on the response of G6P biosensor. Steady state

currents was measured in the range of the pH 4.7 to 8.7,

G6P 0.4 mM, 0.3 V vs. Ag/AgCl, and ca. 2 2 ' ~ .

Figure 5.4 Effect of temperature on the response of G6P biosensor.

Steady state currents was measured in the range of the temperature

1 C , . . - 3 5 OC, respectively, G6P 0.4 mM, 0.3 V vs. AdAgCl, pH 7.4. 7 1

Figure 5.5 Typical voltarnmograms for the G6P elearode containing TCNQ

in presence of 20 mM G6P standard in buffer at pH 7.4, sweep

rate 10 mV/sec, sweep potential O - 0.8 V vs. Ag/AgCI.

(a) In presence of G6P, (b) In absence of G6P

Figure 5.6 Effea of the potential on the response of G6P biosensor.

Steady state current was measured in the range of the potential

0.1 to 0.4 V . vs. Ag/AgCI. respectively, G6P 0.4 mM, pH 7.4 and

Ca. 22°C.

Figure 5.7 Effect of Mg- ions on the response of G6P biosensor. solid dots

and hollow dots are referred to the response of eiectrodes

containing Mg*- and without Mg-. The operating conditions:

0.3 V vs. Ag/AgCI. pH 7.4 and ca. 2-C.

Figure 5.8 Effect of interference on the response G6P biosensor The iight

color bars show the response to the 0.1 mM interference

respectively. the ark color bars show the response to the blood

sampies where glutathiion and ascorbic acid were incubated in

blood for 4 hours before testing, respectively.

Figure 5.9 Calibration curve for the G6P biosensor. The steady state

current was measured at operating potential + 0.3 V vs.

AdAgCl, pH 7.4 and room temperature Ca. 22°C. Data points

are the average of three measurements with 5 0.07 standard

deviation.

Figure 5.10 Characteristic response of an amperometric G6P biosensor to

the presence of G6P in buffer. Each steady state current increased

was resulted in the addition 0.2 mM of G6P. Sweep rate is

50 sedcm. Current scale in Y axis is 1 nA/ cm.

Figure 5.1 1 Determination K, and V, for G6P biosensor using Lineweaver

Burk Plot.

Figure 5. 12 The stability of G6P biosensor. Solid dots and hollow dots are

xiv

referred to the response of sensor storied in the "dry" state and

in the "wet" state, respectively. The operating conditions: . 0.3 V

vs. Ag/AgCI, G6P 0.4 mM, pH 7.4 and ca. 22°C.

Figure 5.13 Effect of the oxygen on the response of G6P biosensor using

deaerated buffer and non-deaerated buffer. Steady state currents

was rneasured in the range of the pH 4.7 to 8.7, G6P 0.4 mM,

0.3 V vs. AgIAgCI. and ca. 2 2 ' ~ .

Figure 5.14 Determination of G6P concentration in human whole blood

sample using a G6P biosensor based on the standard calibration

rnethod (circular dots) and the standard addition method

(square dots). the operating condition: 0.3 V vs. AdAgCl,

pH 7.4 and ca. 22°C.

Figure 5.15 Determination of G6P concentration in human whole blood

sample using a spectrophotometer based on the standard

calibration method (circular dots) and the standard addition

method (square dots). the operating condition: 0.3 V vs.

Ag/AgCI, pH 7.4 and Ca. 22°C.

Figure 5.16 Lineweaver Burk plot for the determination of 1,. Two

electrodes containing G6P-DH 20 U and 10 U were calibrated

respectively .

Figure 5.17 Modeling of the response of G6P biosensor. The line referred

to the simulating response by the mode1 for G6P-DH in 20 U

and 10 U. The dots referred to the experimental renilts from

G6P-DH 20 (tnangular) and 10 U (square) respectively.

Figure 5.18 Effect of the pH on the response of nitrate biosensor. Steady

state current was measured in the range of the pH 4.5 to 9

respectively. NO3 1 mM 0.2 V vs. Ag/Ag/CI, and Ca. 22°C. 95

Figure 5.19 Effen of the temperature on the response of nitrate biosensor.

Steady state current was measured in the range of the

temperature 18 to 35°C respectively, NO3 1 miM, 0.2 V vs.

Ag/Ag/Cl. and pH 7.5.

Figure 5.20 Effect of the operating potential on the response of nitrate

biosensor. Steady state current was measured in the range of

the potential - 0.2 to + 0.3 V vs. Ag/Ag/CI, respectively,

pH 7.5, NO3 1 mM, and ca. 22°C.

Figure 5.2 1 Effect of the enzyme loading on the response of nitrate

biosensor. Steady state current was measured in the range

of the enzyme loading 0.05 to 1 unit. respectively, 0.2 V vs.

Ag/Ag/Cl, pH 7.5, NO3 1 mM, and Ca. 22°C.

Figure 5.22 Stability of the nitrate biosensor. The response of biosensor

was monitored in an average of three times a day over a

period of 7 days. Operating conditions: 0.2 V vs. Ag/Ag/CI,

pH 7.5, No3 1 mM, and Ca. 23°C.

Figure 5.23 Calibration curve for the nitrate biosensor. the steady state

current at operating condition: 0.2 V vs. Ag/Ag/CI, pH 7.5, NO,,

and ca. 22°C.

Figure 5.24 Characteristic response of an arnperometnc nitrate biosensor

to the presence of NO,' in buffer. The steady state current

increased was resulted in the addition of 0.5 mM and 1 rnM

NO3' (noted). Sweep rate is 50 seckm and current scale in

Y axis is 5 &cm.

CHAPTER 1

INTRODUCTION

1.1 SCOPE OF THIS THESIS

Biosensors are useful analytical devices which have potential applications in medicine.

environmental protection and bioprocess control. With their high specificity. high

sensitivity, portable size and low cos& biosensors hold considerable promise and potential

for various analytical purposes.

The development of biosensor technology has been rapid in the last twenty years. Much

progress has been made in developing biosensors based on oxidase enzymes as biological

sensing elements. Oxidase enzymes are extemal cofactor-independent enzymes and most

of them are quite stable. For example, the glucose biosensor uses the enzyme, glucose

oxidase [E.C. 1.1-3.41, for the determination of glucose concentration in blood (Clark,

1962). This type of biosensor has now been commercialized and is being used for routine

blood tests in the medical laboratones (Owen, 1987). With the demonstrated, successfd

application of such cofactor-independent enzymes in the biosensor area, attention is

tuming to biosensors based on more complex cofactor-dependent enzymes such as

dehydrogenases. There are a large number (over 250) of dehydrogenases which can be

used as sensing materials in biosenson. Biosensors based on these enzymes can provide

the detection of a wide range of biologically important molecular species in

biotechnology and medical analy sis (Appelquia et al., 1 985). However, unlike oxidases,

dehydrogenases require nicotinamide cofactors (coenzymes), Le. P-Ncotinamide adenine

dinucleotide (NADH) or P-nicotinamide adenine dinucleotide phosphate (NADPH), as

CO-reactants in the redox reactions (Dixon and Webb, 1979; Willner and Riklin. 1994).

Therefore, the technology for developing this type of biosensor is more difficult than that

for biosensors based on oxidases. The oxidation of NADH (or NADPH) to enzymatically

active NAD' (or NADP') does not occur at clean electrodes unless a high overpotential is

provided and this will result in high risk of interfenng reactions (Elving et al., 1976).

Many attempts have been made to overcome these problems by using either conducting

organic salts or conducting poIymers to mediate the electrochemical oxidation of NAD-

(or NADP-) so that the requirement of high potential becomes unnecessary (Cass et al..

1984; Lobo et al.. 1995). It was reported (Cass et al., 1984) that electrodes prepared using

these methods have probably been the most successful ferrocene-rnediated glucose

sensors to date. The electrode was operated at +160 mV vs. SCE (saturated calomel

electrode) and low variation in output current is seen with variation of the oxygen tension

of the analyte solution.

Two compounds of importance for which amperometnc biosensors could be developed

are glucose-6-phosphate and nitrate ions. Glucose-6-phosphate (G6P) is an important

metabolite involved in nearly al1 marnmalian metabolism. It can be found in varying

concentrations in the liver, skeletal muscle and adipose tissues. Specifically, G6P is a

substrate of both glucose-6-phosphate dehydrogenase (Gap-DH, E.C. 1.1.1.49) and

glucose-6-phosphatase (GoPase, E.C. 3.1.39), which play a key role in the production of

NADPH and in blood glucose homeostasis (Mcgilvery, 1983; Villar-Palasi and

Guinovart, 1997). G6P concentration directly reflects the relative activities of the

enzymes in the metabolic pathways. In recent studies, many hematologists, geneticists.

and biochemists found G6P-DH and G6Pase to be invaluable tools to study a variety of

fûndamental biological problems (Y oshida and Beutler, 1985). A low concentration of

G6P indicates a decrease of glucose transport and is a defect in the pathogenesis of non-

insuiin-dependent diabetes mellitus w D M ) and insulin dependent diabetes (Yoshida

and Beutler, 1995; Shi et al., 1994). An absence of G6P activity uptake and hydrolysis

was observed in liver microsome fiorn a glycogen storage disease Type la (GSD la)

patient (St-denis el al, 1995 and Waddell et al.. 1989). in those studies. the kinetics of

G6P-DH were investigated by monitoring the concentration of G6P in the samples. The

determination of G6P has been based on radioactive, c hromatographic and

spectrophotometic methods. These traditional methods are precise and suitable for many

applications, but they can be complicated and time consurning, requiring many reagents

and costly equipment. Biosensor technology may provide an alternative for the rapid

measurernent of G6P in biological samples. However, to the author's knowledge, no

amperometnc biosensor has been reported to date for the measurement of G6P in whole

blood.

The determination of nitrate ions in drinking water is currently one of the most important

aspects of analytical chemistry. Human beings may metabolize nitrate finally to convert it

into nitrosamines which are suspected carcinogens Nitrate is also highly toxic to the

fetus. Several methods for the determination of nitrate based on photometry are currently

cornrnercially available (Arnold et al., 1988). However, they have disadvantages such as

high detection limit, low precision high cost, and the operation can only be performed in

the laboratory. Another method for determination of nitrate concentrations is with ion-

selective electrodes (ISEs) (Zuther and Cammann, 1994). Unfortunately, these kinds of

sensors suffer extensively from interferences, and the detection lirnit is high. There is an

urgent need for the determination of the nitrate concentrations in drinking water by an

easy but exact and quick method. A limit of 10 ppm nitrate as unit concentration for

drinking water is recommended by the US Community (Greenberg et al 1990).

1.2 OBJECTIVES

The objectives of this thesis were: (1 ) to investigate the developrnent of novel biosensors

based on dehydrogenase enzymes for the determination of glucose-6-phosphate in

medical applications and for nitrate monitoring in environmental applications; (2) to

optimize the performance of the biosensors with respect to operating conditions; (3) to

apply the biosensors in "real sarnple" systerns.

CHAPTER 2

REVIEW OF LITERATURE

2.1 HISTORICAL BACKGROUND OF AMPEROMETRIC BIOSENSOR

DEVELOPMENT

Amperometnc biosensor technology has only evolved over the last twenty years.

Significant progress of this technology has been made and many types of biosensors have

been developed for various analytical purposes. Today, arnperometnc biosensors are

emerging as important analytical tools for routine tests in clinical laboratones. bioprocess

and environmental monitoring sites (Owen, 1987; Kambe, 1994 and Rogers. 1995).

The first amperometric biosensor was a glucose biosensor developed by Clark and Lyons

( 1962). This sensor relied on the enzymatic reaction:

Glucose + O, GOD > Gluconolactone i- Hz 0, (2.1 )

and used an oxygen electrode. with glucose oxidase (GOD) entrapped at its surface, to

measure the local decrease in the oxygen tension at the electrode surface which is

proponional to the concentration of glucose in the solution. The problem with this

arrangement was the dependence of the output current on the dissolved oxygen tension.

The current became proportional to the oxygen concentration rather than the glucose

concentration when the dissolved oxygen tension was below a certain level.

Subsequently. another glucose sensor was developed by Cass et al. (1984). In this sensor,

the enzyme performs the tira redox reaction with its subarate, and is then reoxidized by a

mediator as opposed to oxygen. The mediator, in its tuni, is oxidized by the electrode:

in the solution

Glucose+ GûD/ FAD + Gluconolactone+ GOD/ FADH,

GOD/FADH,+2Mm +GOD/FAD+2Mr,+2H-

At the electrode

2M, --+2M,+2e- (2.4)

In this scheme flavin adenine dinucleotide (FAD) represents a flavin redox center in

glucose oxidase and M.,, / M d is the mediator which has been assumed to be an electron

couple. Many chernical compounds have been found to be efficient mediators such as

ferrocene F e (CN& N-methylphenazinium (NMP-), tetrathiafùlvalene (TTF). and

7,7,8,8-tetracyanoquinodimethane (TCNQ) (Cardosi and Turner, 1 987).

