Enzyme-induced Fenton reaction coupling oxidation of o-phenylenediamine for sensitive and specific immunoassay

In this paper, a simple and feasible immunoassay protocol with signal amplification for sensitive determination of alpha fetoprotein (AFP, used as a model) by using new enzyme-induced Fenton reaction (EIFR) accompanying the oxidation of o-phenylenediamine (OPD) system. We discovered that glucose oxidase (GOx) has the ability to stimulate in situ Fenton reaction coupling the oxidation of OPD. GOx-catalyze of glucose results in the formation of hydrogen peroxide (H 2 O 2 ). It can catalytically oxidize Fe 2+ to Fe 3+ , meanwhile producing hydroxyl radicals (•OH). The latter effectively initiated the catalytic oxidation of o-phenylenediamine (OPD) to 2,2-aminoazobenzene (DAP), resulting in the production of an electrochemical signal. On the basis of EIFR-OPD system, a new immunoassay protocol with GOx/anti-pAb 2 -conjugated gold nanoparticle (Ab 2 -AuNP-GOx) detection antibody can be designed for the detection of target AFP on capture antibody–functionalized transparent 96-well polystyrene microplate, monitored by recording the current peak of the generated DAP. The electrochemical current shows to be positively correlated with the concentration of target AFP. Under optimal conditions, the proposed assay exhibited good electrochemical responses for detecting target AFP in the range of 1.0 pg mL − 1 to 100 ng mL −1 with a detection limit (LOD) of 0.5 pg mL −1 (0.5 ppt) estimated at the 3 S blank level. Additionally, the precision, reproducibility, specificity, and method accuracy were also investigated with acceptable results. Importantly, the EIFR-OPD system can be further extended for the determination of other protein or biomarkers by controlling the specific recognition system (antibody or aptamer).