ZEPTOMOLE-DETECTING BIOSENSOR FOR ALKALINE-PHOSPHATASE IN AN ELECTROCHEMICAL IMMUNOASSAY FOR 2,4-DICHLOROPHENOXYACETIC ACID

A bienzyme substrate-recycling biosensor in a flow injection analysis system is described for the sensitive measurement of alkaline phosphat ase (ALP) and applied to the fast readout of a competitive immunoassay for the widely used pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). The phenol-indicating biosensor consists of a Clark-type electrode co vered by a membrane with coentrapped tyrosinase and quinoprotein gluco se dehydrogenase. ALP dephosphorylates phenyl phosphate to phenol (K-m = 36 mu M) outside the flow system. Phenol is oxidized in the sensor membrane by the oxygen-consuming tyrosinase via catechol to o-quinone. The quinone is reconverted to catechol by glucose dehydrogenase. This substrate cycling results in a 350-fold amplified sensor response to phenol, The oxygen consumption of the enzyme couple in the presence of phenol is monitored as a decrease in current. A total of 3.2 fM ALP ( 320 zmol/100 mu L) has been detected after a 57.5 min incubation with phenyl phosphate, All involved reagents are stable over the time of me asurement. The sensor does not produce any measurable blank signals. T he immunoassay detects 0.1 mu g/L 2,4-D, the maximum concentration for pesticides allowed in drinking water by European Community regulation s. The applicability of this biosensor for fast immunoassay readout is demonstrated by a 2 min incubation, By comparison, a standard photome tric method (p-nitrophenyl phosphate) requires overnight incubation.