Potential-resolved electrochemiluminescence multiplex immunoassays with isolated cathodic and anodic co-reactants and pre-oxidized Ag-doping methionine-stabilized Au nanoclusters.

In this work, a potential-resolved electrochemiluminescence (ECL) multiplex immunoassay (MIA) was developed using Ag-doping methionine-stabilized Au nanoclusters (Met-AuAgNCs) with immobilized co-reactant as the anodic ECL tag and nanocomposite of gold nanoparticles/graphene oxide/N, N'-dicaproate sodium-3,4,9,10-perylene-dicarboximide (AuNPs/GO/PDI) as the cathodic ECL tag. Compared with methionine-stabilized Au nanoclusters (Met-AuNCs), the ECL of Met-AuAgNCs was enhanced 5.61-fold. When anodic co-reactant of N,N-diethylethylenediamine (DEDA) was connected to Met-AuAgNCs, the ECL of DEDA-Met-AuAgNCs was 11.3-fold of that of Met-AuAgNCs in DEDA solution due to the shorter charge transfer distance between Met-AuAgNCs and DEDA. After a pre-oxidation at 0.95 V for 60 s, the ECL of DEDA-Met-AuAgNCs was further enhanced by 10.6- and 27.9-fold in the cyclic voltammetric and potential step modes, respectively. The pre-oxidation ECL enhancement was demonstrated by an immobilized co-reactant promoters mechanism. In a potential-resolved ECL-MIA, carbohydrate antigen 125 and carbohydrate antigen 19-9 were adopted as model analytes, with the detection limits of 0.029 and 0.076 mU mL^-1, respectively. The work provides a proof of concept using self-ECL luminophores with immobilized co-reactant promoters in situ formed for potential-resolved ECL-MIAs with isolated anodic and cathodic co-reactants.