Another glucose electrode proposed by Albery and Bartiett (1985) was based on an even

simpler and more direct method without mediator and on an electrode material on which

the reduced enzyme GOD/FADH2 can be directly oxidized.

In the solution, one has

Glucoset GODI FAD+Giuconolactone+ GODI FADH,

and at the electrode, one has

GODIFADH, -GOD/FAD+2H'+2e-

The three types of electrodes described above reflect a trend in the development of

amperometnc biosensor technology. Moreover, the fundamental properties of biosensor

behavior must be understood both in terms of its constituents and in the cornple'rities of

their interrelationships in order to optimize criticai criteria such as response time.

selectivity. and stability. Irnmobilization technologies and new membrane materials may

profoundly affect the end performance of a particular biosensor. In the next section. a bnef

review of techniques that have been used for the development of amperometnc biosensors

by many biosensor researchers is given.

2.2 PRINCIPLES OF AMPEROMETRIC BIOSENSORS

An amperometnc biosensor consists of a biologicai sensing element and an

electrochemical transducer. The biological element provides the specific recognition of

analyte relied on their specificity of binding to the analyte. The transducer generates the

signal associated with the specific recognition under an applied potentiai. A generic

scheme of the principle of an arnperornetric biosensor is given in Figure. 2.1.

0 S E P 1 Binding v

O -Cornplex mixture

O

7- Binding site

Signal

.c -

STEP 2

Figure 2.1 Principle of un Amperometric Biosemor (ixkp~edfrom Harwood and Pouton

1996).

\ Imrno bilized biosensing molecules

/ Transduce

* - - Analyte recognition \ and signal transduction

Most amperometnc biosensors involve enzymes such as oxidoreductases which catalyze

redox reactions whose rates are made proponional to the analyte (substrate)

concentration. Typically the progress of the reaction is monitored arnperometncally by

measunng the rate of formation of a product or the disappearance of a reactant. If the

product or reactant is elearoactive, then its concentration may be monitored directly If

the product or reactant is not elearoactive, an incorporation of an electroactive species in

sensors would be needed for shuttling the electrons between the redox center of the

enzyme and the transducer.

In sumary, the basic requirements for an amperometric biosensor are: (i) an enzyme

which acts on its substrate to produce (or consume) a molecule which is capable of being

reduced or oxidized (directly or indirectly) at a suitable electrode; (ii) a method for

immobiliring the enzyme in close proxirnity of the electrode which retains the activity of

the enzyme; (iii) an electronic systern capable of controlling the potential of the electrode

and measuring the current produced by the oxidation or reduction.

2.3 THE BIOLOGICAL SENSING COMPONENT

In biosensors, biological sensing components are used to target appropriate substrates in

the samples so that the determination of the anaiytes can be achieved. Different biologicai

components may be combined with various kinds of transducers provided that the reaction

of the biological elernent with the substrate can be monitored. A number of biological

cornponents have been used to constxua diRerent arnperometric biosensors and as Listed in

Table 2.1.

Table 2.1 Biologi.cal components commonly used in the construction of mperometric

biosensors f C h k 1962; Rzedel et al.. 1989. Wang and Lin, 1988; and Di Gleria et al..

f 986).

Biological components Biosensors - Examples

Enzymes

Micro-organisms

Plant and animal tissues

Enzyme-labeled antibodies

Glucose biosensor

BOD biosensor

Mixed plant tissue-Carbon paste

biosensor

Lidocaine biosensor

The most cornmon biological components applied in biosensor technology are enzymes

because they are usually reasonably stable. soluble in the water and c m be easily purified.

Al1 enzymes can be categorized into six main classes (Enzyme Commission Classification)

as given in the Table 2.2. Oxidoreductase enzymes are widely used in constructing

arnperometnc enzyme electrodes because they are involved in redox reactions where they

transfer H atoms, O atoms or electrons fiom one substrate to another, causing electronic

transportation which cm be detected by the amperometnc transducers. Some

oxidoreduct ase enzymes need ext emd cofactors (coenzymes) to help cat alyze the

reactions and are temed cofactor-dependent enzymes, some of them have tightly bound

cofactors which they have no requirements and are termed cofactor-independent enzymes.

Typically extemal cofactors are nicotinamide adenine dinucleotide (NAD?, nicotinamide

Table 2.2 Enyme cl"srrfiatio~z (Palmer. 1995,)

E.C . Nurnber Enzyme class Type of reaction catalyzed

Oxidoreductases

Transferases

Hydrolases

Lyases

Isomerases

Ligases

Oxidation/reduction reactions

Transfer of an atom or group between

two molecules (excluding reactions in

other classes)

Hydrolysis reactions

Removal of a group from substrate (not

by hydrolysis)

Isomerization reactions

The synthetic joining of two molecules.

coupled with the breakdown of

pyophosphate bond in a nucleoside

triphosphate

adenine dinucleotide phosphate (NADP-) and flavin-adenine dinucleotide (FAD).

There are considerable numbers (over 250) of CO-factor dependent dehydrogenase

enzymes which they cm be used to construa biosenson for monitoring a variety of

important molecular species in biological samples. These types of enzymes have been

routinely used in many enzymatic assays in laboratones but hardly used in biosensors

because of two major problems. Firstly, an high overpotentid is required for the

electrochemicai oxidation of NAD(P)H which bruigs a hi& risk of interference responses

during the measurement. Secondly, the formation of produas such as dimers causes

fouling of the electrode surface (Moirouix et al.. 1980). Thus, development of a biosensor

based on an NAD(P-) dependent enzyme requires overcorning additional problems

compared to those based on oxidase enzymes.

Enzymatic reactions are essential during the process in biosensors. The reactants of

enzyme-catalyzed reactions are tenned substrates and each enzyme is quite specific in

character, acting on a particular substrate or class of substrates to produce a particular

produa or products. The process of enzyme catalyzed reactions cm be modelled by

enzyme kinetic theory. Based on the steady-state assumptions for enzyme-catalyzed

reaction, that is. by assuming that the rates of formation and breakdown of the complex

are equai, a rate equation is developed. The enzyme-catdyzed reaction is:

where E, S and ES refer to the enzyme, substrate and enzyme-substrate complex. ki, kz

and k, are the rate constants.

Mathematical expressions of enzyme-catalyzed reactions are based on the weU known

Michaelis-Menten equation:

where VmK = L, e, and L, kt are the Michaelis-Menten constant and turnover number.

S the concentration of substrate, V,, the maximum rate of reaction. and e the

concentration of enzyme. The Michaelis-Menten equation serves as an adequate example

for the curent purposes and fully accounts for the reaction sequences encountered in

enzyme catalysis. The methods for the analysis of enzyme catalysed reactions are used to

characterize the two important parameters, K, and V-,. By rearranging the equation

(2.9), the Lineweaver-Burk equation (2.10) is derived in the following for the

detennination of Km and V,,.

The plot, being linear, can be extrapolated and fiorn the extrapolated plot the values of Km

and V,, can be determined as s h o w in Figure 2.2.

2.4 Electrochernical Aspects

Amperometric biosensor technology involves a series of concepts of electrochernistry

(Oldham, 1994) which is v e q important for the design and construction of arnperometnc

biosensors. In the foiiowing, a brief introduction regarding basic electrochemical

tntercept = - Z / K , I

Figure 2.2 Lineweaver-Burk PIor

knowledge used in arnperometric biosensor construction is presented.

2.4.1 Three-electrode ce11

Electrochemical experïments are usually conducted in a three-electrode ce11 in which three

electrodes, i.e. a working electrode, a reference electrode and a counter electrode. are

placed with the sample solution to be tested. The working electrode is the biosensor. The

potential of the working electrode is maintained against the reference electrode. The

counter electrode provides a way of completing the cell circuit and allows the current flow

to be driven through the counter electrode instead of the reference electrode. In this way,

the potential drops due to high solution resistance between the working electrode and

reference electrode are minimized. A simple three electrodes ceIl circuit is shown in

Figure. 3.3.

I R E

Figure 2.3 A simple three elecrrode cell circuit (Hill and Sanghera. 1990). WE: Workmg

elecirode. RE: reference elec*ode. CE: Counter elecnode.

2.4.2 Redox reactions

In an amperornetric biosensor, both the enzymatic reaction and the electrochernicai

oxidation or reduction are carried out (Figure 2.4) and can be described by the following

reaction:

where O is the oxidized species and R is the reduced species.

Figure 2.4 Schematic representation of redox reactions ocairring on the nu-jace of the

workmg electrode (HE) in an elec~ochemicuI cell. FE stands for working electrode, R

and O are referred to zhe reduced and oxidiredfonn of species conveyed on the electrode

surface during the process.

The eiectrochernical redox reaction requires an extemally applied potential to overcome

thennodynamic or kinetic constraints. in an electrochemical ceU an applied potential

actually is the dzerence of potential (AE) between a working electrode and a reference

electrode. The appiied potential, AE, wili control the concentration of the two redox forms

in accordance with the Nernst equation:

where CR, C. are concentrations of the reduced and oxidized electroactive species in the

solution and E' is standard electrode potential of the working electrode. When AE = 0. 1

= O, there is no current flow. If AE > O, oxidation may occur, and when AE < O, reduction

occurs.

The reaction process involving chernical species and electrons carried on electrodes cm be

interpreted by Faraday's laws. Faraday's Iaw States that the amount of chernical change

occumng at an electrode is proportional to the quantity of electricity passing through the

cell, and can be described as follows:

Q -= -ANR = AN, nF

where No. NR denote the amount (number of moies) of oxidized and reduced species

present, AN. (or ANR) is the change in oxidiied (or reduced) amount, Q is the amount of

electricity needed to oxidize or reduce aü of the species i in the electrolyte solution of an

electrochemical ceil and F is Faraday's constant (96500 C mol-').

2.4.3 Cyclic voltammetry

Cyclic voltammetry (CV) is a waveform produced by linearly scanning the potential from

an initiai value, Ei, to a second value and then back to the initial d u e . Ei. It is comrnonly

used as the initial electrochemical technique to characterize redox systems and provide

important information on the enzyme-rnediator interactions. The important parameters of

a cyclic voltammograrn are the magnitudes of the anodic peak current (i,). the cathodic

peak current (i,), the anodic peak potentiai &,), and the cathodic potential &). Cyclic

voltammetry is usually performed in a three-electrode ce11 and a typical cyclic

voltammograrn (CV) is given in Figure 2.5. The information that can be extracted from a

CV as follows:

the separation of peak potentials determines the number of electrons transferred in the

electrode reaction for a reversible couple derived 6om Nernst equation (equation

2.15).

Where E, is the anodic peak potentiai. E, is the cathodic peak potential, F is the

Faradic constant and n is the number of eiectrons.

the faradic current (if) indicates the concentration gradient of the redox couple formed

at the electrode surface according to equation 2.16

where Do is the d a s i o n coefficient of the electroaaive species and A is the area of

the electrode. For a reversible couple, i, is approxhnately equal to i, or i, 1 iF n 1

where i,, i, are the anodic and cathodic peak currents in the CV.

The rate constants cm be determined for evaluaiing the performance of mediaton in a

aven enzyme systern (Hill and Sanghera, 1990).

Figure 2.5 TjpicaI Cyclic Voltmmognmr showing method of ewtrapolating baselines and

&tenniningpeak mrrentr. @im?taiytid System, Corn. LI,. 1984)

2.5 CONSTRUCTION OF AMPEROMETRIC ENZYME ELECTRODES

Increasing research efforts have developed a variety of approaches in biosensor

technology. There are many excellent general reviews (Connolly, 1995; Vadgama and

Crumpl. 1992; W t h s , 1993; Wring and Hart. 1992) and monographs (Schmid and

Scheller. 1989; Scheller and Schrnid. 1991) now available. Conventional methods based

on carbon paste techniques have been practicdly used in the construction of cofactor-

independent enzyme electrodes (Cass. 1990). Emerging techniques using chernically

modified carbon paste for constnicting cofactor-dependent enzyme eiectrodes are

currently under active investigation (Harwood and Poutoa 1996). The ability of

controlling the molecular structure of the eiectrode surface is an important advance. and it

allows tailoring of electrodes to meet the requirements of a particular biological redos

system. A variety of approaches developed for modification of biosensors are show in

Figure 2.6 (Bartlett, 1987). These snidies provide the basis for the developrnent and

investigation of G6P and nitrate biosensors in this study, and some are discussed in the

following sections.

