| Research Article |
Open Access |
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| Glucose Oxidase Modified Electrodes of Polyaniline and Poly (aniline-co-2-anilinoethanol) as a Biosensor: A Comparative Study |
| Ali Keyhanpour, Seyed Mohammad Seyed Mohaghegh* and Ahmad Jamshidi |
| Iron Polymer and Petrochemical Institute, Tehran, Iran |
| *Corresponding author: |
Seyed Mohammad Seyed Mohaghegh
Iron Polymer
and Petrochemical Institute
P.O. Box: 14965-115, Tehran, Iran
Tel: 48662444
Fax: 44580026
E-mail: s.m.mohaghegh@ippi.ac.ir |
|
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| Received October 23, 2011; Accepted January 13, 2012; Published January 16,
2012 |
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| Citation: Keyhanpour A, Mohaghegh SMS, Jamshidi A (2012) Glucose Oxidase
Modified Electrodes of Polyaniline and Poly (aniline-co-2-anilinoethanol) as a
Biosensor: A Comparative Study. J Biosens Bioelectron 3:116. doi:10.4172/2155-
6210.1000116 |
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| Copyright: © 2012 Keyhanpour A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited. |
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| Abstract |
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| A simple technique is described for constructing a glucose sensor so that enzymes, glucose oxidase (GOD)
was immobilized by cross-linking via glutaraldehyde 0.1% at 0.1 M phosphate buffer with 7.0 pH in a polyaniline and
poly(aniline-co-2anilinoethanol) thin films, which were electrochemically deposited on a platinum plate in phosphate
and acetate buffer. The results of EIS and SEM indicated the successful immobilization for enzymes in the polymers
film. The maximum current response was observed for the electrodes at pH 7 and potential 0.65 V (versus Ag/
AgCl). The poly (aniline-co-2anilinoethanol)/GOD electrode gives high stability and fast response as compared to
polyaniline/GOD electrode in amperometric measurements so results show that the poly (aniline-co 2anilinoethanol)
electrode is more suitable for biological systems. |
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| Keywords |
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| Glucose oxidase; Poly (aniline-co-2anilinoethanol);
Immobilization; Polyaniline; Biosensors |
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| Introduction |
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| Glucose is a bio-molecule that is known to play a variety of roles
in the welfare of mankind. The development of amperometric glucose
biosensors is extensively investigated research area because of its
importance in the treatment of diabetes mellitus health care, food
and environmental monitoring and process industries, etc [1-6]. To
date, the most commonly used amperometric glucose sensors use the
specific recognition of glucose oxidase (GOD). The determination of
biological compounds with biosensors has several advantages, e.g.
ease of manipulation, rapidity and simple pretreatment of samples
and the establishment of analytical methods based on biosensors [7].
Conducting polymers have also found applications in amperometric
enzyme electrodes with the intention to couple the electron transfer
reaction between enzyme and electrode via the ramified conducting
network of the polymer for example, polypyrrole [8], polyaniline
[9,10], poly (o-anisidine) [11], poly (o-toluidine) [12] and etc.
Among others, the poly-conjugated conducting polymers have been
recently proposed for biosensing applications because of a number
of favorable characteristics such as: (i) direct and easy deposition on
sensor electrode by electrochemical oxidation of monomer, (ii) control
of thickness and (iii) Redox conductivity [11]. Conducting polymers
have attracted much interest as a suitable matrix for entrapment
of enzymes [13,14] forming an enzyme electrode. These enzyme
electrodes are a reliable, accurate and low-cost biosensors widely used
in biomedical analysis. The immobilization of enzymes in electrode
materials is achieved either by physical or chemical methods [15-18].
Immobilization of enzymes is under taken either for the purpose of
basic research or for use in technical processes of commercial interests.
Immobilization means that enzymes, while retaining their catalytic
activity, is confined within a certain space or is bound to solid carriers
or to one another. Immobilization of enzymes has several advantages.
