SEPARATION-FREE SANDWICH ENZYME IMMUNOASSAYS USING MICROPOROUS GOLD ELECTRODES AND SELF-ASSEMBLED MONOLAYER IMMOBILIZED CAPTURE ANTIBODIES

A novel separation-free sandwich-type enzyme immunoassay for proteins is performed by designing an electrochemical detection system that enables preferential measurement of surface-bound enzyme-labeled antibody relative to the excess enzyme-labeled reagent in the bulk sample solution. In this initial model system, the assay is carried out using gold-coated microporous nylon membranes (pore size 0.2 mu m) which are mounted between two chambers of a diffusion cell. The membrane serves as both a solid phase for the sandwich assay and the working electrode in the three-electrode amperometric defection system. The capture monoclonal antibody is immobilized covalently on the gold side of the membrane via a self-assembled monolayer of thioctic acid. In the separation-free sandwich assay, both model analyte protein (human chorionic gonadotropin; hCG) and alkaline phosphatase labeled anti-hCG (ALP-Ab) are incubated simultaneously with the immobilized capture anti-hCG antibody. Surface-bound ALP-Ab is spatially resolved from the excess conjugate in the bulk sample solution by introducing the enzyme substrate (4-aminophenyl phosphate) through the back side of the porous membrane. The substrate diffuses rapidly through the porous membrane where it first encounters bound ALP-Ab at the gold surface. The enzymatically generated product, aminophenol, is detected immediately by oxidation at the gold electrode (at +0.19 V vs Ag/AgCl), and the magnitude of current is directly proportional to the concentration of hCG in the sample. The response time after substrate addition is less than I min, although maximum response toward the analyte protein requires a sample/conjugate preincubation time of 30 min with the porous electrode. The assay is demonstrated to function effectively in both buffer and whole human blood with a defection limit of 2.5 units/L hCG (in blood), which is comparable to most of heterogeneous EIAs that require multiple washing steps.