Flow-injection determination of catechol with a new tyrosinase/DNA biosensor

Abstract

Biosensors find application in flow analysis due to their high selectivity and sensitivity. Decrease in the response during extended use, originated by degradation, inhibition or structural changes of the enzyme or leaching of active components by the flow, is the prevailing problem. As an alternative to additives and preparation techniques cited in the literature, it is proposed to use DNA as a matrix for improving preservation of the activity of a diphenol-sensor-based tyrosinase, Tyr, (EC 1.14.18.1). The Tyr-DNA mixture was incorporated into carbon paste, CP-DNA-Tyr, or applied on glassy carbon, GC-DNA-Tyr. The CP-DNA-Tyr, covered by a membrane -of Cuprophan, presented superior performance in amperometric operation under flow conditions (electroreduction of the products of the enzymatic oxidation of diphenols in the presence of O2). In comparison with paste electrodes without DNA, CP-Tyr, a current increase of one order of magnitude was observed for catechol FIA peaks, with good repeatability during several hours of operation. The response decayed ca. 50% after every 3 to 4 days of use (with dry storage at 4°C overnight). Original performance was recovered by simply substituting the used paste for a new portion of stock paste, stable for 2 months under refrigeration. Evaluation of 18 different substrates and potential interferents indicated that, at the adopted potential of -0,15 V vs. Ag/AgCl, only p-cresol gives a response comparable to catechol. Flow-injection determination of catechol samples was conducted at a frequency of 30 injections/h, with linear response from the detection limit of 1×10-6 up to 5×10-5 mol l-1.