These are: (i) the stability of the enzyme by protecting the active
material from deactivation; (ii) repeated use; (iii) significant reduction
in the operation cost; (iv) easy separation and recovery of the enzymes.
Advantages of immobilization of enzymes in conducting polymer
by electro polymerization are an easy one-step procedure, accurate
control of the polymer thickness via the electrical charge passed during the film formation process, localization of the electrochemical reaction
exclusively on the electrode surface allowing precise modification of
microelectrodes and surfaces of complex geometry and the possibility
to build up multilayer structures. The immobilization of enzymes or
proteins in such polymers can be achieved by several techniques like
physical entrapment, chemical cross-linking and covalent coupling.
The physical entrapment covalent coupling and other methods suffer
from leaching problems of the enzyme [19,20]. However this problem
can be significantly overcome by using chemical cross-linking method
of immobilization [21-23] via glutaraldehyde. GOD has been the most
frequently used molecular recognition element, due to its high binding
specificity, high turnover rate and relatively high stability. GOD can
selectively catalyze the oxidation of glucose in the presence of oxygen
to form hydrogen peroxide (H2O2), which can be electrochemically
detected by using electrodes [e.g., platinum (Pt) and gold (Au)] at
potentials positive to around +0.6 V (vs. Ag/AgCl). The reactions can
be described as follows: |
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 And  |
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| Experimental |
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| Reagents |
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| The monomers aniline (Merck) and 2-anilinoethanol (Merck) were distilled twice before use. The thin films of polyaniline and poly
(aniline-co-2-anilinoethanol) were synthesized electrochemically on
platinum substrates under cyclic voltammetric conditions in a single
compartment glass cell. A three electrode geometry was employed
during the electrochemical polymerization in which a platinum
substrate functioned as the working electrode (0.4 cm2 ⇓ 0.4 cm2), a Pt
rod as counter electrode and Ag/AgCl as the reference electrode. The
films were electropolymerized in aqueous solution containing 0.1M
monomer(s) and 1M H2SO4 (Merck) as electrolyte. Glucose oxidase
(EC 1.1.3.4, 5 80 Umg−1 protein, from Merck) and D- glucose were
used without further purification and glucose solutions were stored
overnight at room temperature before use. The GOD was immobilized
by cross-linking via glutaraldehyde (0.1 %) (Merck) on films, thus
restricting the leaching of the enzyme film. These films were left for 30
min and washed with phosphate and/or acetate buffer. Buffers including
the HAc + NaAc system and the KH2PO4 + Na2HPO4 were always
employed as supporting electrolyte. Then both enzyme electrodes were
washed thoroughly with 0.1 M phosphate buffer solution and stored at
4°C. All solutions were made up with twice distilled water. |
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| Apparatus |
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| Amperometry, cyclic voltammetry (CV) and Faradaic impedance
spectroscopy measurements were performed with a CHI 660B
electrochemical workstation (CH Instruments, USA) in a conventional
three-electrode cell. Pt plate electrode was used as working electrode. An Ag/AgCl electrode (saturated KCl) and a Pt rod were used as reference
and counter electrode, respectively. The scanning electron micrograph
was recorded using JEOL, JSM-6360A SEM machine. The Faradaic
impedance measurements were carried out in a background solution of
in solution 0.5M Na3PO4 at a normal potential. The alternating voltage
was 5mV and the frequency range was 0.1 Hz to 100 kHz |
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| Synthesis of polyaniline and copolymer of aniline/2-
anilinoethanol |
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| Polyaniline and poly (aniline-co-2-anilinoethanol) film was
synthesized from an aqueous solution of distilled 0.1 M aniline and
0.1 M 2-anilinoethanol (Merck) and 1.0 M of sulfuric acid (Merck)
using electrochemical deposition method. It was carried out by cyclic
a voltammograms technique at 27°C in one compartment, threeelectrode
glass cell. The platinum plate was used as working and Pt
rod was used counter electrode and Ag/AgCl was used as reference
electrode. The electrolyte solution was prepared in distilled water. To
control the thickness of the films, electro polymerization of this solution
was carried out using a number of voltammetric cycle’s: The first cycle
was applied to induce the polymerization process and the following
cycles to achieve the overall coating of the electrode. After synthesis,
polymer coated electrodes were rinsed thoroughly in distilled water
and dried in cold air and then use for subsequent characterization.
