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
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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
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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. Literature Cited 1. Handbook of Conducting Polymers, Vol. I; Skotheim, T. A., Ed.; Dekker: New York, 1986. 2. Turner, A. P. F.; Karube I.; Wilson G. Biosensors; Oxford University Press: Oxford, 1987. 3. Handbook of Biosensors and Electronic Noses, Medicine, Food, & the
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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Environment; Erica Kress-Rogers, Ed.; CRC: Boca Raton, FL, 1997. Zotti, G. Synth. Met. 1992, 51, 373. Bidan, G. Sensors Actuators 1992, B6, 45. Lyons, M. E.; Fitgerald, C.; Bannon, T. Analyst 1993, 118, 361. Murray, R. W. In Electroanalytical Chemistry, Vol. 13; Bard, A. J., Ed.; Dekker: New York, 1983. Sadik, O. A. Anal. Methods Instrum. 1995, 2, 293. Sadik, O. A.; Van Emon J. M. Biosens. Bioelectron. 1996, 11, 1. Diaz, A. F.; Bargon, J. In Handbook of Conducting Polymers, Vol. I; Skotheim, T. A., Ed.; Dekker: New York, 1986; p 81. Sadik, O. A., Wallace, G. G. Electroanalysis 1993, 5, 555. Daiz, A. F.; Castillo, J. L.; Logan, J. A. J. Chem. Soc., Chem. Commun. 1980, 397. Swoboda, B.; Massey, V. J. Biol. Chem. 1965, 240, 2209. Kanatzidis, M. G. Chem. Eng. News 1990, 68(49), 36. Masila, M.; Sargent, A.; Sadik, O. A. Electroanalysis 1998, in press. Symposium on Electronic Nose and Artificial Neural Networks; Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, 1998, New Orleans, LA, Abstract Numbers 524–528.
Journal of Chemical Education • Vol. 76 No. 7 July 1999 • JChemEd.chem.wisc.edu
