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 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.
Bauer
C G
CG
Institute for Biochemistry and Molecular Physiology, University of Potsdam, Berlin-Buch, Germany.
Eremenko
A V
AV
Ehrentreich-Förster
E
E
Bier
F F
FF
Makower
A
A
Halsall
H B
HB
Heineman
W R
WR
Scheller
F W
FW
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
D006540:Herbicides; D015084:2,4-Dichlorophenoxyacetic Acid; D000469:Alkaline Phosphatase