For preparing of copolymers, We were used the volume percentage
90/10 aniline and 2-anilinoethanol, because there are two major factors in
producing biosensors, the first high conductivity and the second reduced contact resistance between the metal electrodes and the polymer film. It is
shown that with increase of 2-anilinoethanol in copolymer, conductivity
and resistance are decreased, but the processability and adhesion are
increased. Hydrophilic film polymer obtained has increased with
increasing (2-anilinoethanol) content. Considering the figure 1 is observed
with increasing monomer 2-anilinoethanol in copolymer, and the
number of pair’s peaks is reduced. This shows that poly (2-anilinoethanol)
and poly (aniline-co-2-anilinoethanol) are formed [26]. The PANI films
showed a typical redox response with three redox couples (Figure 1). The
first responses occur without loss or gain of protons (leucoemeraldine/
emeraldine) and third redox electrochemical response involves
protonation and deprotonation (emeraldine/pernigraniline) couples are
due to the interconversion reactions of PANI upon varying the potential.
The middle peaks can be related to the formation of quinones (mostly
benzoquinone) as a consequence of a hydrolysis reaction in water [27]. |
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|
Figure 1: Cyclic voltammograms recorded during the synthesis of (a) polyaniline
and the poly (aniline-co 2 anilinoethanol) with the volume percentage [(b)
90/10- (c) 85/15- (d) 50/50] and (e) Poly (2-anilinoethanol) films in aqueous
solution of H2SO4 as electrolyte. |
|
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| SEM studies polyaniline and poly (aniline-co-2-
anilinoethanol) |
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| The scanning electron micrograph was recorded using JEOL, JSM-
6360A SEM machine. The SEM micrograph of synthesized polyaniline
and poly (aniline-co-2-anilinoethanol) is shown in Figure 2. Poly
(aniline-co-2-anilinoethanol) structure show higher uniformity and
porosity than polyaniline structure, which is suitable for immobilization
of biocomponent [28]. |
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Figure 2: The scanning electron micrograph of synthesized (a) polyaniline and
(b) poly (anilineco-2-anilinoethanol) with optimized electrochemical process
parameters. |
|
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| Immobilization of GOD on poly (aniline-co-2-anilinoethanol) |
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| Conducting polyaniline and poly (aniline-co-2-anilinoethanol)
films were prepared using cyclic voltammograms technique in a solution
of 1M aniline, 1M 2-anilinoethanol and 1M H2SO4. Pt plates were used
as working electrodes and Pt rod was used as the counter electrode.
The films were thoroughly washed with phosphate buffer prior to use.