2.5.1 Immobilization of enzymes on the biosensor surface

Immobiiization of enzyme is a method for retaining enzymes on electrodes without loss of

enzyme activity. This is an essential step in the construction of a successful enzyme

electrode. There are four methods commonly used for enzyme imrnobilization on

biosensors (Figure 2.7, Barker, 1987 ). They are:

a) Adsorption

b) Entrapment

Biosensors

Multilayer Monolayer

Adsorption Covalent

7 Vapor phase

attachrnent deposition

Reversible Irreversibie

Polymers Cyanuric Carbon Silanization chioride fbnctionali-

zation

Electrochemical Dip or Covalent polymerization dropcoat cross-linking

Conducting Redox polymers polymers

Figure 2.6 Various qprmches in the construction of biosensors @mtIerf, 198 7)

(a) Adsorption

(d) Cross-linking

(b) Adsorption-cross-linking

( c ) Entraprnent

(e) Covalent binding

Figure 2.7 Meth& of enzyme irnmobilization (amker, 1987)

c) Cross-linking

d) Covalent binding

A&orpfio>r

In this technique, enzymes are physically adsorbed ont0 the substances such as silica gel,

glass, hydroxyapatite, and collagen. These substance with enzymes c m be extended to ion

exchangers such as DEAE cellulose, CM-Cellulose, DEAE-Sephadex, and a variety of

phenolic resins (Figure 2.7% b). Adsorption is less disruptive to enzyme protein than

chernical methods of attachent. Because the binding forces of absorbed substances are

hydrogen bonds, multiple salt M a g e s and Van der Waal's forces. they are appropriate to

the electron transition complexes. However, the binding forces are more susceptible to

change in pH, temperature, ionic arength, or even the presence of the substrates (Cabral

et al., 1984).

Ennapment

Enzyme imrnobilization by physical entrapment (Figure 2 . 7 ~ ) has the benefit of

applicability to many enzymes and may provide relatively smdl perturbation to the enzyem

native stmcture and function. The enzyme is entrapped within the polymer network such

as polyacrylamide gel where the enzyme in solution is retained by a membrane permeable

to substrates and reaction products. However, there are two drawbacks in this method:

large diffusion bamiers to the transport of substrate and produa leading to reaction

retardation, particularly with high molecular weight substrates; and continuous loss of

enzyme activity since some pore sizes permit escape of the enzyme. Nevertheless, cross-

linking entrapped protein with glutaraldehyde can ofien overcome the latter problem

(Barker, 1987).

Cross-linking

Immobilization by cross-linking molecules of enzyme is most cornrnonly brought about by

the action of glutaraldehyde, whose two aldehyde groups form Schiffs base link with tiee

amino groups. Suice several free amino groups are likely to be present on each enzyme

molecule. a cross-iinked network will be formed (Figure 1.7d). This method can well

protect enzyme from leaking but cm sometime cause a large Ioss of enzyme aaivity

(Barker 1987).

Covulerzî bonding

The enzyme funaional groups cm be linked by covalent bonds to the support rnatrix to

irnmobilize the enzyme on the surface of electrode (2.7 e). It is essential that conditions

used for the formation of covalent bonds are sufficiently rnild so that liale catalytic activity

is Iost (Barker 1987).

2.5.2 Electron transfer mediators

Amperometric biosensors use a number of conducting chernical compounds called

mediators to effectively transfer electrons between the redox center of the enryme and the

interface of electrodes. An effective mediator mua be chemicdy stable in both reduced

and oxidized forms, and must have a redox potential which it shows Little or no change in

conditions of varying pH. It should be easy to imrnobilw at electrode surfaces, and should

operate at a reiatively low electrode potential (less than 0.4 V vs. reference potential). in

order that potentially interfering molecules cannot electrochemically be oxidized at the

enzyme electrode (Harwood and Pouton, 1996). Many researchers have studied the use of

such electron mediation with varying degrees of success. The most successfÙ1 exarnple

was the ferrocene mediated glucose biosensor based on dimethylfemcinium ion (Cass rr

al., 1984). Performance of this mediated biosensor has shown that the observed anodic

current was the response to glucose in the concentration range 1-30 mm01 dm" of glucose

at an operation potential of 160 mV vs. the Saturated Calomel Electrode (SCE). The

electrocatalytic process of this mediated electrode is given in the Figure 2.8. Other studies

on mediators for biosensor such as tetrathiafùlvalene (Tm) and tetracyanoquinodimethane

(TCNQ) have also been reported (Mulchandani and Bassi 1995; Kulys et al. 1984). TTF

mediated arnperometnc enzyme electrodes were developed for the monitoring of L-

glutamine and L-glutamic acid in growing marnmalian ce11 culture. Under the optimal

operating conditions, these electrodes have demonstrated low detection limit, broad tinear

range, excellent stability, and accurate response at a potential of 0.15 V vs. Ag/AgCl. A

tyrosinase-TCNQ based enzyme electrode for the determination of phenol in water

showed linear cathodic current response against phenol concentration at an applied

potential of 0.13 V vs. AdAgCl.

2.5.3 Techniques of chernical modification based cofactor-dependent enzyme

biosensors

NADH or NADPH dependent enzyme electrodes suffer from the problems of high

overvoltages and side reactions. Many attempts have been made, using various mediators,

Figure 2.8. Schematic diagrum of charge transfer process for the detemination of

glucose at a ferrocene-mod~fied electrode (Cms er al., 1984).

to decrease the overvoltages so that electrochernical reactions can bt used to regenerate

one redox form of the cofactor when dehydrogenase reaction is used in synthesis (Gonon

et al., 1992). Dorninguez and CO-workers (1993) reponed a new approach for carbon

paste enzyme electrodes chemicdly modified with insoluble phenothiazine polymer

derivatives for eIectrocata1ytic oxidation of NADH. This technique made possible the use

of mediators entrapped into polymers that can be cast on solid electrodes or mixed into

carbon paste electrodes, both forms revealing high catalytic efficiency for electrocatalytic

NADH oxidation at low potentials. A cornparison of three dEerent techniques worked

out for the construction of three amperometric biosensors for ethanol based on NADH

dependent yeast alcohol dehydrogenase (ADH) was conducted by testing the biosensor's

response. The response of the biosensor made using this new approach (chemically

modified carbon paste) was highest and fastest for ethanol in Flow Injection System.

Advantages of this technique for constniction of NADH dependenr enzyme biosensors

were that: (1) an effective contact was estabiished between the enzyme, the mediator and

the CO-factor NAD- (Figure 2.9); (2) this design allows the use of an increasing arnount of

enzyme immobilized in the electrode and results in increased reaction rate.

The development of an arnperometnc biosensor utilizing the NAD(P)*-cofactor-dependent

enzyme based on an electropolymer film was descnbed by Willner and Riklin (1994). In

this approach, pyrroloquinolinequinone (PQQ) was covalently linked to the enzyme and a

self-assembled monolayer (SAM) attached on the electrode surface. Regeneration of the

native NAD(P)H cofactor by PQQ could lead to electrical communication between the

electrode and the enzyme redox center. Addition of NADH or NADPH to the PQQ SAM

modified electrode resulted in anodic currents at a potential of - 0.06 V vs. AdAgCl that

depend on the concentration of the added NAD(P)H. No anodic currents couid be

detected in this voltage region when NAD(P)H interacted with the unmodified electrode.

Reaction schemes of this novel technique are given in Figure. 2.10.

2.5.4 Bienyme based biosensors

Amperometric biosensors based on bienzyme systems have been reported in a few

biosensor applications (Schubert et al., 1985; Minitani et al., 1985; Compagnone et al.,

Figure 2.9 Description of the alcohol dehydkogenase- nicotinamide adenine

dinucIeotide-po&ethylenzrnine cornplex (ADH-NAD--PEI) within the chemicaiiy modzfied

cmbon paxte eleclrodes (Dominguez et al. 1993).

S ubstrate

-PQQ -C -NH

NAD(P)H Product

Figure 2.10 (a) Oxidation of NADH by the SAM PQQ electrode. (5) Eleciron-manger

communication of a SAM of PQQ and enyme by a dxfisional NAD(7) ' cofactor

(WNner and Rzkfzn, 199;O.

1977). In these studies, a pair of enzymes were used in enzyme electrodes. Based on the

recyclization reactions catalyzed by a two-enzyme system the amplification of sensor

response was increased 8-40 fold. The sensitivity was increased compared to the

unamplified reactions (Schubert. 1985). The study by Compagnone ( 1977) provides a

glucose oxidasehexokinase electrode for the detemination of ATP. Glucose was

catalyzed by the glucose oxidase reaction and produces hydrogenperoxîde (Hz02) which it

is measured at the electrode surface. When ATP is present in solution, glucose is partially

consumed by the hexokinase reaction decreasing the arnount of H202 produced. The

change in the current is related to the concentration of ATP. The reactions involved in the

measurement are the following :

Gtu cmc audase Glucose + O, -Gluconic acid + H.0, - - (2.1 7)

(S. 18)

The communication of bienzyrne systern may be a prospective approach that c m be

considered to be a usefùl in the development of biosensor based on dehydrogenase

enzyme.

2.6 APPLICATIONS OF BIOSENSORS

2.6.1 Medical applications

There are many opportunities for the application of biosensors in clinical diagnostics.

However, as of now, very few biosensors have been commercialized and used in the

medical applications. The two main reasons are: (1) lacking the developments of suitable

and "robua" biosensors. (2) finding a suitable "niche" in the diagnostic market to displace

current established analytical methods. Several arnperometnc biosensors which they have

been reported for medical application are show in Table 2.3.

Table 2.3 Amperomeiric biosemors in the rnedzcal m&sis (Jm~chen el al.. 1989:

Hanvood et al.. 1995)

Sensor Analyte Type of solution

Glucose Glucose

Hydrogen peroxide lactate, uric acid

Glut m a t e Glutamate

ATP ATP

Bilinibin BiIirubin

W arfarin Warfârïn accohols

Ketone Ketone groups

Blood, urine

Urine, sweat

Blood

BIood

Blood

Drug, urine

Drug, urine

Biosensor technology in this area faces a very big challenge from the well established

measurement systems. Large number of highly-automated diagnostic machines fiom major

instmmentation manufacturers are placed in centralized hospital laboratories for routine

andysis and for the provision of emergency measurements on a state basis (Connolly,

1995). Therefore, the successfbl introduction of biosensors requires that the sensor either

meets a need that the automated machines cannot supply or gives a distinct advantage in

patient care or assessments. In order to exploit more biosensor applications it is imponant

to carefully identie the target users and the required performance for biosensor research.

The opportunities for biosensors based on a particular need in clinical diagnostics are

considered to exist in the following areas (Connolly, 1995):

Direct electron transfer in proteins

Optimization of molecular interactions in the solid phase

Micrometer and nanometre scale behavior of diagnostic devices

Device design for whole blood sensors

Biocompatibility studies for in vivo sensors

Packaging for in vivo sensors

Microfabrication techniques for mass fabrication of sensors

Irnmobilization and protection of biomolecules on multi-anaiyte devices

Signal interference in biological samples

Bed-site monitoring

In home test kits

2.6.2 Environmental monitoring

Consideration of environmental monitoring for the detection of pollutants is becoming

increasingly important to regdatory agencies, the regulated community, and the general

public. Monitoring of hazardous poiiutants in the environments concerning industrial

releases has been legislated, e-g., the Resource Conservation and Recovery Act (RCRA),

the Toxic Substances Control Act (TSCA), the Clean Water Act, the comprehensive

Environmental Response, etc. (Rogers, 1995). Two hundreds and seventy five pollutants

are listed as priority hazardous substances based on their potential deleterious effects on

human health or eco-systems. Driven by the need for fast, ponable and low-cost methods

for environmental monitoring, a number of biosensors are currently being developed.

Table 2.4 lists arnperometric biosensors developed in the field of environmental

monitoring.

Table 2.4 A mperorne~ic biosensors de veloped in the environmental monitoring (Kanrbr .

f 994; Hansen et al.. 1989)

Sensor Andyte Type of solution

BOD

Nitrite

Ammonia

cetylcholinestera

Sulfite

Cyanobactena

BOD oxygen

Nitnte

Ammonia

se Pesticides

Sulfite

hazardous substances

Waste water

Waste water

Waste water

River, well water

Air

W aterway s

2-6.3 Bioprocess control

Biosensors have potential applications in biotechnological in-line or on-line process

control and on-line measurements of the concentrations of bioproduas or reactants. The

main advantages of using biosenson in bioprocess are: (1) on-line bioprocess control: ( 3 )

fast availability of results from the process; (3) handy and easy to operate for workers; (4)

safe and clean working environment; (5) low cost. Some measurernents using biosensors

for various biotechnology substances in a process are given in the table 2.5.

