In the Laboratory
Electropolymerized Conducting Polymers as Glucose Sensors
An Undergraduate Analytical Chemistry Laboratory Experiment
Omowunmi A. Sadik,* Sharin Brenda, Patrick Joasil, and John Lord
Department of Chemistry, State University of New York at Binghamton, P.O. Box 6016, Binghamton, NY 13902-6016;
*[email protected]
The use of electropolymerized conducting polymers for the
analysis of glucose is suitable for undergraduate instrumental
analysis laboratory. Conducting polymer film has been of
considerable research interest (1), and its application for glucose
analysis can be used to introduce undergraduate students to some
contemporary electrochemical and biosensor principles. Moreover, the preparation of conducting polymer films requires
only inexpensive starting materials and low-cost equipment.
In recent years, the development of analytical techniques
based on small sensing devices for use in clinical analysis,
environmental monitoring, and bioprocess control has become
an area of tremendous interest (2, 3). Using electropolymerized
polypyrrole films, sensing devices based on the immobilization
of oxidoreductase enzymes (enzymes involved in the oxidation
and reduction of biological molecules) and glucose sensors as
well as other biological sensors have been developed (4–6 ).
The suitability of conducting polymer films in sensing applications is due to their electrochemical sensitivity to the presence
of selected ions in a solution. For example, glucose assay uses
a film containing glucose oxidase (GOx), an enzyme which
catalyzes the oxidation of glucose by oxygen to produce gluconic
acid and hydrogen peroxide (eq 1). During the catalytic cycle,
the flavin prosthetic group of the GOx is first reduced by
the glucose and then reoxidized by the molecular oxygen.
GOx
β-glucose + O2 + H2O → β-gluconic acid + H2O2 (1)
The amount of glucose present in the solution is determined either by observing the rate of oxygen consumption
in the solution, or the rate at which hydrogen peroxide is
produced. The peroxide produced is amperometrically determined by electrochemical reduction at 0.8 V vs Ag/AgCl
reference electrode (eq 2):
+2e{ (0.8 V)
2H2O2 + 2H+ → 2H 2O
(2)
An alternative determination of the H2O2 is by the Mo(VI)catalyzed reaction with iodide (7); this is followed by the
amperometric reduction of iodine at a potential of < 0.2 V
versus saturated calomel electrode (SCE) (eqs 3 and 4).
Mo(VI)
H 2O2 + 2H+ + 2I { → I2 + 2H2O
0.2 V
I 2 + 2e{ → 2I {
(3)
(4)
The electrochemical reduction of H2O2 results in a flow of
current. The magnitude of this current is linearly proportional
to the concentration of glucose over the range 2–30 mM.
The conducting polymer plays the triple role of enzyme host,
charge transducer, and permselective membrane. Perhaps the
most critical role of the biomolecule–polypyrrole interface is
to insure efficient electron transfer and hence a high signalto-noise ratio, as depicted in Figure 1.
Medox
Glucose
GOxred
Pt Ppy
Ppy-immobilized
Medred
GOxox
Gluconolacton
Figure 1. Reaction scheme depicting the oxidation of glucose at
polypyrrole/GOx interface.
The advantages of conducting polymer films in glucose
sensing may also be attributed to their tight adherence to solid
electrode substrates (e.g., platinum, gold, glassy carbon), the
ability to generate analytically useful signals upon the application of electrical potential, and the possibility of introducing
various functional groups into the polymer matrix. Moreover,
the ease of preparing the films by simply changing the electrochemical polymerization conditions results in sensors with
low costs, making this an effective approach to introducing
undergraduate students to conventional methods of analysis—
especially in departments with limited resources. The approach
may also introduce students to undergraduate research. Ongoing projects in our laboratory are focused on the development
of polymeric coatings for solid electrodes with the goal of
creating electrochemical sensors that respond selectively to
certain biochemical species of interests (8, 9). Some of these
works have been extended to cover part of our undergraduate
analytical chemistry research and teaching programs. Subsequently, our students have expressed a great deal of enthusiasm
for these experiments.
