Anal. Chem. 1996, 68, 796-806
Electroanalytical Detection of Glucose Using a Cyanometalate-Modified Electrode: Requirements for the Oxidation of Buried Redox Sites in Glucose Oxidase Guoqiong Du, Chao Lin,† and Andrew B. Bocarsly*
Frick Laboratory, Department of Chemistry, Princeton University, Princeton, New Jersey 08544-1009
Modification of a nickel electrode with a mixture of iron and ruthenium cyanometalates allows one to efficiently turn over glucose oxidase in the presence of its substrate. A limit of detection for glucose of 25 µM can be obtained with an observed saturation concentration of 10 mM. Glucose detection is found to be very sensitive to the cationic environment of the electrolyte. The largest currents for glucose oxidation are observed in the presence of nonelectroactive multiply charged cations. The solid state nature of the surface-confined cyanometalate redox mediator argues against the widely held mechanism for enzyme oxidation in which the redox mediator is required to enter the enzyme active site. An enzyme-based electrochemical sensor requires some form of electrical communication between the active center of a redox enzyme and the electrode surface, since the active site of the redox enzyme is typically within an insulating protein shell.1 Glucose oxidase (GOX), with a crystallographically determined size of 60 Å × 52 Å × 77 Å, contains two “buried” redox-active flavin adenine dinucleotide (FAD) centers.2 In the natural system, diffusion of O2 through the protein matrix is thought to supply oxidizing equivalents to the FAD centers. The reaction sequence is illustrated by eqs 1 and 2,
GOXox + glucose f GOXred + gluconolactone
(1)
GOXred + O2 f GOXox + H2O2
(2)
where GOXox is the oxidized state of glucose oxidase and GOXred is the reduced state of glucose oxidase. Oxidation of purified GOX has been observed at several metal electrodes.3 However, in these cases, the observation of GOX electroactivity has been associated with chemisorption of the enzyme leading to denaturation and exposure or removal of the FAD subunits. Under such conditions, the activity of the GOX with respect to glucose oxidation is significantly reduced. However, the denatured enzyme apparently has a limited capability to electroanalyze the oxidation of intact GOX. † Current address: I-Stat, Princeton, NJ 08540. (1) Murray, R. W.; Dessy, R. E.; Heineman, W. R.; Janata, J.; Seitz, W. R. Chemical Sensors and Microinstrumentation; ACS: Washington, DC, 1988. (2) Hecht, H. J.; Kalisz, H. M.; Hendle, J.; Schmid, R. D.; Schomburg, D. J. Mol. Biol. 1993, 229, 153. (3) Szucs, A.; Hitchens, G. D.; Bockris, J. O. M. J. Electrochem. Soc. 1989, 136, 3748.
796 Analytical Chemistry, Vol. 68, No. 5, March 1, 1996
To date, most analytical strategies for the electrochemical determination of glucose involve detection of the H2O2 reaction product (eq 2) or, alternatively, monitoring decreased O2 concentrations.4,5 However, fluctuations in ambient O2 concentrations introduce analytical difficulties with these approaches. Thus, methods that are not dependent on O2 as the electron mediator have been sought. Several soluble electron mediators, which include ferrocenes,6 quinones,7 ruthenium amines,8 ferricyanide,9 and hexacyanoruthenate8,10 have been employed to accelerate electron transfer between GOX redox centers and electrode surfaces. In such systems, reaction 1 is followed by
GOXred + mediatorox f GOXox + mediatorred + 2H+ (3) where the reduced form of the mediator is subsequently electrooxidized. It is this electrooxidation process which is utilized in the analytical detection of glucose. When assembling a real sensor, a membrane is usually required to confine the enzyme and the mediator near the electrode surface.11 This added complexity can be circumvented by developing integrated enzyme/mediator systems, as demonstrated by Hale et al.12 and Bourdillon and Majda.13 Alternatively, it has been demonstrated that certain electrode-chemisorbed heterocyclic compounds, such as a 4,4′-bipyridine, allow for the oxidation of GOX at gold electrodes.14 Based on this type of chemistry, Heller has demonstrated that GOX can be “wired” with redox subunits by partial denaturation, followed by covalent attachment of several redox centers (ferrocene, for example) and renaturation.15 The synthetic redox centers provide a self-exchange pathway between the enzyme surface and the FAD units, making electrochemistry possible at a variety of metal electrodes. More recently, partial denaturation (4) Carr, P. W.; Bowers, L. D. Immobilized Enzymes in Analytical and Clinical Chemistry; Wiley: New York, 1980. (5) Lin, C. Ph.D. Thesis, Princeton University, Princeton, NJ, 1992. (6) Cass, A.; Davis, G.; Francis, G. D.; Hill, H. Anal. Chem. 1984, 56, 667. (7) Kulys, J. J.; Cenas, N. K. Biochim. Biophys. Acta 1983, 744, 57. (8) Crumbliss, A. L.; Hill, H. A. O.; Page, D. J. J. Electroanal. Chem. Interfacial Electrochem. 1986, 206, 327. (9) Schlapfer, P.; Mindt, W.; Pacine, P. Clin Chim. Acta 1974, 57, 283-289. (10) Taniguchi, T.; Miyamoto, S.; Tomimura, S.; Hawkridge, F. J. Electroanal. Chem. 1988, 240, 333. (11) Heller, A. Acc. Chem. Res. 1990, 23, 128. (12) Hale, P. D.; Inagaki, T.; Kraran, H. I.; Okamoto, Y.; Skotheim, T. A. J. Am. Chem. Soc. 1989, 111, 3482. (13) Bourdillon, C.; Majda, M. J. Am. Chem. Soc. 1990, 112, 1795. (14) Holt, R. E.; Cotton, T. M. J. Am. Chem. Soc. 1989, 111, 2815. (15) Degani, Y.; Heller, A. J. Phys. Chem. 1987, 91, 1285. 0003-2700/96/0368-0796$12.00/0
© 1996 American Chemical Society
and renaturation of GOX in the presence of a redox polymer has been shown to provide a similar benefit.16 Related schemes have been developed in which a redox polymer such as poly(vinylferrocene) or a conducting polymer such as polypyrrole is coated onto a solid electrode in order to catalyze GOX oxidation via a mediation-type process.17,18 Common to such studies is the assumption that GOX electrocatalysis is achieved by bringing a redox-active center into direct charge transfer contact with the FAD centers of the enzyme, thereby opening up a redox-mediation channel. In this paper, we will show that certain solid state redox mediators, based on interfacial structures containing bridging cyanometalates, are effective catalysts for GOX electrochemistry and thus glucose detection. To the best of our knowledge, this is the first report of redox catalysis to GOX via the general class of Prussian Blue and its analogs. While mediated charge transport through the cyanometalate is demonstrated, the solid state nature of these systems precludes intimate contact between the buried FAD centers and the catalyst. This observation brings into question the long-standing assumption that the mediator must physically shuttle (via diffusion or by electron exchange “effective” diffusion) between the electrode and the FAD centers. Rather, it would appear that orientation of the enzyme with respect to the electrode is of primary importance for the charge transfer channel. The current studies are based on the modification of Ni electrodes with a solid-state mixture of nickel ferrocyanide and nickel ruthenocyanide, systems we have previously studied separately as interfacial charge transfer mediators.19 EXPERIMENTAL SECTION Chemicals. Glucose oxidase from Aspergillus niger was obtained from Sigma. Contamination of sample by free FAD was assayed for by UV-visible spectroscopy. This analysis set the limit of potential FAD contamination at