Table 2.5 Amperomeîric Bzosemors for Bioprocess Controi (Kmibr. 199-1)

Sensor Analyte Type of solution

Glucose

Assimilable sugars

Acetic acid

Ammonia

Methanol

Glutamic acid

Formic acid

Methane

Short chain fatty acid

Amino acid

Urea

Fructose, sucrose

Acetic acid

Arnmoni a

Methanol, ethanol,

alcohol

Glutamic acid

Formic acid

Methane

short chah fatty acid

amino acid

Urea

Molasses broth

Animal ce11 culture

Molasses broth

Acetic acid culture broth

Nitrifymg media

Yeast fermentation broth

Food fermentation broth

Aeromonas fomicans culture media

Methane culture media

Raw milk

Food fermentation media

Urea culture media

2.7 PRINCIPLES OF THE AMPEROMETRIC G6P BIOSENSOR

A new arnperometnc biosensor for the determination of glucose-6-phosphate (G6P)

concentration in bIood incorporates a NADPH-dependent enzyme, a TCNQ mediator. and

NADP- and was constructed using chernicaiiy modified carbon paste based on novel

technique developed by Dorninguez et al. (1993). The reaction sequences to be expected

are as follows:

G6P i NADP + ,6 - phosphate - Gluconate + NADPH

G6P and NADPF are catalyzed by the enzyme. G6P-DEI, to generate NADPH and the

product 6-phosphate gluconate (Eq. 2.19). Oxidation of NADPH is chernically

undertaken with mediator, TCNQ (Eq. 7-20), while T CNQ is electrochernically oxidized

at low operation potentials resulting in the production of two electrons at the electrode

sudace. Anodic current response presented by a G6P electrode should be proportional to

the arnount of G6P utilized for the reaction under appropriate operating conditions. The

reaction scheme of the G6P biosensor is depiaed in Figure 2.1 1

2.8 PRINCIPLES OF AN AMPEROMETRIC NITRATE BIOSENSOR

A mediated amperometric nitrate biosensor is proposed in this thesis which uses a

NADPH-dependent enzyme, nitrate reductase w), a mediator, and an electropolymer

for its construction of this biosensor. The reaction sequences are descnbed as follows:

Electrode surface

Figure 2.1 1 Reaction scheme for a rnedialed amperometrzc glucose-6-phoqhate

biosensor where G6P-DH is the enzyme; NADP- is the CO-factor and TCNO,. TCN&

are the oxidized mtd reduced fm of the mediator.

N O j - i N . 4 D P H > N 0 2 - + N A D P -

Med, + NADP- -+ Med, + NADPH

Med ,, t e LW > Med ,,

NR catalyzes the chernical reaction to produce nitrite and NADP- (Eq. 2.22). a mediator is

expected to regenerate NADPH while it is electrochemicaiiy reduced under an operating

potential. A cathodic current response should be obtained which is proportional to the

concentration of nitrate in the solution. The reaction scheme for a mediated amperometric

nitrate biosensor is presented in the Figure 2.12.

Electrode surface

Figure 2.12 Reaction scheme for a rnediated amperometric ninate biosensor where NR is

the enzyme, nitiwte redzictase; NADPH ir the co-jactor and M e d , Med,, are the

o x i ~ t i o ~ ~ and reduction fonns of mediators.

It may be noted that the nitnte formed is unstable and under appropriate conditions it too

can be electrochernicaiiy oxidized (Lin and Wu 1997). In this study, an oxidation current

at an operating potential of + 0.2 V vs. Ag/AgCI, was also observed with the nitrate

biosensor. This can be accounted for by the following proposed mechanism:

KN02 + TCNQ, TCNQ,, + O2

In the studies of nitrate reductase Nason and Evans (1953), Garrett and Nason ( 1969) and

Donald and Alan (1974) have reported that nitrate reductase contains groups of oxidation-

reduction components. They proposed the foiiowing electron transport sequence for

nitrate reductase for the enzyme activities:

NADPH +AD +cytochrome b S n 40 -O7- J.

cytochrome c

where Mo is the enzyme active site. Genetic studies (Cove, 1 966; Beers and Sizer. 1 95 1)

have revealed considerable complexities in the regdation of the synthesis of the enzyme.

Mutations in any one of at least six independently segregating genetic loci lead to the

absence of a functionai nitrate reductase although in many cases a defective protein is

produced which retains the ability to catalyze either one of the two associated reactions. A

conclusion has been stated in the study of Donald (1974) in that the kinetic data indicate

that a lack of interaction between the NADPH and the nitrate binding sites could also

suggest a physicai as weii as a functional separation of the two ends of the electron

transport sequence. Thus the evidence is compatible with the assumption that the nitrate

reduaase molecule consisting of a iinear series of electron carriers capable of being

altematively oxidized and reduced d u ~ g the transport of electrons from one end of the

sequence to the other. On the other hand, the magnitude of the response given by nitrate

electrodes with Merent mediators may be associated with the structure of the mediator

compounds used.

2.9 SUMMARY

A bnef literature review has been given about several aspects of biosensors: concepts of

biosensor constituents and electrochemiary, conventional techniques used to produce

arnperometnc biosensors, novel techniques using chernical modifications for NAD(P)H-

dependent enzymes electrodes, and applications of arnperometric biosensors. With these

knowledge and technology about biosensors in mind, two proposais for the development

of arnperometnc biosensors for the determination of giucose-6-phosphate in blood and

nitrate in solution were presented and carried out in this study.

CHAPTER 3

MODELLING OF STEADY STATE RESPONSE OF BIOSENSORS:

APPLICATION TO G6P BIOSENSOR

3.1 MODEL DESCRIPTION

The modeling of processes in enzyme electrodes is important in order to achieve an

understanding of biochemical kinetics for a biosensor and for the optimization of

operating parameters. Using a model to evaluate experirnental data the rate limiting steps

in the transaction of the analyte concentration into a sensor response can be established,

and the relevant mass transport and enzyme kinetic rates cm be determined. Recently,

research has shown increasing interest in this area. Many models relating the biosensor

response to various rate processes have been proposed (Meil and Maloy, 1975:

Schulmeister and Schubert, 1989). The model proposed by Chen and Tan (1995)

descnbed the steady-state sensing characteristics of a biosensor based on biooxidation of

organic solutes by dissolved oxygen. Leypoldt and Gough (1984) have similarly reponed

a model for predicting the immobilized enzyme arnount in the membrane of a glucose

biosensor in order to control the range of glucose detectability. This model is very helpfùl

for the design of sensors when the CO-substrate can become the limiting substrate for the

enzyme reaction.

In this chapter, the mathematicai modelling of processes in the operaiion response of a

G6P biosensor is proposed. The following assumptions are made: (1) one substrate, one

produa enzyme, which converts substrate S to Produa P, using the NADP'MADPH as

cofactor. (2) It is assumed that there is suficient NADP- presenr so that the concentration

of free enzyme, E, is much smaller than the concentration of enzyme bound to NADP-. It

is also assumed that the kinetics of the binding of the enzyme to NADP- is sufficiently

rapid so that equilibrium is established between E and E-NADP*: The reaction schemes

are as foilows:

where S and P is the substrate and product, E-CO. and E'-CR are the enzyme-cofactor

complexes. CO and CR are the oxidized and reduced cofactor (NADP* or NADPH). N

and M are the oxidized and reduced mediators. KI, kz, k3 and kE is the rate constant (MI

-2 -1 S-'1, or (mol cm s / (M mol cm'*)).

3.2 MODEL EQUATIONS

Considering the case of an enzyme electrode the kinetic mechanism is illustrated in

Figure 3.1. Three layers are presented in the figure (1) A bulk solution difision layer (b),

(2) An enzyme-mediator layer (a), and (3) the surface of an electrode (O).

Assuming steady state conditions have been reached:

Rate of substrate supply: J, = D, (So - Sa) (b - a)

Rate of reduced mediator suppiy: J , = - D , ( N a - N o ) / a

Rate of oxidized rnediator supply: J , = D , @ f o - ~ )

Rate of mediator electrochernical oxidation: J,,, = k- No

The output current density: 1 = n Fe, es Jbulk

where n is the charge number, F = 96500 C 1 mol, e, is charge transfer eficiency

At steady state:

Based on these equations Tatsuma and Watanabe ( 1 992) derived the following equation

for the characteristics of the steady state response of a multi-layer modified enzyme

biosensor in the linear region.

where D,, D, are the diffiision coefficient of mediator and substrate. b is the thickness of

the diffision layer in the bulk soIution, a is the distance fiom the electrode surface to

enzyme layer. and r is the total enzyme surface density.

Bulk solution

Carbon paste electrode \ layer

Figure 3.1 Model of biosenror rnechanzsm. Mo and No are the oxidized and reduced fonn

of mediator in the surfce of electrode. M . and Na me the oxzd id and reduced form of

rnediator in the enzyme-mediator layer. SO and Sa me the ~bs t rare in the bulk solution

and enzyme-medialor kayer.

3.3 MODEL FORMULATION

To easily determine the model parameters, the following alternative model equation is

proposed:

Defining k' = Dn / ka b. k" = 1 / (b - a), equation (3.12) equals to equation (3.1 1). The

lumping o f parameters in equation (3.11) leads to modification of the model of Tatsuma

and Watanabe. The model parameters may now be more easily determined using the

procedure described as follows:

3.4 DETERMINATION OF MODEL PARAMETERS

Equation (3.12) can be rewritten in the form of

where

which is the dope of the linear curve represented by equation (3.13), and c m be

determined by the dope fiom the cuwe fitting of the linear range of the experimental

i - Sb, response. k , r can be evaluated by equation

where I,, is the maximum current density and can be obtained from the intercept of the

fitting curve using Lineweaver-Burk equation (Figure 3.2). For a given electrode with a

certain enzyme amount:

where Et is the total enzyme amount. and A the surface area of electrode. k , can be

calculated using Eq. 3.17

Given the value of n, F and D, the two model parameters k and kW can be determined

f?om experimental data for which the steady aate equation (3.12) applies.

The expenmental results obtained in this study were employed to develop a model for the

G6P biosensors. The details of the experimental methods and results will be reponed in

the next two chapters.

CHAPTER 4

MATERIALS AM) METHODS

4.1 CHEMICALS AND APPARATUS

4.1.1 Chernicals

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49, Aspergillus species. from yeusts. E.

d i ) , glucose-6-phosphate. nicotinamide adenine dinucleotide phosphate (NADP ' ),

giycylglycine, G6P-DH assay kit, nitrate reducatase (EC 1.6.6.3, arpergjIIz~s nidu Ians.

from yrart), nicotinamide adenine dinucleotide phosphate (NADPH), bovina serum

albumin, polypyrrole, glutathione, ascorbic acid were purchased from Sigma Chernical

Company (S t. Louis, MO, USA). 7,7,8,8 -Tetraqanoquinodimethane, polyethylenirnine,

1,3 -p henylenediamine, 1 , 1 -dimethyLferrocence, tetrathiafulvalene, resorcinol,

glutaraldehyde, graphite powder, minera1 oil were supplied fiom Aldrich Chernical

Company (USA). Potassium nitrate, sodium phosphate, potassium phosphate, magnesium

chloride, sodium hydroxide were purchased from Fisher Scientific Company (USA).

Human whole blood samples were obtained h m Victoria Hospital, London, Ontario.

4.1.2 Electrochemical apparatus and accessories

Most of electrochemical apparatus used in experiments are from Bioanaiyticai Systems inc

(BAS) (Lafayette, Indiana, US A). They include electrochemical carbon paste Teflon

electrodes with a cavity in size of 3 mm diameter, 5 mm depth, glassy carbon Teflon

electrodes, Ag/AgCl reference electrode, platinum wire counter electrode, electrochemical

cell, Faradic cage, Voltammograph (Model CV-27), low current module (Model PAI), X-

Y-t chart recorder.

4.2 EXPERIlMENTAL TECHNIQUES

4.2.1 Preparation of enzyme and chemical solutions

4.2.1.1 Phosphate buffer

Phosphate buffers (0.1 M pH 7.4) were made as follows: to make 1 liter of phosphate

buffer, 13.6 g of -PO4 and 14.2 g of NazHP04 were dissolved in the deionized water to

make 1 liter of solution each. 160 mL of KWzPOj was added to 840 mL of Na2HP04. .4

pH meter was used for adjusting pH until it read 7.4. The dserent pH phosphate buffers

made for testing the effect of pH on the response of electrodes were followed with similar

procedures with respect to pH. Sirnilar procedures were also followed for making 0.25 M.

pH 7.4 Glycyglycine buffer using 0.1 M sodium hydroxide as required.

4.2.1.2 Enzyme solutions

G6P-DH was prepared using 0.25 M pH 7.4 glycylglycine buffer. 200 pL of glycylglycine

buffer was added to a bottle containing G6P-DH 100 unit to make 0.5 unit/pL

concentration of G6P-DH solution. The activity of the enzyme was detemiined by an

enzyrnatic assay to determine the arnount of enzyme needed for the production of a G6P

biosensor. The assay method used was enzymatic assay of glucose-6-phosphate

dehydrogenase (Sigma Chernical Company). Nitrate reductase solution was prepared

using 0.1 M, pH 7.5 phosphate buffer. Two hundred microliters of buffers were added to

the bottle containing nitrate reductase crystals to form 0.05 Unit/w of enzyme stock

solution. The aaivity of nitrate reductase was assayed before construction of the

biosensor. The assay method used was enzyrnatic assay of nitrate reductase WADPH)

provided by Sigma Chernical Company.

