Biossensor enzimático para detecção de fungicidas ditiocarbamatos: estudo cinético da enzima aldeído desidrogenase e otimização do biossensor

Abstract

Recebido em 13/7/05; aceito em 3/4/06; publicado na web em 11/8/06 ENZYMATIC BIOSENSOR FOR THE DETECTION OF DITHIOCARBAMATE FUNGICIDES. KINETIC STUDY OF ALDEHYDE DEHYDROGENASE ENZYME AND BIOSENSOR OPTIMIZATION. Initially, all major factors that affect the rate of the AldH-catalyzed reaction (enzyme concentration, substrate concentration, temperature and pH) were investigated. Optimal activity was observed between pH values of 7.5 and 9.5 in the temperature range of 25 to 50 o C. Kinetic parameters, such as K m (2.92 μmol L -1 ) and V max (1.33 10 -2 μmol min -1 ) demonstrate a strong enzyme-substrate affinity. The sensors were based on screen-printed electrodes modified with the Meldola BlueReinecke salt (MBRS) combination. Operational conditions (NAD + and substrate contents, enzyme loading and response time) were optimized. Also, two enzyme immobilization procedures were tested: entrapment in poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) and crosslinking with glutaraldehyde. Chronoamperometry was employed to observe the biosensor responses during enzymatic hydrolysis of propionaldehyde and also to construct inhibition curves with maneb and zineb fungicides. Best results were found with the following conditions:; enzyme loading = 0.8 U per electrode; response time = 10 min, and inhibition time = 10 min. Current intensities around 103 ± 13 nA with the sensors and good stability was obtained for both immobilization procedures. Detection limits, calculated using 10% inhibition were 31.5 μg L -1 and 35 μg L -1 for maneb and zineb, respectively. Results obtained with other MBRS-modified electrodes consisting of mono and bi-enzymic sensors were compared. The ability to catalyze NADH oxidation by MB was also highlighted.Keywords: AldH-based biosensors; dithiocarbamate fungicides; enzymatic kinetic. INTRODUÇÃOA investigação dos pesticidas constitui um caso delicado. A necessidade de analisar resíduos de pesticidas depara-se com problemas analíticos complexos, devido à gama de substâncias existentes em classes distintas e que devem ser determinadas em diferentes tipos de matrizes, sejam elas ambientais ou de alimentos 1 . Entre os agrotóxicos mais difundidos atualmente, encontram-se os fungicidas ditiocarbamatos (DTC's). Tais compostos formam a mais importante classe de pesticidas empregados no controle de diversas doenças fungais em sementes, frutos e vegetais. Embora a toxidade aguda dos DTC's seja relativamente baixa, existe uma urgente necessidade de se encontrar um método de detecção sensível, confiável, e que permita rápidas tomadas de decisão, no caso de intoxicação em trabalhadores rurais e/ou contaminação ambiental ou alimentar. Isto devido ao fato de que, dependendo dos compostos encontrados, estes podem vir a ser carcinogênicos 2 , goitrogênicos 2 , mutagênicos 3-5 , teratogênicos 3,6,7 , provocar distúrbios neurológicos 8 e, também, afetar a biota aquática 9 . Há uma grande preocupação com relação a certos compostos como, por ex., os fungicidas maneb e ditane, que contêm manganês, precursor d...

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