The enzyme solutions were prepared in 0.1M phosphate buffer (pH
7.0) with the working concentration of 20 U/ml for GOD. The enzyme
GOD was immobilized by cross-linking via (1 %) glutaraldehyde on
polyaniline and poly (aniline-co-2-anilinoethanol) films and left 30
min and then washed with phosphate and acetate buffer to remove any
loosely bound enzyme. Because physical adsorption of an enzyme onto
a solid although is probably the simplest way of preparing immobilized
enzymes but the method relies on non-specific physical interaction
between the enzyme protein and the surface of the matrix, brought
about by mixing a concentrated solution of enzyme with the solid. In
addition, because of the weak bonds involved, desorption of the protein
resulting from changes in temperature, pH, ionic strength or even the
mere presence of substrate, is often observed. Another disadvantage is
non-specific further adsorption of other proteins or other substances
as the immobilized enzyme is used. This may alter the properties of
the immobilized enzyme or, if the substance adsorbed is a substrate for
the enzyme, the rate will probably decrease depending on the surface
mobility of enzyme and substrate. But cross-linking an enzyme to it is
both expensive and insufficient, as some of the protein material will
inevitably be acting mainly as a support, resulting in relatively low
enzymatic activity. Generally, cross-linking is best used in conjunction
with one of the other methods. |
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| Faradaic impedance spectroscopy |
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| Impedance spectroscopy is an effective method to probe the features of the surface modified electrodes. These studies were employed for
modification of the electrode with individual and mixed components
used for the fabrication of the biosensor. The complex impedance
can be presented as the sum of the real Z’ (ω) and imaginary Z’’ (ω),
components that originate mainly from the resistance and capacitance
of the cell, respectively. The general electronic equivalent scheme
(Randles and Ershler model, inset of Figure 3, include the ohmic
resistance of the electrolyte solution, Rs, resulting from the diffusion
of ions from the bulk electrolyte to the electrode interface, the double-
layer capacitance, Cdl and the electron-transfer resistance Rct that exists
if a redox probe is present in the electrolyte solution. The electron-
transfer resistance, Rct, controls the electron-transfer kinetics of the
redox probe at the electrode interface. From the shape of an impedance
spectrum, the electron-transfer kinetics and diffusion characteristics
can be extracted from spectra. |
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Figure 3: Nyquist plots of the electrochemical impedance spectroscopy (EIS)
for (a) pt bare (b) polyaniline and (c) poly (aniline-co-2-anilinoethanol) and (d)
polyaniline/GOD, (e) poly (anilineco-2-anilinoethanol) film. |
|
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| In addition, Figure 3 Shows the Faradaic impedance spectra, presented
as Nyquist plots (Z′ (ω) vs. Z′′ (ω)) upon the modification of the electrodes
polyaniline and poly (aniline-co-2-anilinoethanol). The electron-transfer
resistance decreased, due to the increase in the hydrophilic with increase
in content of 2-anilinoethanol, making it easier for the electron transfer to
take place. That confirmed the successful synthesis of poly (aniline-co-2-
anilinoethanol) film for the biosensor. The response current of the active
device depends on the contact resistance between the metal electrodes and
the polymer film [11]. |
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| Current response of the polyaniline and poly(aniline-co-2-
anilinoethanol) GOD electrodes |
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| The change in response current of the active device glucose oxidase is
the parameter of interest for sensor applications. The response potential
of the device depends on several factors such as 1-the contact resistance
between the metal electrode and the polymer film, 2-the geometric
factor of the film and 3-the film conductivity. The film conductivity is
depends on several factors, such as analyte pH, temperature, polymer
film potential, substrate concentration and enzyme loading. The GOD
was immobilized on electrochemically synthesized polyaniline and poly
(aniline-co-2-anilinoethanol) film by cross-linking via glutaraldehyde.
The potential-time relationship of polyaniline /GOD and poly (aniline-
co-2-anilinoethanol)/ GOD electrode when the current of the enzyme
was set 0.65V in phosphate and acetate buffer is as shown in figure 4,
figure 5 and figure 6 respectively. |
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|
Figure 4: Current–time curves for the glucose oxidase electrode of (a) polyaniline and (b) poly (aniline-co-2-anilinoethanol) at 0.65V. Glucose solution (1) 1 mM, (2) 3
