Sensitive detection for enzyme-linked immunosorbent assays based on the adsorptive stripping voltammetry of indigo in a flow system

The suitability of carrying out sensitive electrochemical detection in conventional enzyme-linked immunosorbent assays (ELISAs) that use alkaline phosphatase (AP) as label was demonstrated. Studies were carried out for pneumolysin (PLY) and interleukin-10 (IL-10) ELISAs. The supports of this sensitive detection were the use of 3-indoxylphosphate (IP) as substrate and the employ of both an appropriate electrochemical technique (alternating current adsorptive stripping voltammetry (ACAdSV)) and a flow system. The adsorption of the enzymatic hydrolysis product on a carbon paste electrode permitted its pre-concentration and the combination with a flow system allows performing the measurement in fresh electrolyte, eliminating possible interferences. Calibration curves obtained in optimised conditions indicated an increase in sensitivity (70 times for PLY and 10 times for IL-10) when compared with conventional spectrophotometric detection. Limits of detection as low as 0.4 pg PLY per well and 0.2 pg IL-10/ml were obtained. Validation of the methodology was made by determining samples of a proteic matrix spiked with standards.