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 degrees C. Kinetic parameters, such as K-m (2.92 mu mol L-1) and V-max (1.33 10(-2) mu mol min(-1)) demonstrate a strong enzyme-substrate affinity. The sensors were based on screen-printed electrodes modified with the Meldola Blue-Reinecke 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: [NAD(+)] = 0.25 mmol L-1; [propionaldehyde] = 80 mu mol L-1; 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 mu g L-1 and 35 mu g 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.