mM, (3) 5 mM, (4) 7 mM, (5) 10 mM, (6) 15 mM, (7) 20 mM, (8) 30 mM, (9) 40 mM in 0.1M phosphate buffer, pH 7. |
|
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Figure 5: Current–time curves for the glucose oxidase electrode of (a) polyaniline and (b) poly (aniline-co-2-anilinoethanol) at 0.65V. Glucose solution (1) 1 mM, (2) 3
mM, (3) 5 mM, (4) 7 mM, (5) 10 mM, (6) 15 mM, (7) 20 mM, (8) 30 mM,(9) 40 mM in 0.1M acetate buffer, pH 7. |
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Figure 6: The relationship between response current and glucose concentration for the GOD electrode of polyaniline (♦) and poly (aniline-co-2-anilinoethanol)
(■), in 0.1M phosphate buffer, pH 7 (a) and in 0.1M acetate buffer, pH 7 (b). |
|
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| Determination of the kinetic constants |
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| The kinetic parameters, Km and Imax values of the free and
immobilized invertase were determined by measuring initial rates of
the reaction with sucrose as substrate in acetate buffer (3mM, pH 7)
at 35°C. The Km and Imax values for the free and immobilized invertase
were calculated from Lineweaver–Burk plots by using the initial rate of
the enzymatic reaction. |
| |
 |
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| Where [S] was the concentration of substrate, I and Imax represented
the initial and maximum rate of reactions, respectively. Km was the
Michaels constant. The value of Km depends on immobilization of
enzyme and lesser the Km gives the faster response of the electrode to
glucose [31,32] (Figure 7, Figure 8, Table 1). |
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|
Figure 7: Determination of apparent Michaelis–Menten constant (Km) for
the GOD electrode of polyaniline (♦) and poly (aniline-co-2-anilinoethanol)
(■) in 0.1M phosphate buffer, pH 7. |
|
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Figure 8: Determination of apparent Michaelis–Menten constant (Km) for the
GOD electrode of polyaniline (♦) and poly (aniline-co-2-anilinoethanol) (■) in
0.1M acetate buffer, pH 7. |
|
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Table 1: Apparent Michaelis–Menten constant (Km) and maximum current (Imax) of
polyaniline and poly (aniline-co-2-anilino)/GOD films in 0.1 M phosphate and 0.1
M acetate buffer at pH 7. |
|
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| Effects of temperature value of the polyaniline and of the poly
(aniline-co-2-anilinoethanol) GOD electrodes |
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| Solutions containing glucose may differ in temperature. It is
important to understand how the electrochemical sensing performance
of the two electrodes responds to the temperature changes. Our test
solution was 0.1M phosphate buffer, pH 7 and acetate buffer, pH
7 containing 3mM glucose. During the glucose measurements, the
temperature of the solution was controlled. For each of the electrodes,
there were a total of six measurements between 35 and 60°C with increments of 5°C. Figure 9 shows the responses of the biosensors to
the change of temperature. The response patterns of the two biosensors
were analogous. The measured current signals were proportional to
the temperature increase and reached their heights between 50 and
55°C. The reasons for slower and faster reaction rates are because
the temperature is directly proportional to the kinetic energy of the
enzyme and surrounding molecules. Higher kinetic energy makes for a
higher chance for the enzyme to bump into molecules that it will react
with. According to the Arrhenius formula, |
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Figure 9: Effect of temperature on the responses (a) of the polyaniline/GOD and (b) of the poly (aniline-co-2-anilinoethanol) GOD, glucose sensors studied by amperometric
method for 3mM glucose in (♦) phosphate buffer, pH 7 and (■) acetate buffer, pH 7. |
|
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| I (T) = I0 exp (-Ea/RT) |
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| lnk=lnA-(Ea/RT) |
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| lnI=lnI0 – Ea/RT |
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| where k is the rate constant and E, the activation energy. Since the
electrode surface areas, the quantity of the enzyme and the concentration
of substrate are constant, the steady-state current is proportional to the
rate constant k [33]. We replaced k with I in the Arrhenius formula. The
relationships of log I vs 1/T are plotted in Figure 11 and two straight
lines were obtained. The activation energies were calculated from the
slopes. The values of E of the polyaniline/GOD, for the phosphate buffer
and acetate buffer are 45.3 kJ/mol and 49.5 kJ/mol and values of E poly
(aniline-co-2anilinoethanol) GOD for the phosphate buffer and acetate
buffer are 41.9 kJ/mol and 46.2 kJ/mol, respectively From the above
