Development of a separation-free sandwich type electrochemical enzyme immunoassay for measuring proteins in undiluted whole blood.

Abstract

A novel separation-free sandwich type enzyme immunoassay (EIA) for measuring proteins in undiluted whole blood is described by designing an electrochemical detection system that enables preferential measurement of surface bound enzyme-labeled antibody relative to the excess enzyme conjugate in the bulk sample solution. 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 3-electrode amperometric detection system. The capture monoclonal antibody is immobilized covalently on the gold side of the membrane via use of self-assembled monolayer of thioctic acid. In the separation-free sandwich assay, both model analyte protein (human chorionic gonadotropin (hCG) and (or) prostate specific antigen (PSA)) and alkaline phosphatase labeled antibody (ALP-Ab) are incubated simultaneously with the immobilized capture anti-analyte 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 backside 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, 4-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 analyte protein in the sample solution. The response time after substrate addition is less than 1 min, while maximum response toward the analyte protein requires a sample/conjugate pre-incubation time of 30 min with the porous electrode. The assay is demonstrated to function effectively in both buffer and whole human blood (using hCG and PSA as models) with detection limit of 0.50 ng/ml and 0.37 ng/ml for hCG and PSA respectively. These values are comparable to most existing heterogeneous EIAs that require multiple washing steps. By employing two co-existing microporous gold electrodes on the same membrane, this system can be modified for the non-separation simultaneous assays of two proteins (hCG and PSA) in whole blood. The new assay format is also applied for the non-separation sandwich assay of viruses using potato virus A as the model analyte. The proposed approach may provide a promising avenue for the development of compact, portable, and inexpensive test systems capable of quickly testing diagnostically important proteins (e.g., cardiac marker proteins) in whole blood.