Novel, reagentless, amperometric biosensor for uric acid based on a chemically modified screen-printed carbon electrode coated with cellulose acetate and uricase

Abstract

Amperometry in stirred solution has been used for the systematic evaluation of modified screen-printed carbon electrodes (SPCEs) with a view to developing a reagentless biosensor for uric acid. The developed system consists of a base cobalt phthalocyanine (CoPC) electrode tailored to the electrocatalytic oxidation of H₂O₂ by means of a cellulose acetate (CA)–uricase bilayer. Uricase was immobilized by drop-coating the enzyme onto the CA membrane covering the CoPC-SPCE. The device exploits the near-universal H₂O₂-generating propensity of oxidases, the permselectivity of the CA film towards H₂O₂ and the electrocatalytic oxidation of this product at the CoPC-SPCE. The electrochemical oxidation of the resulting Co⁺ species was used as the analytical signal, facilitating the application of a greatly reduced operating potential when compared with that required for direct oxidation of H₂O₂ at unmodified electrodes. The time required to achieve 95% of the steady-state current (t₉₅i_SS) was 44 s [relative standard deviation = 7.5%(n= 10)]. Amperometric calibrations were linear over the range from 13 × 10^–6 to 1 × 10^–3 mol dm^–3, with the former representing the limit of detection. The CA membrane extended the linear range of the biosensor by over two orders of magnitude, when apparent Michaelis–Menten constants (K_m′) of immobilized and free enzymes are compared. This suggests that the process is diffusion-controlled and not governed by the kinetics of the enzyme. The precision of electrode fabrication was determined by cyclic voltammetry to be 4.9%(n= 6). Successive amperometric calibrations (n= 7) over 7 d using a single sensor revealed only a 14.0% diminution in sensitivity from the original response. Sensor stability and the dependence of the steady-state current on the pH, ionic strength and temperature of the supporting electrolyte were studied and the results are presented. The functioning of the biosensor is indifferent to a wide range of potential interferences studied in a synthetic sample and results correlate favourably with those of a standard hospital method.