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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vi
xi
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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-poðylenzrnine 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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