4.t.l.3 Standard solutions

A O. 1 M glucose-&phosphate standard solution was made for testing the response of G6P

biosensor. A 0.03 + 0.002 g G6P-Na was dissolved into 1 rnL of deionized water A O 1

M KN03 standard solution was made for nitrate biosensor by adding 0.01 k 0.002 g

KNOj to 1 rnL deionized water. Glutaraldehyde and Bovine sexum albumin stock

solutions were used at the sarne concentration for immobilization of the enzyme on the

surface of the biosensor. A total of 10 pL of giutaraidehyde regent was dissolved in 100

pL of deionized water to form glutaraldehyde stock solution and 5 k 0.5 mg BAS was

dissolved in 100 jiL deionized water to make BAS stock solution. Glutathione and

Ascorbic acid as potential interference are required for testing response of G6P biosensor.

O. 1 M each solution was made with deionized water.

4.2.1.4 Solutions of PEI + NADP' and PEI + NADPH

A 50 % Polyethyienimine solution was diluted in deionized water and made up to 1 mL

0.2 % PEI diluted solution. 5 t 0.5 mg of NADPt was added into 0.2 % PEI solution and

well mixed at room temperature. In a similar way, using 5 + 0.2 mg of NADPH to make 1

rnL solution of PEI + NADPH was made.

4.2.2 Electrochemical apparatus description

Cyclic voltamrnograph system used in this study is shown in Figure. 4.1. In this syaem,

Figure 4.1 Experimentd sel up for amperornetrzc biosensor studies

the working electrode, the AdAgCl reference electrode and the counter electrode were

placed on a 10 mL cell. A magnetic nimng bar in the ce11 provided uniform mixing (Figure

4.2) during the operation. The voltammogram was connected to three elenrodes to gîve a

readout of the current at a certain applied potential. A low current module (signal

amplifier) was connected to the voltammogram for readout when the electric signai was

very low (e.g. current in nA level). Current-time curves were recorded in a chart with a

XY-t Recorder.

4.3 THE G6P BIOSENSORS

4.3.1 Construction of the G6P electrodes

Three diEerent protocols were investigated for the construction of G6P electrodes. The

first protocol was as follows: carbon paste was prepared with 50 mg graphite povier and

T CNQ in minera1 oil. This mixture was packed into the Teflon electrode (BAS) and a 0.5

mm dent was made on the surface of electrode. A 50 pL of mixed solution containing PEI

and NADP- was dropped on the top of the paste. 20 of enzyme G6P-DH solution (0.5

Unit1p.L) was then added. The electrode surface was dried for 4 - 5 hours. Finally, an

electropolymer film of 1, 3-phenylenediamine-resorcinol was formed on the surface of

electrode using procedure in Section 4.3.2. The rnodified electrode was then washed in the

stirred buffer container for a few minutes and then stored in the buffer, ready for testing.

In the second protocol, carbon paste was prepared in a similar way to that described

above. The enzyme was immobilized by cross-linking with glutaraldehyde and bovine

Counter

Working Electrode

Reference Electrode

Electrode

Nitrogen Maint enance Nitrogen Purge Tube

Teflon Electrode Body

Tube

Figure 4.2 Elecbochemicol cell and a Tefin elecrode (BAS Chem. Cm.)

semm albumin (BSA). One and a haif milliliter of giutaraldehyde and BAS were added on

an electrode respectively and the mixture was dned for an hour. M e r lmmobilizatio~ the

electrode was washed with the buffer and stored in the buffer.

In the third protocol, the electrode was made using 4: 1 mixture of unrnodified carbon

paste (CP) and chernicaily modified carbon paste (CMCP). The CP was made by

thoroughly mixing with 40 mg carbon graphite, 10 pl of mineral oil until a uniform paste

was formed in a g l a s dish. The CMCP was made by combining enzyme (e.g. G6P-DH).

CO-enzyme (NADP3, mediator (TCNQ), activator (MgClz) and polyrner (PEI). Five mg

TCNQ was previously dissolved in 100 pl of toluene and the toluene totaily vaporized a

few minutes later. Dried fine TCNQ crystals were mixed with 10 mg carbon graphite. 100

pl of solution containing 0.2% PEI and 6.5 x 105 M NADP* and 20 units G6P-DH were

added into the paste with TCNQ to form CMCP. The g l a s dish containing CMCP was

placed in a desiccator and allowed to dry under vacuum for approxirnately three hours.

Dried CMCP was gentiy combined with 4 pl of minerd oil until a unifonn paste was

obtained.

Figure 4.3 shows the constniction of G6P electrode based on this procedure. Firstly, CP

was tightly pressed into the cavity of the Teflon electrode to fil1 3/4 of volume and CMCP

was added to the top. Then the electrode was poiished on weighng paper to produce a

flat, shiny surface with an area of about 7 x 104 m2. The electrode was then washed with

the buffer and stored in "dry" or "wet" state as describeci in Section 4.3.5 unti

Teflon Electrode

L

CMCP

'b

Teflon electrode 'G~P \ G6P-DH, NADP'. PEI

TCNQ

(b)

Figure 4.3 (a) Consiruciion of G6P electrode bmed on carbon paste (CP) and chernical

rnodifed carbon paie (CMCP). (6) Conceptual view of entrapped biosenszng materials.

4.3.2 Immobilization of enzyme behind an electrochemical polymer fùm

Immobilization by an electropolymer film on the surface of the biosensor was carried out

Oas follows: The working electrode with absorbed enzyme, reference electrode AdAgCl

and a platinum wire counter electrode were placed into an electrochemicai cell. The ce11

contained a deaerated solution of 1.5 mM each of resorcinol and 1.3-phenylenediamine in

10 rnL 0.1 M (pH 7.4) phosphate buffer. The working elearode potential was cycled

between O and 0.8 V vs. Ag/AgCl at a scan rate of 20 mV/s for a total of 8 cycles to

deposit a polyrner film behind which the adsorbed enzyme was prepared. Cyclic

voltammograrns were recorded on the X-Y recorder. Findly, electrode was washed

immersing its tip into a phosphate buffer for 15 minutes and any unbound materials and

monomers were removed.

4.3.3 Experimental procedure for the characterization of the G6P biosensor and

nitrate biosensor

The procedures for cdibration of G6P electrodes are described as foiiows: the working

electrode, AdAgCl reference electrode, and platinum wire counter eiectrodes were

inserted into the electrochernical ceU containing 10 mL phosphate buffer (O. i M, pH 7.4).

A magnetic stir bar was used to mix the contents of the electrochemical ceiI. A constant

potentid (e.g. 0.3 V vs. Ag/AgCI for G6P) was applied to the working electrode and the

background current was dowed to stabilize. Standard solutions (0.1 M) of either G6P or

NO,' were consecutively injected into the buffer with a 20 pL increment for each addition.

The response current to each injection was measured when a steady state current was

attained. Caiibration curves for the G6P biosensor were prepared based on the results

obtained from experiments described above. A net increasing current was calculated by

subtraction of the steady state current in addition of G6P to the background current (Zb )

in absence of G6P or to the steady state current corresponding to the previous addition of

G6P Thus (1-Ib) versus concentration (C) were plotted. A linear concentration range was

determined by the results (Km) presenting in a linear relationship on the calibration

graph. Standard derivations were based on three sets of expenmentai data. The constant

Km of the reaction rate was cdculated based on Lineweaver-Burk method.

The response time was determhed by measuring the time intervai from the injection of

G6P to the point where the following steady state current was reached. The limit of

detection was determined by comparison of background signal fluctuations and signal

response. A signdnoise ratio of 3 was employed and the detection limit was determined

to be 50 ph4 for the G6P electrodes.

The stability of G6P electrodes was examined by testing the response of severd electrodes

to the G6P standard in buEer solution respectively. The results fiom these tests were

plotted by using the response currents to the 0.4 mM of G6P against tirne. Besides, a

comparison of the effect of the response on storage by dBerent method was also

presented on this plot, and results are discussed in chapter 5.

î h e effect of operating potential, pH, and metal ions (Mg-) on the response of the G6P

biosensor were examined. Experiments for examinhg temperature effects on the biosensor

response was conducted in a jacketed ceil comected to a water bath with a temperature

controîîer.

4.3.4 Cyclic Voltammetry of the G6P biosensor

Cyclic voltammetnc midies were carried out by vaqhg the potentiai between -0.2 - 0.6

V at 10 mV/s for 1 cycle. This procedure was repeated after 30 rnM G6P was added to

the cell for another cycle.

4.3.5 Storage of the G6P biosensors

It is very important to use appropnate methods to store biosensors. A conventional

method named "wet" method for storing biosensors is used to insert biosensors into the

appropriate buffer and keep then at the appropnate temperature. in the "dry" method, the

tip of the G6P biosensor was tightly covered by a cap and stored at the temperature of

4°C. The results based on the two storages of the biosensor will be discussed in the

Section 5.1.4. The nitrate biosensors were stored by immersion in phosphate buffer (0.1 M.

pH 7.5) at 4°C.

4.3.6 Assays for the examination of interferences

The effect of interferences on the response of the G6P biosensor was investigated. The

fust experiment was performed to examine the response of the G6P electrode in the

standard solution of buffer containhg an interferencing substance. The second experiment

was conducted to measure the response in the blood sample solution where interferencing

substances were added to blood and samples of blood were previously incubated for four

hours. Glutathione and ascorbic acid, which are electrically active and nomaily exist in

blood (Luuatto. 1993), were used as interferences in these assays. The results of these

assays are discussed later.

4.3.7 Determination of G6P in human blood using the G6P biosensor

The new developed G6P biosensors were applied to the "reai samples". human whole

blood for the determination of G6P concentration. Standard Calibration Method (SCM).

was employed for the experirnents. To ven@ the resuits obtained fiom SCM an

altemiative method, Standard Addition Method (SAM), was also employed for the

determination G6P concentration in blood sarnples. In addition, the same human whole

blood sample tested by a biosensor was tested again using SCM and SAM by a

spectrop hotometer. The procedures of these methods in testing are described in the

following.

P r e p m i o n of soiutions

Six standard solutions of G6P were prepared in the range from 0.1 M to 0.6 M. Blood

samples were kept in room temperature for a while before testing.

Procedure based on Standard Cufibration Technique

Experimental procedure for blood sample assays were:

1. Construction of G6P biosensor based on carbon paste and chernical modified carbon

paste techniques. The fieshly making sensor was stored in a refigerator over night.

2. The caiibration of G6P biosensor was camïed out using the prepared G6P biosensor

under an optimal operation condition where phosphate buffer (O. lm pH 7.4), applied

potential 0.3 V vs. Ag/AgCl and room temperature. A standard calibration graph was

constructed based on the response of the G6P biosensor to the additions of prepared

standard solutions of G6P in each 10 pL aliquots.

3 . The buffer in the ce1 was changed but the condition was identical to what is described

above. A fi@ pL human blood sample was directly pipetted fiom the blood sample

bottle and was injected into the stirring buffer after the background current reached a

steady state. A net increasing anodic current was measured after current arrived in

next steady state.

4. Determination of G6P concentration in blood was measured based on a calibration

curve (Figure 5.14) constructed as described previously. The G6P concentration in

human whole blood was calculated based on the calibration.

Procedure based on standard Aadition Method

Blood sarnples were also assayed using a standard addition method (Harris, i 991; Bories

and bories, 1995). The expenments were conducted using the sarne G6P biosensor and on

the same day. In the standard addition method, calibration curves for G6P were prepared

as descnbed earlier with one dinerence. Each increasing concentration of G6P contained

the sarne amount of blood, e-g. the first injection was 10 pL of buEer (no G6P) and 50 pL

of blood, subsequent injection were 10 pL b a e r (containhg increasing concentration of

G6P) and 50 pL blood. The concentration of G6P in human blood was determined by the

extrapolation of the caiibration curve.

43.8 Determination of G6P in human blood using spectrophotometer

The principle of the reaction is that glucose-6-phosphate is oxidized by oxidized

nicotinamide-adenine dinucleotide phosphate (NADP*) to 6-phospho-gluconate in the

presence of the enzyme glucose-6-phosphate dehydrogenase. The chernical reaction

equation is:

Glucose - 6- Phosphate + NADP- G6P-DH.Llg'- b 6 - Phospho - giuconate + NADPH

The arnount of NADPH formed during the reaction is stoichiometric with the arnount of

glucose-6-phosphate. The increase in NADPH is measured by means of its absorbance at

340 m.

Preparation of solutzom:

a. Phosphate buffer: (0.1, M pH7.4)

preparation described in section 4.2.2.2

b. G6P-DH solution (1 0 U/ mL):

Diluted stock suspension (G6P-DH fiom yeast) using 0.25 M Glycylglycine buffer, pH

7.4.

c. Standard soiution of G6P was made in 0.05 M, 0.1 M, 0.15 M., 0.2 M. 0.3 M. and

0.4M using deionized water.

Magnesium chionde (MgCl2 0.3 M) was made by dissolving 0.02 g magnesium

chloride in deionized water and made up to I mL.

Nicotinarnide adenine dinucleotide phosphate (NADP' 0.02 M) was made by

Dissolving 0.03 g NADP- in deionized water and made up to 2 mL

Prepurution of sample solution

Human blood was obtained from Victoria Hospital, London, Ontario and stored at 4°C.

Before assay blood sample, solution was ailowed to stand for 30 min at room temperature

to avoid the effea of temperature on readouts.

