A homogeneous enzyme-free ratiometric immunoassay for the determination of C-peptide

In this study, a homogeneous enzyme-free ratiometric (HOMO- EF-RA) immunoassay was developed for the sensitive detection of C-peptide. In the immunoassay, there have been a miscible detection system by mixing with the fluorescent quantum dots conjugated antigen (QD-Ag conjugates) and the dylight dye conjugated antibody (DL-Ab conjugates). When connecting between Ag-QD conjugate and Ab-DL conjugate by specific recognition, the system emitted fluorescence resonant energy transfer (FRET). The target C-peptide can inhibit the connection and FRET formation between QD-Ag conjugates and DL-Ab conjugates, thus changing the dual fluorescence. By measuring the ratio dual fluorescence changes of the system, the content of C-peptide was evaluated without any enzyme used and multiple incubation and washing steps. This immunoassay realized the highly sensitive (as low as 0.12 ng mL−1), selective and rapid (as less as 6 min) detection of C-peptide. Furthermore, the simple and convenient immunoassay was applied successfully to the determination of C-peptide in real serum samples. [Display omitted] •A homogeneous enzyme-free ratiometric immunoassay was developed for the detection of C-peptide.•The immunoassay was composed of a miscible detection system by mixing QD-Ag conjugates and DL-Ab conjugates.•The detection processes had the detection signals self-correction effect based on the dual-fluorescence-output.•With this immunoassay the highly sensitive, selective, rapid and stable detection of C-peptide was achieved.