Some of the synthetic approaches to producing most
conducting polymers are via chemical and electrochemical
routes. Simply, these polymers can be chemically prepared
by exposing the monomers to strong oxidants, typically
Fe(III), giving rise to polymer products in the form of black
powders. The polymer thus formed is simultaneously oxidized
to the doped state, the incorporated anion serving as the
dopant. The electrochemical synthesis of conducting polymers
can be initiated through the application of a constant current
or constant potential and a cyclic potential scan. For example,
the formation of a polypyrrole coating on solid electrode in
the presence of a counterion (C {) or supporting electrolyte is
shown in Scheme I.
H
H
n
N
+
C–
Eox
H2 O
+
e–
1/ H
2 2
+
C–
N
N
H
+
N
N
H
H
n
OH –
Scheme I
JChemEd.chem.wisc.edu • Vol. 76 No. 7 July 1999 • Journal of Chemical Education
967
In the Laboratory
The electroinitiated polymerization is characterized by the
oxidation (or reduction) of a soluble initiator on the electrode
surface. This is accompanied by the formation of active species
(anions, cations, or radicals) while the polymer precipitates
directly on the electrode surface in the form of a cohesive film.
The apparent stoichiometry for the polymerization reaction,
which includes the stoichiometry for the formation of the
polymer chain plus the charge associated with the oxidation
of the polymer, is in the range 2.06–2.5 F/mol of polymer
(10). The electrochemical method is advantageous in terms of
the easy control of the growth rate and the film thickness, the
enhanced electrical properties due to the conductivity of the
polypyrrole film, and the relatively inexpensive polymerization
procedure. Therefore, the objectives of the undergraduate
student experiment are to (i) perform cyclic voltammetry on
electropolymerized conducting polymers, (ii) observe the effect
of various parameters on the voltammogram obtained, and
(iii) perform quantitative analysis of glucose.
Figure 2. A batterypowered 2-electrode
undivided cell for the
electrochemical deposition of conducting
polymers.
Electrolyte
Anode
Cathode
Experimental Procedure
Preparation and Characterization of EnzymeImmobilized Polypyrrole Film
A conducting polypyrrole film containing an immobilized
enzyme can be conveniently prepared using a simple electrochemical setup with either a 2-electrode battery-powered cell
(Fig. 2), or a 3-electrode (Fig. 3) one-compartment cell (or
even a beaker). The best films have been prepared using three
electrodes, with the auxiliary electrode separated from the
working and reference electrodes. Films prepared using a simple
2-electrode undivided cell are of poorer quality because of
the complications that this cell configurations presents.
In our setup, a reagent-grade pyrrole (Aldrich) was distilled
before use and deoxygenated with nitrogen for 5 min. The
reagent-grade β-D-glucose and GOx types II and VII (Sigma)
were used without further purification. The GOx type II had
an activity of 26,500 units/g, and type VII had an activity of
125,000 units/g. Both were stored desiccated below 0 °C.
Stock glucose solutions were allowed to mutarotate overnight
at room temperature before use and were stored at 4 °C.
In a typical undergraduate student experiment, either
platinum (0.2 cm2) or glassy carbon (0.08 cm2) can be used as
a working electrode with an aqueous solution of KCl (0.1 M),
pyrrole (0.5 M), and GOx (150 units/mL) in a small beaker.
Platinum serves as the auxiliary electrode. A silver/silver
chloride (Ag/AgCl) electrode or saturated calomel electrode
(SCE) may be used as reference, and the electrical leads from
a potentiostat or galvanostat can be attached to the electrodes.
In our setup, electrolysis at a current density of 1–2 mA/cm2
for a period of 5–10 minutes was employed. This resulted in a
deposition of a smooth film of polypyrrole containing immobilized glucose oxidase (PP/GOx/Pt) at the working electrode.
