de Albuquerque Yaico D Tanimoto, Ferreira Lucas Franco
Laboratório de Eletroquímica Aplicada, Instituto de Química, Universidade Federal de Uberlândia, CEP 38.400-089, CP: 593, Uberlândia-MG, Brasil.
Anal Chim Acta. 2007 Jul 23;596(2):210-21. doi: 10.1016/j.aca.2007.06.013. Epub 2007 Jun 13.
A tyrosinase (Tyr) screen-printed biosensor based on the electroreduction of enzymatically generated quinoid products was electrochemically characterized and optimized for determination of carbamates and organophosphorus pesticides. A composite electrode prepared by screen-printing a cobalt (II) phthalocyanine (CoPc) modified cellulose-graphite composite on a polycarbonate support was employed as electrochemical transducer. The Tyr biosensor was prepared by immobilization of enzyme on the composite electrode surface by cross-linking with glutaraldehyde and bovine serum albumin. Parameters affecting the biosensor response such as response time, enzyme loading, concentration and pH of the buffer solution were optimized utilizing catechol as substrate. The maximum response for o-quinone enzymatically generated was obtained after 2 min of reaction. A good reproducibility and high operational stability were found for Tyr biosensor (60 units) at 50 mM phosphate buffer, pH 6.50. Under these conditions, the useful lifetime of biosensor was 10 days. After 15 days, the biosensor could be used with 20% of the initial value. Inhibition studies on the o-quinone steady-state current (at -0.20 V versus Ag/AgCl) were performed to investigate the inhibition kinetics of the pesticides in the enzymatic activity of mushroom tyrosinase. The results shown that the methyl parathion and carbofuran can lead to competitive inhibition process of the enzyme, while diazinon and carbaryl act as mixed inhibitors. Linear relationships were found for methyl parathion (6-100 ppb), diazinon (19-50 ppb), carbofuran (5-90 ppb) and carbaryl (10-50 ppb). Analysis of natural river water samples spiked with 30 ppb of each pesticide showed recoveries between 92.50% and 98.50% and relative standard deviations of 2%.
基于酶促生成的醌类产物电还原的酪氨酸酶（Tyr）丝网印刷生物传感器进行了电化学表征，并针对氨基甲酸酯类和有机磷农药的测定进行了优化。通过在聚碳酸酯载体上丝网印刷钴（II）酞菁（CoPc）修饰的纤维素-石墨复合材料制备的复合电极用作电化学换能器。通过用戊二醛和牛血清白蛋白交联将酶固定在复合电极表面制备Tyr生物传感器。以邻苯二酚为底物，优化了影响生物传感器响应的参数，如响应时间、酶负载量、缓冲溶液的浓度和pH值。反应2分钟后，酶促生成的邻醌获得最大响应。在50 mM磷酸盐缓冲液（pH 6.50）中，Tyr生物传感器（60个单位）具有良好的重现性和高操作稳定性。在这些条件下，生物传感器的使用寿命为10天。15天后，生物传感器仍可使用初始值的20%。对邻醌稳态电流（相对于Ag/AgCl为-0.20 V）进行抑制研究，以研究农药对蘑菇酪氨酸酶酶活性的抑制动力学。结果表明，甲基对硫磷和克百威可导致酶的竞争性抑制过程，而二嗪农和西维因则作为混合抑制剂。发现甲基对硫磷（6-100 ppb）、二嗪农（19-50 ppb）、克百威（5-90 ppb）和西维因（10-50 ppb）呈线性关系。对添加了30 ppb每种农药的天然河水样品进行分析，回收率在92.50%至98.50%之间，相对标准偏差为2%。
