A novel approach to prepare a stable charge transfer complex (CTC) electrode for the direct oxidation of flavoproteins and the fabrication of a third generation amperometric biosensor (Koopal, C.G.J.; Feiters, M.C.; Nolte, R.J.M. Bioelectrochem. Bioenerg. 1992, 29, 159-175) system is described. Tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ), an organic CTC, is grown at the surface of a shapable electroconductive (SEC) film (a polyanion-doped polypyrrole film) in such a way that it makes a tree-shaped crystal structure standing vertically on the surface. Glucose oxidase (GOx) is adsorbed and cross-linked with glutaraldehyde to fix at the surface of the CTC structure. The space between crystals is filled with cross-linked gelatin to ensure the stability of the treelike crystal structure as well as the stability of the enzyme. Because of the close proximity and the favorable orientation of the enzyme at the CTC surface, the enzyme is directly oxidized at the crystal surface, which leads to a glucose sensor with remarkably improved performance. It works at a potential from 0.0 to 0.25 V (vs Ag/AgCl). The maximum current density at 0.25 V reaches 1.8 mA/cm2, with an extended linear range. The oxygen in the normal buffer solution has little effect on the sensor output. The current caused by interference contained in the physiological fluids is negligible. The working life as well as the shelf life of the sensor is substantially prolonged. The sensor was continuously used in a flow injection system with a continuous polarization at 0.1 V, and the samples (usually 10 mM glucose) were injected at 30 min intervals. After 100 days of continuous use, the current output dropped to 40% of the initial level. No change in the output of the sensor was observed over a year when the sensor was stored dry in a freezer. The electrochemical rate constants and the effective Michaelis constant of the system are reported.

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