The deposition current was monitored as a function of time to
determine approximate film thickness. The film thickness was
calculated on the assumption that 20–25 mC/cm2 of charge
yielded a 0.1-µm layer (11) and a desirable film thickness of
10 µ m is appropriate.
Glucose can be determined indirectly via an electrochemical determination of hydrogen peroxide. The PP/GOx/Pt
electrodes were first preconditioned by setting the potentiostat
at 0.0 V for several minutes (up to 15 min) to allow background
current to diminish to a constant value. Aliquots of a stock
968
Figure 3. A divided 3-electrode cell for the electrochemical deposition of conducting
polymers.
solution of glucose were added to a solution of KI, reagentgrade (NH4)6Mo7O24?4H2O, phosphate buffer, and GOx to
give a final glucose concentration of 3 × 10{3 M, final iodide
concentration of 0.1 M, a final Mo(VI) concentration of 1 ×
10{3 M, and a final phosphate buffer concentration of approximately 0.15 M (pH 6.5). The reagent solutions for glucose
determination were saturated with O2 and stirred rapidly.
The analytical signals obtained were based on the slope
of the current-time responses immediately after the addition
of glucose. The data analysis involved (i) determination of
E °′ and n for the ferricyanide/ferrocyanide couple in 0.1 M
KCl at the polymer-modified platinum electrode; (ii) measurement of anodic and cathodic peak currents (ipa and ipc,
respectively) for the voltammogram obtained; (iii) plotting
of ipa and ipc versus the square root of the scan rates; and
(iv) use of a linear least squares analysis for both plots. For the
quantitative analysis of glucose, a plot of the peak current was
obtained from the current–time plots at different concentrations. The values of the current were plotted against the
glucose concentration using linear least square analysis, thus
enabling the determination of unknown glucose concentration.
Discussion
Electrodes coated with polypyrrole containing immobilized
glucose oxidase were prepared and characterized to determine
their suitability as working electrodes for glucose determination.
Figure 4 is a cyclic voltammogram of the PP/GOx/Pt electrode
showing the typical pseudo reversible oxidation of the conjugated polypyrrole backbone (12). The electron transfer properties of the electrodes were examined using cyclic voltammetry.
Aqueous solutions of K3[Fe(CN]6 gave a Fe(II)/Fe(III) wave
with E °′ at 0.465 V and a 70-mV peak separation (at 20 mV/s),
Journal of Chemical Education • Vol. 76 No. 7 July 1999 • JChemEd.chem.wisc.edu
In the Laboratory
3
Current / µA
2
1
0
-1
-2
- 0 .8
- 0 .6
- 0 .4
- 0 .2
0 .0
0 .2
0 .4
E / Volts
Potential applied
8
6
i / mA
Glucose injected
Figure 4. Cyclic voltammogram of a platinum disk electrode coated
with polypyrrole glucose oxidase versus Ag/AgCl reference electrode, showing typical pseudo reversible oxidation of the conjugated pyrrole backbone ( v = 20 mV/s, phosphate buffer saline).
4
(a)
(b)
(c)
(d)
2
0
-0.8
-0.4
0
0.4
E/V
Figure 5. Amperometric determination of glucose in the presence
of 1 unit/mL GOx at 0.0 V using a PP/GOx/Pt electrode. Current
vs potential curves at (a) 1 × 10{3 M, (b) 5 × 10 {4 M, (c) 2 × 10 {4
M, (d) 1 × 10{4 M.
10
Current / (A/cm2 )
9
8
7
6
0
2
4
6
8
10
[Glucose] / (mmol/L)
Figure 6. Plot of current (normalized for 1 µm of polymer) vs glucose
concentration, illustrating the reproducibility of the enzyme immobilization and electrode preparation. The current reponses are for
electrodes prepared from three GOx-modified polymers (series 1–3).
indicative of a reversible one-electron transfer reaction. Hence
the PP/Pt electrode behaved well as a working electrode for
single redox couples.
