title: Zeptomole-detecting biosensor for alkaline phosphatase in an electrochemical immunoassay for 2,4-dichlorophenoxyacetic acid.
abstract: A bienzyme substrate-recycling biosensor in a flow injection analysis system is described for the sensitive measurement of alkaline phosphatase (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 covered by a membrane with coentrapped tyrosinase and quinoprotein glucose dehydrogenase. ALP dephosphorylates phenyl phosphate to phenol (K(m) = 36 microM) 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 microL) has been detected after a 57.5 min incubation with phenyl phosphate. All involved reagents are stable over the time of measurement. The sensor does not produce any measurable blank signals. The immunoassay detects 0.1 microgram/L 2,4-D, the maximum concentration for pesticides allowed in drinking water by European Community regulations. The applicability of this biosensor for fast immunoassay readout is demonstrated by a 2 min incubation. By comparison, a standard photometric method (p-nitrophenyl phosphate) requires overnight incubation.
authors.affiliation: Institute for Biochemistry and Molecular Physiology, University of Potsdam, Berlin-Buch, Germany.
authors.forename: C G
authors.initials: CG
authors.lastname: Bauer
authors.forename: A V
authors.initials: AV
authors.lastname: Eremenko
authors.forename: E
authors.initials: E
authors.lastname: Ehrentreich-Förster
authors.forename: F F
authors.initials: FF
authors.lastname: Bier
authors.forename: A
authors.initials: A
authors.lastname: Makower
authors.forename: H B
authors.initials: HB
authors.lastname: Halsall
authors.forename: W R
authors.initials: WR
authors.lastname: Heineman
authors.forename: F W
authors.initials: FW
authors.lastname: Scheller
mesh_terms: D015084:2,4-Dichlorophenoxyacetic Acid / Q000032:analysis*; D000469:Alkaline Phosphatase / Q000032:analysis*; D015374:Biosensing Techniques; D002138:Calibration; D004563:Electrochemistry; D006540:Herbicides / Q000032:analysis*; D007118:Immunoassay
chemical_list: D006540:Herbicides; D015084:2,4-Dichlorophenoxyacetic Acid; D000469:Alkaline Phosphatase