Procedures of experimenf based on the S t u n h d Calibrarion Method

1 . Tumed on spearophotometer and allowed it to w m up for 30 min, set UV at 340

nm

2. Adjusted readout of absorbance to zero against air

3 . Pipetted 1.9 rnL phosphate buger, 0.9 mL NADP-, 0.1 mL MgCh, and 0.1 mL G6P-

DH into a cuvette and rnixed well,

4. moved cuvette into spearophotometer, read absorbance (&) when absorbance was

stable (about 5 min later)

5 . added 3 pL 0.05 standard soIution of G6P and 1 pL buffer into cuvette and read

absorbance (A,) when it was stable

6 . recorded readouts of absorbance &, Ai and used AAio = Ai - & to construct

calibration graph

Repetition of steps above and readouts of A3, AID, were obtained frorn the

additions of other different concentration solutions of G6P used to construct calibration

graph.

This experimental procedure using standard addition method followed steps 1-4 as

descrobed in section 4.3.7, but in step 5, absorbance readouts were based on each addition

of a 1 pL blood sample with a 3 pL different concentration solution of G6P.

The methods used in spectrophotometry for the determination of the G6P concentration in

human blood samples based on SCM and SAM were similar to that employed in biosensor

measurement and were described in section 4.3.7.

4.4 THE NITRATE BIOSENSORS

4.4.1 Constructions of nitrate electrodes

Similar protocols as described for the G6P biosensor were for the construction of the

nitrate biosensor. These are surnmarized in Table 4.1 to avoid repetition.

4.1.2 Optimization of the biosensor for nitrate response

Optimization of the response for the nitrate biosensor was perfonned by charac te~ng the

biosensor response to the variable parameters: pH, temperature. operating potential.

enzyme loading, and method of construction. The stability of the response of the nitrate

electrode was also evaiuated. The cdibration curve for the nitrate biosensor was plotted

with respect to the substrate concentration (NO3) at the optimal operating conditions.

Most experiments were carried out using the methods that were employed for the G6P

biosensors. The nitrate biosensor was constmaed in the previous day of testing and stored

in the phosphate buffer (pH 7.5, 0.1 M) at a temperature of about 4 ' ~ during the rest of

the time. The sarne eiectrochernical system was employed to conduct al1 the optimization

tests.

Table 4.1 Summary of protocols for the construction of nitrate biosensors

Protocol Technique

CP + CMCP containing TTF or DMF, electropolymer film of

Resorcinol and 1 -3-Phenylenediamine on the surface of the

electrode.

CP + NR, PEI-NADPH, mediator on surface of the biosensor.

electropolymer film of Resorcinol and 1.3-Phenyienediarnine on the

electrode surface, (mediators used are TCNQ. DMF and TTF)

CP + CMCP (NR PEI-NADPH, Tm), Cross-finking, membrane of

glutaraidehyde and bovine semm albumin

CP + PEI-NADPH, DMF and NR above the CP, (NR used of 0.5,

1, or 2 units respectively for tests), poiymer film of resorcinol and

1,3-phenylenediarnine on the electrode surface

CP + CMCP (PEI-NADPH. DMF, and TBP), polymer film of

resorcinol and 1.3 -p heny lenediamine on the electrode surface

Using glassy carbon electrode, mixture of NR, PEI-NADPH and

mediator diredy added on the electrode surface, polypyrrole film

above the mixture,

CHAPTER 5

RESULTS AND DISCUSSION

5.1 DEVELOPMENT OF TEi'E G6P BIOSENSOR

This section descnbes the research undertaken towards the development of the G6P

biosensor. The effect of the method of construction and operating conditions. pH.

temperature, enzyme loading is first considered. The linearity, minimum detection lirnit

and stability are described. The application of the biosensor to measurement of G6P in

human blood is presented.

5.1.1 Optimuation of G6P biosensor response

The optimization of G6P biosensor response was investigated which included the method

of the electrode constniction, effects of pH, temperature, operating potemial. activators.

biosensor stability, interference, and expenmental characterization of various sensor

parameters.

5.1.1.1 E f k t of the construction techniques

The construction of the G6P electrodes has been studied in three different protocols. The

performance of electrodes constructed using these protocols is shown in Table 5.1. It can

be seen that electrodes constructed by the method of protocol 3 show a higher response,

shorter response time and longer lifetime. The electrodes made by the methods of protocol

1 and protocol 2 appear to be less sensitive, and have longer response tirne and shorter

lifetirne.

Table 5.1 Response of G6P elecmde b d on d%feent cons~~ctzon methmis

Protocols Response Response time Life time

(MmM) (sec) (day

Protocol 1 and protocol 2 were designed according to the previous studies on the

development of amperomentric glutamine electrode (Mukhandani and Bassi, 1995).

trnrnobilization of G6P-DH and NADP- on the surface of TTF modified carbon paste

electrode was completed by entrapment behind an electrochernicalIy deposited film of 1,3-

phenylenediamine and resorcinol copolymer and glutaraldehyde cross-linking. However, it

was found in this study that the elearodes constructed by these methods did not show

sensitive and stable response. Effort for improving the response of electrode was made in

designing protocol 3 based on the previous work of Dorninguez et al. (1993), and it was

observed that this type of electrode exhibited a more sensitive and stable response to the

substrate, glucose-6-phosphate.

The problems with protocols 1 and 2 may be explained as foilows: (1) large diffusion

barrier of polymer membrane to the transport of substrate, and the additional barrier to the

substrate due to the accumulation of product on the surface of the electrodes; (2) leaking

of biosensing molecules with low molecular weight, for example, NADP- or mediators:

(3) enzyme and PEI being the macromolecules having big molecular weight so that it may

be too heavy for them to be held by the film of PDA-resocinol polymer or the membrane

of glutarahylde-BS4 resulting in a fouling problem on the surface of elearodes; (4)

immobilization of cross-linking using glutaraldehyde and BSA deactivated G6P-DR

resulting in the lower response of biosensors (Barker, 1987). In conctusio~

immobilization of polymer film and cross-linking are not suitable for the construction of

the G6P-DH electrodes.

The technique of protocol 3 used to construct G6P electrodes overcomes the problems

associated with the methods used in protocols 1 and 2. As shown in Figure 4.3. the CF

above the CMCP in protocol 3 provides an effective electric pathway to transfer the

reaction signal from CMCP to voltarnmograph, and provides good contact with the

CMCP. In the CMCP, enzyme. CO-factor, mediator and PEI are intimatety combined. The

PEI acts as a polymer backbone binding ail biosubstances to prevent loss. Thus, there is no

need of an additional extemal membrane for the retention of biosubstances on the

electrode surface so that substrates are able to directly react with biosensing materials

resulting in faster response.

The reproducibility of the G6P electrode based on the constmction of protocol 3 was

studied by measuring the steady-state current of three electrodes as shown in Figure 5.1.

The weii reproducible responses were obtained in the iïnear range nom these electrodes

under optimal operating conditions. The study on the effect of the enzyme loading on the

Figure 5.1: n e reproduction of the respunse of ~ h e G6P biose~~sor from three electrodes constmctrd using the CP - C M ï P technique. Steadv state airrems were measured at pH 7.4

0.3 1' vs. Ag A g / . and room temperuttire @a. 22" C) respectiveiy.

output signal of G6P biosensor was found that the more sensitive and higher response

were observed fom the electrodes containing the higher Ioading of enzyme (Figure 5.2) .

This is due to the fact that an increased arnount of G6P-DH is available to catalyze the

reaction.

5.1.1.2 Effect of pH

The effect of pH on the response of G6P electrode was studied using a phosphate buffer in

the range of 5 to 8.5 with 0.4 mM glucose-6-phosphate standard soiution. It was found

that the response of the electrode was a strong function of pH featuring a response curve

of current against pH shown in Figure 5.3. A maximum response was observed at the pH

of 7.4. This result indicates that the enzyme activity of G6P-DH can be well presented at

this condition. This value of pH is found to be consistent with that used to perform the

assay of G6P-DH by the spectrophotometnc method (Sigma Chem., 1993).

5.1.1.3 Effect of temperature

The expenment for examining the effect of temperature on the response of G6P electrode

was performed in a jacketed elearochernical ceU which was comected to a water bath of

controlled temperature. The G6P electrode was tested at temperature arranging fiom 1 8 ' ~

to 35'~ and the results are shown in Figure 5.4. It can be seen that the current response of

the G6P electrode increases with the Uicrease of temperature in presence of 0.4 mM G6P

standard solution. The maximal responses were exhibited at a temperature of 30'~. The

higher response rnay be a result of increasing activity of the enzyrne at higher temperature.

However, fiom the previous study (Palmer, 1995), the activity of some enzymes may

G6P-DH (Unit)

Figure 5.2 Eflect of the enzyme (G67P-DH) Ioading on the response of G6P biosensor. Stea& state ciment war measirerd al pH 7.4. G6P 0.4 mM, 0.2 vs. Ag A@, and ca. 22 C.

Figure 5.3: Eflect of pH on the respome of G6P biosensor. Steady srare mtrenl was meanrred in the range of the pH 4.7 to 8.7,

respectively. 0.3 C F vs. Ag AgCI, G6P 0.4 mM. and CU. 22 C.

Temperature (OC )

Figure 5.4: Eflect of the temperature on the response of G6P biosensor. S t r a 4 smte cun-ent wos meanrred in the range of the

temperature I 5 - 35 " C. respective&, G6P O. 4 mM. 0.3 V vs. Ag AgCI. pH 7.4.

decrease under very hi& temperanires. This was also found in our study: even higher

temperatures led to a decreased and unaable response. In addition. a shoner lifetime with

the electrodes tested at above 25°C was observed. As a result, al1 further experimentation

was conducted at room temperature (ca. 22" C).

5.1.1.4 Effect of operating potential

EIectrochernicai behavior of G6P biosensors was investigated to find the electrochemical

potential for optimal performance of the G6P biosensor. A cyclic voltammograrn (CV) in

Figure 5.5 shows the electrochemical behavior of G6P biosensor in the range of potential

fiom -0.1 to 0.6 V vs. Ag/AgCI. The curve b describes the response in the absence of

substrate G6P in buffer, and the curve a represents the response in the presence of G6P 20

mM in buEer. An anodic current peak is observed around potential 0.4 vs. AdAgCl on the

CV due to the oxidation of mediator TCNQ to TCNQ cations occumng at the surfâce of

G6P electrode. It can be seen in the curve b that a significant increase of anodic current

(about 80 pA net increase) was results from the addition of 20 mM G6P standard solution

in buffer due to the increase of TCNQ cations. Based on the observations presented in the

CV graph (Figure 5 . 9 , the optimal potential for G6P biosensor was selected Le. 0.3 V vs

AgIAgCl. Expenments were also conducted using various constant potentials. The results

are presented in Figure 5.6. It is seen that in the range of potential 60m 0.1 to 0.4 V vs.

AdAgCl current response increases with increasing potentials with a smaller increase rate

of current beyond potential of 0.3 V which is in a good agreement with information 6om

the CV.

Potential D(IAgIAgCI]

Figure 5.5 TpicaI voltmntograns for the G6P elecîrode containing TCNQ in presence of20

mM G6P simtdmd in h@er ut pH 7.4. sweep rate IO m Visec, sweep potentiui O - 0.8 V vs.

AgiAgCi. (a) In presence ofG6P. (3) In absence of G6P.

Potential (V)

Figure 5.6: EfJect of porstitiul on the response of G6P biosemor. Steagv siate nrrrent was rneasured in the range of the opera t i~~g pofrntial O. I to 0.4 V vs. Ag,AgCi, respective&,

pH 7.4, G6P 0.4 mM. um'ca. 22" C.

5.1.1.5 Effect o f activator ~ g * on the response of G6P electrodes

Previous studies have indicated that divalent cations ( Mg-) are required as activators for

the G6P-DH catalytic reaction in the liver tissue, yeast and bactena. The involvement of

this activator leads to the modification of protein stmcture during enzymatic reaction

(Wilkinson, 1963; Scheller et al.. 1991). In this study, it was found that the large

increasing current were observed in the presence of 0.4 mM G6P from the biosensors

containing Mg- in cornparison to the biosensors without Mg--. The response of the

biosensors using electrodes with Mg-- was nearly IO-fold the response from the sensors

without Mg--. In Figure 5.7, Curve A represents the response of a biosensor constructed

with 1 x Io4 M Mg-' , and curve B gives for the response of sensors without Mg--. The

higher response of senson containing Mg" may be attnbuted to Mg-- activation in the

reaction.