Thereafter, the PP/GOx/Pt electrodes were used as the
working electrodes to detect the H2O2 produced (via I2) by
the GOx-catalyzed oxidation of glucose in bulk solution.
Using the PP/GOx/Pt electrode, the effect of enzyme concentration was studied. Theoretically, the rates of the sequence
of reactions 1 and 3 should increase with GOx concentrations
as long as reaction 3 is fast in comparison with reaction 1,
and until the reaction becomes saturated with GOx/glucose
(enzyme/substrate) complex. Figure 5 shows a 1 unit/mL
enzyme concentration curve in which t = 2 corresponds to
an injection of 1 × 10{3 M glucose. The effect of varying the
glucose concentration at constant GOx concentration was
studied. The current increased linearly with time, even when
a relatively large enzyme concentration (150 units/mL) was
chosen. Thus the current observed at any given time and the
slope of the curve were directly proportional to the glucose
concentration for a given enzyme concentration. The range
of glucose concentrations measured was between 1 × 10{4 and
5 × 10{2 M. The glucose concentration was linear at low concentrations up to approximately 1 × 10{3 M (Fig. 6). Above
this concentration the response is nonlinear, becoming insensitive to additional amounts of glucose above 5 × 10{2 M.
Using a procedural blank solution with the PP/GOx/Pt
electrode, the limit of detection was estimated at 3 times the
background noise, at current level of 5.54 µA; this corresponds
to 8 × 10{5 M glucose.
Figure 6 shows the current response to glucose additions
of polypyrrole electrodes prepared from three GOx-modified
polymers. These responses, when corrected for the differences
in the polymer loading, deviated by a maximum of 6.6% at
1 × 10 {3 M. Differences in the regularity of the coating were
visually observable using the electrochemical method. Hence,
signal variation could be expected. For larger variations in the
amount of polymer deposited, the signal may be influenced
by the rate of diffusion in the polymer and different electrodes
cannot be compared in this way. The Michaelis–Menten
parameters (Km and Vmax) were calculated for 1.0-unit/mL
GOx solution at rates determined after 5 min, utilizing 10 {3–
10{4 M concentrations of glucose. The Km was calculated to
be approximately 1.5 × 10{3 M, and the Vmax was on the order
of 10{9 m/s, comparable with values cited in the literature
for GOx (13).
Many conducting polymers are being developed for practical applications such as rechargeable batteries, electrolytic
capacitors, display devices, and components of solar energy cells
(14 ). The possibility of using conducting polymers for analytical purposes has resulted in the development of potentiometric
and amperometric sensors, and a sizable number of literature
reviews and publications on conducting polymer sensor applications are already in existence (2–9). Sensing devices utilizing
arrays of conducting polymers have been used in the construction of “electronic noses” and these are now commercially
available (3, 15, 16 ).
Conclusion
The use of an electropolymerized conducting polypyrrole
for the determination of glucose can illustrate the fundamentals
of electrochemical and biosensor concepts. It reinforces the
JChemEd.chem.wisc.edu • Vol. 76 No. 7 July 1999 • Journal of Chemical Education
969
In the Laboratory
underlying principles of dynamic electrochemistry by showing
the potential conducting polymers for analytical applications.
In this experiment, glucose oxidase was chosen as a model
system because the homogeneous enzyme kinetics are well
characterized; the enzyme is readily available in a pure form
and is reasonably stable. However, the experiment can easily
be modified for the analysis of other species (such as anions,
cations, and neurotransmitters), and either the electrochemical
or analytical information or both can be emphasized.
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3. Handbook of Biosensors and Electronic Noses, Medicine, Food, & the
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Sadik, O. A., Wallace, G. G. Electroanalysis 1993, 5, 555.
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Journal of Chemical Education • Vol. 76 No. 7 July 1999 • JChemEd.chem.wisc.edu