Anal. Chem. 1992, 64, 2645-2646
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Biosensor Based on Conducting Polymers D. T. Hoa,?T. N. Suresh Kumar,$N. S. Punekar,§R. S. Srinivasa,?R. Lal,I and A. Q. Contractor*,$ Materials Science Center, Department of Chemistry, Bioscience and Engineering Group, and Department of Electrical Engineering, Indian Institute of Technology, Powai, Bombay 400 076, India
INTRODUCTION We report here a new biosensor based on electronically conducting polymers. The sensor action is based on the change in electronic conductivity in response to changes in the microenvironment such as pH or redox potential of the solution in contact with the polymer. Though the possible use of this concept as a chemical or redox sensor has been suggested,l this is the first proof-of-concept study of a biosensor. It is now well established that the electronic conductivity of this class of polymers shows large changes in response to changes in electrochemical potential1 or pH.2 Pickup has shown3 that the change in conductivity is an exponential function of electrochemical potential. Thus the change in electronic conductivity can be used as a very sensitive probe of changes in redox potential and hence concentration of electroactivespecies in the s ~ l u t i o n Specificity .~ to the desired biomolecule can be obtained by immobilizingthe appropriate enzyme in the polymer matrix. The particular system chosen for investigation was polyaniline-glucose oxidase to detect the concentration of glucose.
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Figure 1. A schematic representation of the sensor structure. Change in resistance ,SL
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EXPERIMENTAL SECTION The sensor described here has a bread/butter/jam configur a t i ~ n .Polyaniline ~ (butter)was deposited on platinum (bread) followed by a thin layer of polyaniline incorporating glucose oxidase (jam). The electrode configuration was similar to that described by Wrighton.' It consisted of two platinum disks embedded in epoxy and separated by a gap of 8pm. Alternatively, the configuration described in ref 2c was also used. A polyaniline film (butter) was deposited on platinum (bread) and grown to a thickness sufficient to bridge the gap between the electrodes. A thin polymer-enzyme film (jam) was deposited on this. The sensor structure is shown schematically in Figure 1. The first polyaniline layer (butter) was deposited according to the procedure of D k 6 The twin electrodes were subjected to potentiodynamic cycling in a solution containing 0.1 M aniline in 0.1 M H2S04 between the potential limits -0.2 and +0.8 V vs SCE. The second layer (jam) was deposited by a similar procedure from a phthalate buffer of pH = 4 containing 0.1 M aniline and 250 units/mL of GOD (EC 1.1.3.4 from Sigma). The potential was scanned between the limits -0.2 and +1.2 V vs SCE. The polymerization was carried out for 2 h. This two-layer structure was required because it was not possible to bridge the gap between the platinum electrodes by growing the film from the second
* Author for correspondence.
+ Materials
Science Center.
* Department of Chemistry.
Bioscience and Engineering Group. of Electrical Engineering. (1) (a) Kittleson, G. P.; White, H. S.; Wrighton, M . S. J. Am. Chem. SOC.1984, 106, 7389-7396. (b) Paul, E. W.; Ricco, A. J.; Wrighton, M. S. J. Phys. Chem. 1985,89,1441-1447. (c) Thackeray, J. W.; Wrighton, M. S. J . Phys. Chem. 1986,90,6674-6679. (2) (a) Jozefowicz, M.; Yu, L. T.; Perichon,J.; Buvet, R. J. Polym. Sei. 1969,C22,1187. (b)MacDiarmid,A. G.;Jin-ChihChiang;WuSong Huang; Humphrey, B. D.; Somasiri, N . L. D. Mol. Cryst. Liq. Cryst. 1985,125, 309-318. (c) Gholamian, M.; Suresh Kumar, T. N.; Contractor, A. Q. Proc. Indian Acad. Sei. 1986, 97, 457-464. (3) Ochmanska, J.; Pickup, P. G., J. Electroanal. Chem. Interfacial Electrochem. 1991,297, 211-224. (4) Wrighton, M. S. Science 1986,231, 32-37. (5) Patent applied for. (6) Diaz, A. F.; Logan, A. J. J . Electroanal. Chem. Interfacial Electrochem. 1980, 111, 111-114.