5.1.1.6. Effect o f interferences

The effect of interferences on the output current of G6P biosensor was investigated. Five

biosubstances, glucose, hctose. sucrose, glutathione and ascorbic acid. were tested for

this purpose. It was found that no interfering response was obtained fiom glucose.

mictose and sucrose but strong interfering responses were found from glutathione and

ascorbic acid, which are electrochernically active metabolites in blood. However. a

significantly reduced response (80% reduction) was observed from the hurnan whole

blood sarnples which were prepared by previously adding glutathione and ascorbic acid to

the blood and by incubating for 4 hours with air (see Figure 5.8). The significant reduction

of the i n t e r f e ~ g response may be attributed to the fact that glutathione and ascorbic acid

Figure 5.7: EHect of Mg * - ions on the response of G6P biosemor. Solid dots a»d hollow dots are referred to the

rrspunse of electrocies cotztai»zing Mg - ' and without Mg - * . 7he

operuring corditions: pH 7.4. 0.3 L' vs. Ag. A g î i , and CU. 22 C.

(yu) asuodsaa

are irreversibly oxidized by oxygen in the erythrocytes (Luzzatto. 1993; Wilkinsoe 1962.

Mathews and Van Holde, I W O ) .

5.1.2 Characterization of the response of the G6P electrodes

A caiibration curve of G6P electrode is shown in Figure 5.9. Three sets of expenmental

data were used to construct the calibration curve and a linear equation was obtained by

curve fitting. The response in the range of G6P concentration was linear from 0.05 to 0.6

rnM. The characteristic parameters of the calibration curve are listed in Table 5 2. It can

be seen that the current response becomes nonlinear and less sensitive to the addition of

G6P in the buffer when concentration of G6P is beyond 0.6 mM. The response time of the

G6P biosensors was found to be ca. 50 seconds corresponding to the injection of 0.2 mM

G6P in buffer (Figure 5.10).

The kinetics of the enzyme reaction of the G6P electrode was analyzed based on the

Michaelis-Menten kinetic equation. Assuming that enzyme reaction is represented by

The rate of reaction is

Figure 5.9: Calibration curve for the G6P biosensor. The steady stute current was measured at pH 7.4. operating potential + O. 3 C.' vs. Ag/AgCl,

and room remperature (22'~). Duta points are the merage of three rneasurments with + 0.0 7 standard deviation.

Table 5.2 Characteristic parameten of G6P biosensor in this study

Parameters Results

Linear range

Slope

R'

Standard deviation

Km

v m

Response time

Detection limit

0.05 - 0.6 mM

13.552

0.9869

0.07 (average)

3.19 (mM)

59.5 (nAhM)

50 Sec

0.05 mM

The Lineweaver-Burk equation based on equqtion (5.2) plotting the reciproca1 reaction

rate (v) against reciprocal subarate concentrations is

This equation is used for the determination of rate constant, K, and the maximum

reaction rate V,. The results are show in Figure 5.1 1.

The stability of the G6P biosensors was aiso evaluated as a fùnction of tirne. It was found

that the stability of the biosensor response was largely dependent on the methods used for

storing them. Two methods employed for this purpose have been described in chapter 4.

In Figure 5.12, the electrodes aored in "dry" state showed 95% of the original response

during the first 5 days (assuming 100 % response on the firn day) and then the response

graduaily decreased in the next 10 days. However, 50% of the original response was still

retained after 14 days. In cornparison, the response of the electrode aored in the "wet"

state was only observed within the first seven days under the same testing condition. The

probiems with these electrodes rnay be attributed to the leaking of biosensing substances

al1 the time during the storage.

The effect of aeration and deaeration on the response of G6P electrodes was also

examined and no significant change in current response was found from the tests under

both conditions (see Figure 5.13).

Figure 5.11: Deiermir~utio~~ uf K , and C', for G6P bioserzsor ushg Lrneweaver B w k Plot.

Time (days)

Figure 5.12: The stability of G6P biosensor. Solid dots and hofZow dois are referred to the response of sensor stored in the "dry'' state and in the "wrt " slate. resprcrively. n e operating corditio~rv: pH 7. -1. 0.3 1' W. Ag AApCi. ca. 22 " C.

14 - A Deaerated

Figure 5.13: Effect of the 0-1 on the response of G6P bioserisor mirg drarrated bufler and non-deaearated bufler. &ad,\? statr cztrrent wax mea.uïred at pH 7.4, 0.2 V vs.

Ag AgCl. ard 22 " C.

5.1.3 Measurement of G6P in "real samples"

The study of the application for the new G6P biosensor for "real" samples was perfonned

in human whole blood using Standard Calibration Method (SCM) and Standard Addition

Method (SAM). The concentration of G6P in a blood sample was firn determined by

SCM. To evaluate the performance and applicability of SCM for this "red" sarnples. SAM

was employed for the same blood to obtain a calibration curve. fiom which the G6P

concentration was determined by the intercept on the x-axis. The result using SAM was

found to be in agreement with that from SCM. Figure 5.14 presents the SAM calibration

curve, together with the one fiom SCM. Three blood samples were teçted in this way and

the results are presented in Table 5.3. To further validate the developed G6P biosensor.

the same blood sarnples were tested using enzymatic spectrophotometnc technique. It was

found that the results were in excellent agreement with the measurements using the new

G6P biosensors (see Table 5.3). Again, both SCM and SAM were employed for this

technique, and the results are shown in Figure 5.15. The overall agreement of the

measurernents for the G6P concentration in the whole blood using different andytical

techniques indicates that there is no or very Little interference on the biosensor response.

This is consistent with the observations discussed previously in section 5.1.3 -6. regarding

the effect of interferences. Additionally, the values of G6P concentration measured in this

study are within the range of G6P concentration in blood mentioned in the literature

(Luzzatto, 1993;) where K,,, value shows 0.070 mM in human blood (Wilkinson, 1962;

Miwa and Fujii, 1985).

Table 5.3 Cornparison of G6P concentrutio~z in human bloocl meanrred &y biosensor md

spec~ophotometer using Stundard Calibrution Method (KI() inxi Standard Additiorz

Method (SAM,).

l Number S pectrop hotometer G6P Biosensor

It was found in these tests that the time for measurements using the G6P biosensor is

much shoner than the time used in the spectrophotometric method. The average time for

analysis with a biosensor is 15 minutes for a complete assay, while a spectrophometric

assay can require 3 hours. Therefore. the new biosensor can provide faster and more

economical measurements for determination of G6P in human blood.

Test resuits measured from G6P biosensor method and spectrophotometry using both the

standard calibration (SCM) and standard addition methods (SAM) are shown in Table 5.3.

It is found that SCM and SAM rnethods provide consistent results for al1 the test samples.

5.2 Determination of model parameters and model simulations

A mathematical model was formulated in this study for the G6P sensor and presented in

Chapter 3. To determine the model parameters, two sets of experimentai data were used

to construct two curves in the relationship of the current vs. concentration using two

electrodes containing 20 units and 10 units of enzyme respectively. The siopes of these

two curves were used to obtain two equations from (3.14). To determine kl in equation

(3- 17). Lineweaver-Burk plot was employed for the same experimentai data (see Figure

5.16). 1 , was obtained from this plot and then kl was calculated with Equation (3.17).

The surface area of the electrode A was taken to be 0.0007 dm2. Having values of n F and

D, based on reference (Tatsuma and Watanabe, 1992), the two equations from (3.14)

were used to determine the two model parameters, k' and k". Table 5.4 shows the values

of ali the constants in the model equation (3.12).

Figure 5.16: Lineweawr Burk plot for the determinatzon of lm, .

Two rfectrodrs conlainittg G6P-DH 20 U and 10 (1 were cnlibratrd respectively.

Table 5.4 Determination of rnodel parameters

Paramet ers Value

9 - 96500 C/mol

1 O-' dms s-'

2.33 dm' mol-' s*'

2.5 x IO-^ dm

1.6 x 10"dm-'

The proposed model was verified using experimental data obtained in this study. For

biosensors with given enzymes, the model results were calculated by equation (3.12) and

presented in Figure 5.17, together with the experimental measurements for cornparison. Lt

is found t hat the proposed model adequately descnbes the steady-state kinetic mechanism

and characteristics of the G6P biosensor in the Iinear region.

The value of the t em k W 1 (k , r) is found to be several times that of 11 D, , depending on

the arnount of enzyme used. It can be inferred that the enzyme kinetics infiuence the i-Sba

response more than dfis ion does. It should be noted that no membrane was used in

constmcting the G6P electrodes and the airred conditions were appiied in the

expenments. The experimental techniques and conditions used in the development of the

G6P biosensor are different from that assurned in the reference (Tatsuma and Watanabe,

ISOOO -

Figure 5.17: Modefling of the response of G6P biosettsor. The line referred to the simiriaiion response by the mode! for G6P-DH in 20 U and 10 U. m e dots referred to zhe experimental rendts Rom G6P-DH 20 U (aimgrdar) and 10 U (square) respective&.

1992). Therefore. km should not be physically interpreted by the definition of b and a in

equation (3.11). it only stands for the influence of the enzyme kinetics in the i-SbuUr

response.

With the use of lumped parameters. the model constants k: k" and kl can be easily

determined using nvo sets of experimental data for a given type of enzyme. The mode1

then allows the prediction of i - S ,, response for biosensors using different enzyme units.

This capability has the potential to provide useful information for optimal design of the

G6P and other biosensors. Further work may be needed to provide experimental data to

validate the model prediction of i -Sb, response for various electrodes with different

enzyme units and to determine the range where the model applies.

5.3 DEVELOPMENT OF NITRATE BIOSENSORS

5.3.1 Optimization of the response of nitrate biosensors

Optimization of nitrate biosensors was conducted for the following parameters: pH,

temperature, operating potentiai, enzyme loading, stability of biosensor and construction

techniques.

5.3.1.1 Effect of p H

The change in current of the nitrate biosensor responding to 1 mM of nitrate ions in buffer

at various pH values was detemiined. The experhental results showed optimal pH to be

about 7.5 in the Figure 5.18. This pH was then set at 7.5 for the following experiments.

Figure 5.18: Ejfecr of rhe pH ~n the response of nitrate biosensor. Steady state m e n t was rneawred in the ronge of the pH 4.5 10 9 respective&, NO I mM. 0.2 V vs. AgiAgCi,

and Ca. 22 C.

5.3.1.2 Effect of temperature

The influence of temperature on the response of the biosensor was also examined at

various temperatures. In Figure 5.19, the response current is shown to increase with

increasing temperature. This change was associated mainiy with the activity of the

enzyme, nitrate reductase. However, the stability of the biosensor decreased quickly with

each use for the test conducted above room temperature. Therefore, room temperature

(ca. 2 2 ' ~ ) was employed for subsequent tests.

5.3.1.3 Effect of operatiog potential

A number of potentials in the range of -0.3 to 0.3 V vs. Ag/AgCI were investigated for

examining the response of nitrate biosensors. The optimal potential was selected, 0.2 V vs.

AgiAgCl, fiorn the experimental results (Figure 5.20) in order to maximize the response

and minimize interferences which may occur at higher potentials. Deaeration was

employed to the buffer solution and nitrate standard solution using nitrogen gas when tests

were conducted under a negative applied potential to avoid the influence of oxygen.

5.3.1.4 Effect of enzyme loading

The output signal of the nitrate biosensors was found to Vary with the enzyme loading. In

Figure 5.21. an increasing response of the nitrate biosensor was observed with the increase

of the nitrate reductase loading on the electrode surface. This indicates that the increasing

amount of nitrate reductase l a d s to increasing activity initiaily, hence results in a higher

response of the electrode to the substrate because the CM + CMCP structure of electrode

Temperature ( O C )

Figure 5.19: Efect of remperaiure on the response of nitrate biosensor. Steady statr airrent was measured in the raqge of the temperature 18 fo 35 C reqectiveiy, pH 7.5. N O , / mM. 0.2 b v vs. Ag ARCI.

Potential (V)

Figure 5.20: Effecr of the potentiai on the response of nitrate biosensor- Steadj state mrrent was measured in the range of the potenfial - 0.2 tu - 0.3 V vs. Ag.AgCI

re~pectiveiy, pH 7.5, NO I mM, and Ca. 2 O OC.

Enzyme loading (unit)

Figure 5.21: Eflecr of enzyme loading on the response of nitrote biosrmoi-. Sieaùj siate current was rneanrred in the range of the emymr hading 0.05 to I units respectively. pH 7.5, NO, 1 mM. 0.2 LvvsAgAgCf. d c a . 22°C.

has a higher capacity of enzyme which allows the loading of more enzyme. However.

overloading should be avoided since it can decrease the response due to access particuiar

interaction of biological macromolecules.

5.3.1.5 S tability of nitrate biosensor

As it can be seen in Figure 5.22, the nitrate biosensor gives the highest response in the first

day and then the response is maintained at about 65 % of the original response during the

second and the third days. A reduction of 88 % to the response was obtained from the

experimental results for a 7 day old nitrate biosensor. The stability of the biosensor was

dependent on the methods of biosensor construction, the storage methods and the

temperature. The technique with carbon paste and chernicaily modified carbon paste used

in the construction provided a more stable structure for the electrodes so that a relatively

more stable response could be maintained. An appropriate environmental condition was

needed for storing the biosenson to protect them from losing biosensor activities during

the storage.

