Enzymatic recycling-based amperometric immunosensor for the ultrasensitive detection of okadaic acid in shellfish

Electrochemical immunosensors based on a competitive indirect enzyme-linked immunosorbent assay (ciELISA) and an enzymatic recycling system were developed for the detection of okadaic acid (OA). OA-ovalbumin (OA-OVA) conjugate was immobilised on screen-printed electrodes (SPEs) and competition of a newly generated monoclonal antibody (MAb) for free and immobilised OA was subsequently performed. Secondary antibodies labelled with alkaline phosphatase (ALP) or horseradish peroxidase (HRP) were used for signal generation. Experimental parameters were firstly optimised by colorimetric ELISA on microtiter wells and on SPEs. The ELISA system was then tested by amperometry at +300 mV vs. Ag/AgCl (detection of p-aminophenol produced by the reaction of p-aminophenyl phosphate with ALP) or -200 mV vs. Ag/AgCl (detection of 5-methyl-phenazinium methyl sulfate, redox mediator in the HRP bioelectrocatalysis). The limits of detection (LODs) with standard solutions were 1.07 and 1.98 microgL(-1) when using ALP and HRP labels, respectively. An electrochemical signal amplification system based on diaphorase (DI) recycling was integrated into the ALP-based immunosensor, decreasing the LOD to 0.03 microgL(-1) and enlarging the working range by two orders of magnitude. Preliminary results with mussel and oyster extracts were obtained and compared with the colorimetric immunoassay, the colorimetric protein phosphatase inhibition assay (PPIA) and LC-MS/MS.