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 described. The NEEIA concept is based on the use of a microporous gold electrode, which serves as both the working electrode, and solid phase for the immobilization of binding protein/antibody through a chemisorbed layer of thioctic acid. Competitive assays are performed by incubating the small molecule of interest and an alkaline phosphatase (ALP) labeled analyte competitor (conjugate) with the modified electrode. Surface bound conjugate is spatially resolved from unbound conjugate by introducing the substrate (p-aminophenyl phosphate) through the backside of the 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 subsequently oxidized at the electrode (+190 mV vs. Ag/AgCl). Due to the competitive nature of the assay, the magnitude of the amperometric signal is inversely proportional to the concentration of analyte in the sample. Detection of biotin, digoxin and digitoxin in buffer is demonstrated with detection limits of 1, 0.1 and 10 nM, respectively. In addition, it is shown that digoxin can be measured in undiluted sheep serum with a detection limit of 1 nM, demonstrating that the proposed competitive NEEIA format can be employed for the detection of small molecules directly in complex matrices without any discrete separation or washing steps.