Flow-injection determination of catechol with a new tyrosinase/DNA biosensor1Presented in part at the VII International Conference on Flow Analysis, Piracicaba, Brazil, 1997.1

Abstract

Biosensors ®nd application in¯ow 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¯ow, 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¯ow conditions (electroreduction of the products of the enzymatic oxidation of diphenols in the presence of O 2). 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 48C 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 À0,15 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 1Â10 À6 up to 5Â10 À5 mol l À1 .

Post-column detection of benzenediols and 1,2,4-benzenetriol based on acidic potassium permanganate chemiluminescence

“…For the FIA-CL methods, the detection limits of the reported were apparently inferior to the current, and these methods were only used for the determination of single phenolic congener in simple matrices [19,25,[16][17][18], whereas the current method had successfully determined benzenediols or benzenetriols in natural water with simple sample pretreatment. As the rest methods cited are concerned, they all had relative high limits of detection compared with the present although some of them, such as biosensor [37,39], were claimed having impressive selectivity. Therefore, the established method offers HPLC or CL detection an alternative, sensitive and simple approach for the determination of some phenolic compounds and is no doubt a potential tool for its routine use in various areas, such as environmental analysis, etc.…”

Biossensores baseados no processo de inibição enzimática

“…75 A tirosinase pode ser inibida por uma gama de poluentes, tais como carbamatos, organofosforados, ditiocarbamatos, atrazinas, tiuréas, ácidos carboxílicos aromáticos, triazinas, herbicidas, clorofenóis e agentes quelantes de cobre. [75][76][77][78][79][80][81][82][83][84][85][86] Desta forma, biossensores baseados em enzimas tirosinases podem ser empregados com biossensores baseados em inibição enzimática, detectando tanto o decrescimento da quantidade de oxigênio no meio reacional, quanto a formação da quinona, em presença do agente inibidor. Eletrodos de carbono, ouro, ouro modificado e eletrodos de Clark têm sido empregados na construção destes sensores.…”

Biossensores amperométricos para determinação de compostos fenólicos em amostras de interesse ambiental

“…Também é descrita na literatura a incorporação de vários aditivos em biossensores a base de tirosinase para aumentar a estabilidade e sensibilidade para a determinação de compostos fenólicos 23,29,34,82,83 . …”