COMPETITIVE NONSEPARATION ELECTROCHEMICAL ENZYME BINDING IMMUNOASSAY (NEEIA) FOR SMALL-MOLECULE DETECTION

A nonseparation electrochemical enzyme binding/immunoassay (NEEIA) for the detection of small molecules via a competitive format is describe d. The NEEIA concept is based on the use of a microporous gold electro de, which serves as both the working electrode, and solid phase for th e immobilization of binding protein/antibody through a chemisorbed lay er of thioctic acid. Competitive assays are performed by incubating th e small molecule of interest and an alkaline phosphatase (ALP) labeled analyte competitor (conjugate) with the modified electrode. Surface b ound conjugate is spatially resolved from unbound conjugate by introdu cing the substrate (p-aminophenyl phosphate) through the backside of t he microporous gold electrode. The substrate diffuses rapidly through the microporous gold electrode where it first encounters surface bound conjugate. The enzymatically generated product (p-aminophenol) is sub sequently oxidized at the electrode (+190 mV vs. Ag/AgCl). Due to the competitive nature of the assay, the magnitude of the amperometric sig nal is inversely proportional to the concentration of analyte in the s ample. Detection of biotin, digoxin and digitoxin in buffer is demonst rated with detection limits of 1, 0.1 and 10 nM, respectively. in addi tion, it is shown that digoxin can be measured in undiluted sheep seru m with a detection limit of 1 nM, demonstrating that the proposed comp etitive NEEIA format can be employed for the detection of small molecu les directly in complex matrices without any discrete separation or wa shing steps. (C) 1997 Elsevier Science B.V.