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Figure 2. The sensor response to solutions containing various concentrationsof (i) D-glucose and (ii) pmannose. Sensor no. PAGx.
bath alone. The low rate of polymerization and the poor conductivity of the film grown from the bath at pH = 4 proved to be the limitations. It was not possible to work at a lower pH due to the rapid denaturation of the enzyme. The immobilized enzyme was checked for activity spectrophotometrically using ABTS.7 The PAn-GOD film (0.25 cm2) was immersed in 2.6 mL of a 0.1 M sodium phosphatebuffer (pH 7.0) containing 2.5 mg of ABTS,.2 units of POD, and 4 pmol of D-g1UCOSe. It was maintained for 20 min with stirring at 298 K and was further incubated for 10 min at 310 K. The change in absorbance was monitored at 600 nm. The immobilized enzyme activity on the film corresponded to 0.12 pmol of D-glucose oxidized in the above assay. Under identical conditions, the control PAn film (no GOD) showed negligible activity.
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RESULTS AND DISCUSSION The sensor was immersed in glucose solutions of various concentrations, and its electronic conductivity measured. The electronic conductivity was measured by forcing a 5-nA peakto-peak AC current (1.33 kHz) and monitoring the in-phase voltage across the sensor with a two-phase lock-in analyzer. The results are shown in Figure 2. Measurements were made 20 s after immersing the sensor in the sample solution. The (7) Kunst, A.; Draeger, B.; Ziegenhorn, J. In Methods in Enzymatic Analysis; Bergmeyer, H . U., Bergmeyer, J., Grabl, M., Eds.; Verlag Chemie: 1984; Vol. VI.
0003-2700/92/0364-2645$03.00/0 0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY. VOL. 64. NO. 21, NOVEMBER 1, 1992
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Number of r u n s Figwe9. The sensor response to a solutlon contalnlng 10 mM glucose, as a function of the number of independent measurements. Sensor no. PAGy. change in resistance of the polymer film is linear with concentration of glucose up to 10 mM. The change in resistance is reversible and reproducible. The resistance of the polymer film decreases with increase in concentration of glucose, the modulus of the change, R[glucosel - R[OI, is plotted in Figure 2 for convenience of visualization. In order to verify that the response is specific to glucose rather than any sugar, a measurement was made with mannose, the results of which are also shown in Figure 2. There is virtually no change in the resistance of the polymer with change in mannose concentration. Thus, the sensor action is a consequence of the enzyme-catalyzed reaction and hence specific to glucose. The sensor was used for repeated measurement of a 10 mM glucose solution in order to examine the reproducibility of the sensor. The results are shown in Figure 3. Each measurement was preceded by rinsing the sensor in glucose-free buffer solution. The results show that after an initial fall in activity, the sensor response is stable for more than 14independent measurements with a standard deviation of less than 10%. The initial fall in activity may be attributed to the leaching out or deactivation of loosely bound enzyme. This device represents a new generic biosensor. The use of conducting polymers such as polypyrrole,s polyaniline? and polyindole10 has been made earlier to immobilizeenzymes to produce amperometric biosensors. However, the sensor described here utilizes the change in electronic conductivity of the polymer in response to changes in the microenvironment. The polymer here acta as the immobilization medium as well as the transducer for converting a biochemical signal to an electronic signal. The enzymatic reaction produces Hz02 (8) (a) Umana, M.; Waller, 3. Anal. Chem. 1986,58,2979-2983. (b) Foulds, N. C.; Lowe, C. R. J. Chem. SOC.,Faraday Tram. 1 1986,82, 1259-1264. (c) Iwakura, C.;Kajiya, Y.; Yoneyama, H. J. Chem. SOC., Chem. Commun. 1988,1019-1020.(d) Belanger, D.; Nadreau, J.;Fortier, G.,J. Electronal. Chem.Interfacial Electrochem. 1989,274,143-155.(e) Kajiye, Y.; Tsuda, R.; Yoneyama, H. J. Electroanal. Chem. Interfacial Electrochem. 1991,301,155-164. (9) (a) Bartlett, P. N.; Whitaker, R. G. Biosensors 1987/88,3,359-379. (b) Shinohara, H.; Chiba, T.;Aizawa, M., Sew. Actuators 1988,13,7984. (c) Shaolin, M.; Huaigno, X.; Bidong, Q.J. Electroanal. Chem. Interfacial Electrochem. 