An electrochemical assay system for peroxidase and peroxidase-couplers reactions based on a fluoride ion-selective electrode.

Abstract

A sensitive and accurate potentiometric reaction-rate method for the assay of peroxidase (EC 1.11.1.7) and peroxidase-coupled reactions is described. The technique is based on the peroxidase-catalyzed rupture of the covalent C--F bond in certain organo-fluoro compounds in the presence of H2O2 and the measurement of liberated F- by a fluoride ion-selective electrode. In a study screening 26 organo-fluoro compounds, 12 were susceptible to C--F bond rupture. However, analytically useful reaction kinetics were observed only with 4-fluoroaniline, 5-fluor-2-methylaniline, 4-fluorophenol, 2,3,5,6-tetrafluorophenol, pentafluorophenol, and 3-fluoro-DL-tyrosine. With this assay system, peroxidase activity as low as 25 mU/L could be precisely measured within 2 min. The reaction was coupled to glucose oxidase and cholesterol oxidase for the determination of glucose and cholesterol. Glucose oxidase activity in solution (to 2 U/L) was measured to demonstrate the feasibility of the assay for oxidase enzymes. Only a few microliters of sample is required and most determinations can be completed within minutes. The assay system offers broad applications and is especially attractive for use in enzyme immunoassays.