An amperometric biosensor based on human cytochrome P450 2C9 in polyacrylamide hydrogel films for bisphenol A determination

Electrochemical behavior of human cytochrome P450 2C9 (CYP2C9) incorporated in polyacrylamide (PAM) hydrogel films cast on glassy carbon electrodes (GCE) was investigated. CYP2C9-PAM film electrodes showed a pair of well-defined and nearly reversible cyclic voltammetric peaks. The electron-transfer rate constant was about 6.96 s-(1) in pH 7.0 buffers, and the formal potential (E-0') was -0.427 V (vs. SCE). The dependence of E-0' on solution pH indicated that one-proton transfer was coupled to each electron transfer in the direct electron-transfer reaction. UV-vis absorption spectroscopy demonstrated that CYP2C9 retained a near native conformation in PAM films at medium pH. The CYP2C9 in the PAM film retained its bioactivity and could catalyze the reduction of dissolved oxygen. Upon the addition of its substrate bisphenol A (BPA) to the air-saturated solution, the reduction peak current of dissolved oxygen increased, which indicates the catalytic behavior of CYP2C9 to BPA. By amperometry a calibration linear range for BPA was obtained to be 1.25-10.0 mu M with a sensitivity of 18.21 mu A mM-(1). And the apparent Michaelis-Menten constant for the electrocatalytic activity of CYP2C9 was estimated to be 3.90 mu M for BPA. (c) 2013 Elsevier B.V. All rights reserved.