1991,304,7-16. (10)Pandey, P. C. J. Chem. SOC.,Faraday Tram 1 1988,84,22592265. (11)Bentley, R.In TheEnzymes;Boyer, P. D.,Lardy, H. A., Myrback, H., Eds.; Academic Press: New York, 1973;Vol. VII. (12)(a) Degani, Y.;Heller, A. J. Am. Chem. SOC.1989,111,2357-2358. (b) Degani, Y.; Heller, A. J. Phys. Chem. 1987,91,1285-1289.(c) Degani, Y.; Heller, A. J. Am. Chem. SOC.1988,110,2615-2620. (d) Schuhmann, W.; Ohara, T. J.;Schmidt, H.-L.;Heller, A. J.Am. Chem. SOC.1991,113, 1394-1397. (13)Orata, D.; Buttry, D. A. J. Am. Chem. SOC.1987,109,3574-3581. (14)Data for Biochemical Research; Dawson, R. M. C., Elliot, D. C., Elliot, W. H., Jones, K. M., Eds.; Clarendon Press: Oxford, 1986. (15)Nilseon, H.;Akerlund, A. C.;Mosbach, K. Biochim. Biophys. Acta 1973,320,529-534.
and D-glucono-&lactonewhich nonenzymatically hydrolyzes to gluconic acid." The enzymatic oxidation consumes dissolved oxygen. The change in electronic conductivity can possibly arise from a change in the chemical potential of the microenvironment due the production/consumption of any one or more of the above species. Independent measurements of polymer conductivity as a function of the partial pressure of dissolved oxygen and concentration of HzOz have shown that the sensor response cannot be explained by these factors. The possibility of the polymer acting as an electron transfer mediator would generallybe considered unlikely as the enzyme is merely physically trapped in the polymer matrix. However, this possibility was also examined in light of a recent report of electron transfer from the polyanionicGOD electrostatically bound to a polycationic redox polymer.12a Polyaniline is known to be positively charged at potentials positive to 0.2 V vs SCE,13which are observedat open-circuit in our solutions. Two types of experiments were performed to obtain evidence of such electron transfer from the reduced flavin center of the enzyme to polyaniline. In one set of experiments the open-circuit potential of the "bilayer" film was monitored in response to the addition of increasing doses of glucose, while in the other set, the film was subjected to cyclic potential scans in the presence and absence of glucose. If there is electron transfer from the reduced flavin to the polymer, the open-circuit potential of the film is expected to drift in the cathodic direction, while the cyclic voltammograms are expected to show anodic currents proportional to the concentration of glucose. The results from both of these sets of studies indicate negligible direct electron transfer from the enzyme to the polymer. However, it may be noted that the use of redox mediators covalently attached to the enzyme has been shown to promote electron transfer to a redox polymercoated electrode.12" Therefore in the present case the response must be attributed to the change in pH of the microenvironment within the polymer film due to formation of gluconic acid (pK, = 3.7614). The use of conventional pH electrodes has been previously made for the determination of glucosel6 by monitoring the change in pH a t the surface of a glass electrode coated with polyacrylamide gel containing GOD. The pH dependence of the conductivity of polyanilinehas been studied earlier, and it is known that the conductivity increases with a decrease in pH.2c The dependence of the logarithm of conductivity on pH is sigmoid, but it can be approximated to be linear over a narrow range of pH. This sensor concept will open up the possibility for miniaturization and integration using microelectronic fabrication technology to produce inexpensive chips (electronic tongues) for sensing a range of biomolecules such as glucose, urea, hemoglobin, and cholesterol in a single operation with microliter volumes of sample.
ACKNOWLEDGMENT D.T.H. would like to acknowledge support from the IndoVietnam Exchange Program while T.N.S.K. would like to thank IIT, Bombay,for a research associateship. A.Q.C., R.L., R.S.,and N.S.P. are members of the Ultramicroscopy and Microstructure Engineering Group, IIT, Bombay.
RECEIVED for review December 23, 1991. Accepted July 30, 1992.
Registry No. D-Glucose, 50-99-7; polyaniline, 25233-30-1; glucose oxidase, 9001-37-0; platinum, 7440-06-4.