results, we can see that the activation energy of the glucose oxidation reaction under enzyme catalysis in phosphate buffer is smaller than
that in acetate buffer. Therefore in acetate buffer, the energy required to
enter transition state decreases, thereby decreasing the energy required
to initiate the reaction and reaction time for reaching the steady-state
current is shorter. In addition we can see that the activation energy of
the glucose oxidation reaction under enzyme catalysis in of the poly
(aniline-co-2-anilinoethanol)/GOD electrode is smaller than that
in of the polyaniline/GOD electrode. Therefore in phosphate buffer
was much preferred for use in amperometric measurements for poly
(aniline-co-2anilinoethanol) glucose sensors (Figure 10). |
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Figure 10: Plots of log I against l/T (♦) phosphate buffer and (■) acetate buffer. |
|
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Figure 11: Effect of pH on the GOD electrode response (a) of the polyaniline and (b) of the poly (aniline-co- 2-anilinoethanol). The
steady-state currents were measured at 0.65 V in a 3 mM glucose solution in a 0.1M (♦) phosphate buffer and (■) acetate buffer, pH 7. |
|
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| Effects of pH value of the polyaniline and of the poly (aniline-
co-2-anilinoethanol)/GOD electrodes |
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| The pH of a solution can have several effects of the structure and
activity of enzymes. Changes in pH may not only affect the shape of
an enzyme but it may also change the shape or charge properties of
the substrate so that either the substrate cannot bind to the active
site or it cannot undergo catalysis. Immobilized enzyme-carrier
complex, the enzyme molecules are subject to the effect of the
micro-environment in the pores of the complex. Surface charges and
other micro environmental effects can create a shift up or down of
optimal pH of the enzyme activity [34]. The preparation of the test
solution was similar to that of the temperature effect experiments,
with the exception of the pH value controls. The effect of pH on the
behavior of the enzyme electrodes was studied with 0.1M phosphate
and acetate buffer solutions containing 3 mM glucose. The steady-state
currents at 0.65 V, as a function of the pH values, are shown in Figure
11. The electrochemical responses were quite good at pH ranging from
4.0-8.0, and the maximum current occurred at about pH 7. Bright and
coworkers studied the pH dependence of solubilized GOD reactions
and found a broad pH range of 4.0-8.0 with a maximum current of
approximately pH 7. |
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| Stability and lifetime of the polyaniline and of the poly
(aniline-co-2-anilinoethanol) electrode |
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| Stability and lifetime of the GOD electrode response of the
polyaniline and of the poly (anilineco-2anilinoethanol) have been
studied. It shows excellent stability and response for 10 days (Figure
12). At the beginning of the test of lifetime, activity decreased rapidly.
After a few days the modification electrode shows stable response.
The poly (aniline-co-2-anilinoethanol)/GOD electrode gives high
stability and fast response as compared to polyaniline/GOD electrode
in amperometric measurements so results show that the poly(aniline-
co-2-anilinoethanol) electrode is more suitable for biological systems
and for two electrode decreases current in the acetate buffer more than
phosphate buffer so results show that phosphate buffer is more suitable
for biological systems. |
| |
|
Figure 12: Stability of the polyaniline /GOD (a) and of the the poly(aniline-co- 2-anilinoethanol) GOD (b) electrode on
storage in (♦) 0.1 M phosphate buffer and (■) 0.1 M acetate buffer, pH 7at 6 °C. |
|
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| Conclusion |
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| We have synthesises polyaniline and poly (aniline-co- 2anilinoethanol) glucose oxidase biosensor for the determination of
glucose. The maximum current response for the polyaniline and poly
(aniline-co-2-anilinoethanol) glucose oxidase electrode is at pH 7, 0.65 V
and 50°C in either phosphate buffer or acetate buffer. The poly (anilineco-
2-anilinoethanol)/GOD electrode gives high stability and fast
response as compared to polyaniline/GOD electrode in amperometric
measurements. Results show that the poly (aniline-co-2-anilinoethanol)
electrode is more suitable for biological systems. According to the rate
constant and the activation energy measurements in both electrodes,
the poly (aniline-co-2-anilinoethanol)/GOD electrode is much more
preferable for use in amperometric measurements in both buffers than
polyaniline/GOD electrode due to the comparative fast response. |
| |
|
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