5.3.1.6 Performance of nitrate electrodes based on the différent designs

The constmction of nitrate biosensors has been proposed in severai protocols (in section

4.4.1). Electrodes constructed by protocol 1 were to have shown a more stable response

than that constructed by protocol2. The design of protocol 1 using carbon paste (CP) plus

chernically rnodified carbon paste (CMCP) is also suitable for the construction of nitrate

biosensor.

O

O O - 7 4 6 8 10 12

Time (day)

Figure 5.22: Stability of the rtitrate biosensor. The response of biosemor was rnonitored in a» average of three tirnes a dq): over a period of 7 -S. Operating condition: NO i

mM, pH 7.4, 0.2 Ci vs. Ag A@, and ca. 22 O C.

In protocol 3. substances on the surface of electrode were expected to bind with cross-

linking solid support. Unformnately, this method seems not to be suitable for this type of

biosensor. During the tests the biosensing layer was found to graduaiiy fa11 off fiom the

electrode surface resulting in unstable current and no response thereafter. The reason for

this may be due to the large amounts of the biosensing substances.

Another attempt was made in the construction of the nitrate biosensor using polypyrrole

(PP). According to previous work (Begum et al.. 1993). a glucose biosensor composed of

FAD-(flavinadenine dinuc1eotide)-enzyme, TTF-TCNQ and PP exhibited an effective

molecular interface where electrons directly transferred between PP and FAD-enzyme and

the transducing electrode proceeded at a low potential. However, elearodes made in this

method suffer from the interference problem because the conductive PP responded to the

chloride ions.

5.3.2 Calibration Curve for nitrate biosensors

The calibration curve in Figure 5.23 showed a linear range of O to 1 rnM of nitrate ions

concentration for a nitrate biosensor. The construction of this biosensor was based on the

technique with carbon paste and chemically mod8ed carbon paste containing 1 unit of

nitrate reductase. An expenment was conducted under optimal operating conditions at

potential 0.2 V, pH 7.5, and room temperature. The increasing current response versus

t h e to the addition of nitrate in buffer is shown in Figure 5.24. The lower detection Limit

of nitrate electrodes was found to be O. 1 m . in the cell.

Figure 5.23: Caiibratiort arme for the nitrate bioset~sor- n e steady state crrrrent was measured at operatzng condition: pH 7.5. poteritid O. 2 Y vs. Ag A@, and ca. 22 C.

5.3.3 Determination of nitrate in waste water

The applicability of the nitrate biosensor for the "real sample" was examined in a waste-

water sample with a given nitrate concentration of ca. 300 ppm. A nitrate biosensor was

constructed two days ago and recalibrated immediately before the "real sample" test.

Experiments were conducted under optimal operating conditions and the nitrate

concentration was found to be about 3 10 ppm. Since the developed nitrate biosensors

were stable only for a few days ahead the nitrate reductase is expensive. fùnher

experiments were not carried out in real sarnple application.

5.4 Cornparison of the G6P and nitrate biosensor

Table 5 .5 shows the summary of important iindings in this study. The major pararneters,

stability, linearity and minimum detection liinits, are compared in this Table. As shown.

due to the relatively higher minimum detection limit and the lower stability for the nitrate

biosensor further research is needed to improve the response.

Figure 5.5 Szmary of inportml findings in the development of G6P und n i m e

b iosertsors

Linear range

Minimum detection limit

Stability

G6P Biosensor Nitrate Biosensor

0.05 - 0.6 mM 0.1 - 1 mM

0.05 rnM 0.1 rnM

14 Days 7 Days

CHAPTER 6

CONCLUSIONS AND RECOMMENDATIONS

In this thesis, an amperometric G6P biosensor and an amperometric nitrate biosensor have

been developed for the determination of glucose-&phosphate and nitrate in the aquatic

phase. Based on the proposed principles of glucose-6-phosphate biosensor and nitrate

biosensor, extensive experimental work has been undertaken to constnict both biosensors

and to charaaerize their response under optimal operating conditions. With the

consideration that developed amperometric biosensors should posses excellent sensitivity,

stability selectivity, and easy operation with a low cost, both the G6P and nitrate

biosensors were operated at room temperature. Important conclusions and discussions for

the development of the glucose-6-phosphate and nitrate biosensors are surnrnarized in the

below.

6.1 DEVELOPMENT OF GLUCOSE-6-PHOSPHATE BIOSENSOR

The procedures for construaing the G6P biosensor were initially investigated in several

approaches to achieve optimal response. -4 novel technique featuring combination of

carbon paste with chemically modified carbon paste showed the best performance for the

G6P electrode. It was then employed for constructing al1 the G6P electrodes in this study.

The techniques employed for experimentation were found to be very useful to examine the

performance of electrodes and to delineate their problems. The cyclic voitammograph

techniques help to identify the redox reaction takùig place on the electrode surface at

cenain potentids. The ultraviolet spectrophotometic method provides accurate results for

identifjmg the reliability of the G6P biosensor measurements in the 'real sarnple'

app lications.

In order to achieve the best performance for the G6P biosensors, the operating conditions

were investigated and optirnized at pH 7.4, operating potential 0.3 V vs. Ag/AgCL. and

room temperature (ca. ZOc) Higher response of the G6P biosensors was achieved by

loading more enzyme. glucose-6-phosphate dehydrogenase. The addition of an activator.

Mg" ions in the construction of glucose-6-phosphate electrodes was found to increase the

response of biosensor in nearly 10 fold. The biosensor showed a nearly stable current

response for â days and a decreasing response with 50 % reduction observed by the end of

second week. This is quite acceptable in biosensor technology due to the highly sensitive

nature of biological enzyme used. An appropriate method for storing the bsensor in the

"dry" state was investigated and the G6P electrode stored by this method showed more

stable response and longer life time.

Glucose-6-phosphate biosensors have been tested in the 'real sarnplet-whole human blood

for the practical applications. Experimental results obtained using the new biosensor were

in excellent agreement with the measurements from the standard spectrophotornetric

method, and aiso were consistent with the of G6P concentration levels in the human whoie

blood reported in the literature. No appreciable electrochemical interference response was

observed from the G6P biosensors in the samples tested. However, interferences do exist

from ascorbic acid and glutathione and if there are presents, steps need to be developed to

remove them.

A mathematical model based on Tatsuma and Watanabe's steady-state formulation was

developed for the G6P biosensor to predict the biosensor response. Simulations using the

developed model will have potential to provide usefbl information for design optimization

of the G6P biosensor.

6.2 DEVELOPMENT OF THE NITRATE BIOSENSOR

The investigation for the development of amperometric nitrate biosensor was conducted in

a way similar to that employed for the development of the glucose-6-phosphate biosensor.

The technique of the combination of carbon paste and chemically modified carbon paste

was found to be more stable and reliable for the construction of the nitrate biosensor based

on the obtained experimental results.

Characterization of the response of the nitrate biosensors was carried out to optimize the

operating conditions. The nitrate biosensors exhibited good performance under the

conditions of pH 7.4, operating potentiai 0.2 V vs. Ag/AgCI, and room temperature (ca.

2 2 ' ~ ) which are more relevant to practical application conditions.

6.3 RECOMMENDATIONS FOR FUTURE RESEARCH

Based on the technicd investigations and expenrnental results presented in this thesis. a

number of recornmendations for future research are listed as follows:

1. The G6P biosensor may be implemented for working in a flow injection system for

rapid monitoring of G6P in blood or other biological samples. The techniques to

remove electrochemical interferences need to be developed as well.

2. More 'real sarnpie' assays may be explored for the G6P biosensor applications. For

exarnple, the G6P biosensor should be able to measure the G6P concentration in liver.

skeleton muscle, and some bacteria. In the biotechnical processing, it can provide rapid

monitoring of G6P in the G6P-DH production fiom yeast.

3. More biochemical information about enzyme (nitrate reductase) types. properties and

cataiytic mechanism is needed to improve the sensitivity of the nitrate biosensor.

4. Future studies for the nitrate biosensor would involve:

test h g new mediators

improving stability by new immobilization techniques

applications to bioprocess monitoring

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APPENDIX I

DATA FOR OPTIMIZATION OF G6P AND NITRATE BIOSENSORS

1. Optimization of G6P biosensor

Table 1. 1 The reproducibof the response of G6P biosensor based on three electrodes

A, B and C

G6P

(mm O

0.2

0.4

0.6

Table 1.2 Effect of enzyme loading

Current A

(W O

3.5

6.8

10

G6P-DH

(V i t )

5

IO

20

30

40

50

Current B

(W O

3.5

6.8

9.7

0.8

1

Current C

(W

O

3.5

6.8

10.1

12

14

12.5

15

Response

(5)

O

15.4

20.2

38

56

1 O0

13.1

15.8

Response II

- 5 -2

6.5

- 19

-

Response I

O

5

6.8

12.5

18

33

Mean response

(nA)

O

5.1

6.65

12.5

18.5

33

Table 1.3 Effect of pH

Table 1.4 Effect of temperature

PH Current

Temperature

ec) 15

18

20

32

25

30

Percent

(%)

I I I 37 6.8 80 1

Current

1.7

2.4

3 -4

6.8

7.3

8.5

Current

(%)

20

28

40

80

86

1 O0

Table 1.5 Effect of operating potential

Table 1.6 Effect of activator Mg* ions

Potential (V)

vs. AdAgCl

o. 1

O. 15

0.2

0.3

0.4 I

Current

(nA)

3.5

4.0

4.4

6.1

7. O

G6P

mM

O

Current

(%)

57

66

72

1 O0

115

Response (nA)

without Mg"

O

Response (nA)

with Mg"

O

Table 1.7 Effect of interference

Table 1.8 Calibration curve

Interference

Glutathione

Ascorbic acid

Glucose

hctose

lSUCroSe I o. 1 I O I -

I

Response (Stan.)

(W 24

120

O

O

G6P

(mM)

O. 1

O. 1

O. 1

O. 1

Response (blood)

(d)

3

30

-

-

Table 1.9 Determination of K, and V,

Table 1.10 Stability of biosensor based on the electrode A stored in "dry" and the

electrode B stored in "wet"

Time

(day)

1

2

3

6

8

9

16

Response A

(nA)

3.8

3.8

3.7

3.5

3 -2

1.9

Response B

3.8

3 -5

3.2

O. 5

Response A

(%)

1 O0

1 O0

97

-

93

84.5

50

Response B

(%)

100

93

87

-

13

- -

Table 1.1 1 Effect of deaeration and aeration on the response of G6P biosensor

Table 1.12 "Real sample" test

G6p (mM)

O

0.2

0.4

0.6

0.8

Biosensor assay S pectrophotometric assay

Current (nA)

O

4

7

12

17

Absorbance 1 Absorbance

Current (nA)

O

4

8

1 1

16

2. Optimization of nitrate biosensor

Table 2.1 Effect of pH

Table 2.2 Effect of pH

1 Temperature Response I Response

Response

(%)

40

65

PH

5

6

Response

5 -6

9.1

Table 2.3 Effect of operating potential

Table 2.4 Effect of enzyme loading

Potential

V vs. Ag/AgCl

-0.2

Current

(nA)

4.9

Enzyme loading

m i t )

Current

(%)

32.5

Response

( n . 4

Table 2.5 Stability of nitrate biosensor

Time (day) I NO3 (mM) 1 Response (%) i

Table 2.6 Calibration curve

L

No3 (MM)

O

O. 1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

1

1.2

1.4

1.9

2.4

2.9

3.9

4.9

Current (nA)

O

O. 5

2.1

3 -5

5.1

6.5

8.5

10.5

13

15

18

19.5

21.5

23.5

25.5

28.5

30.5

l/S (IIniM)

O

O. 1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

1

1/i (l/nA)

O

0.5

2.1

3.5

5.1 ,

6.5

8.5

10.5

13

15

3. Expermental setting up condition for G6P and nitrate biosensors

Table 3.1 Experimental conditions for different assays

I ASSAY

Calibration of G6P

Calibration of nitrate

EIectrochemical

Polyrnerization

--

Eapp: -O. 1 - 0.6 V

Scan rate: 10 mV/s

Gain: O. 1 mNV

Eapp: 0.3 V

Gain: O. 1 mAn/

Eapp - 0.4 V, -0.6

V, -0.8 V

Eapp: O - 0.8 V

Scan rate: 20 mV/s

Gain: O. 1 mPJV

PA- 1

off

Gain: 1000 nA/V

Multiplier: x 1

off

off

Axis Y: 1 V/cm

Avis X: 1 V/cm

Axis Y: 1 0 mV/cm

A i s X: 1 mV/cm

Axis Y: 10 mV/cm

Axis X: 1 mV/cm

Axis Y: 0.05 V/cm

Axis X: 1 V/cm

APPENDlX II

R4W DATA FOR MODELLING OF BIOSENSORS

Table 1. Response of two G6P electrodes containing 10U and 20U of enzymes

Table 2 Determination of Imm using Lineweaver-Burk plot using data in Table 1

TabIe 3. Mode1 simulation for the G6P biosensors containing 10 units and 20 units

of enzyme

1 Biosensor 1 Biosensor 1 Modelling 1 ~ e d e l l i n ~

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