Fluorescent enzyme-linked immunoassay strategy based on enzyme-triggered in-situ synthesis of fluorescent copper nanoclusters

The routine enzyme-linked immunesorbent assay (ELISA) with colorimetric readout is unsuitable for detecting low concentrations of target molecules. Herein, we proposed one sensitive fluorescence immunoassay strategy using alkaline phosphatase (ALP) as the labeling enzyme and copper nanoclusters (CuNCs) as fluorescent indicator. In this strategy, ALP specifically catalyzes the hydrolysis of ascorbic acid 2-phosphate (AAP) to yield ascorbic acid, and then the resultant ascorbic acid interacts with Cu2+ in the presence of DNA template to in-situ synthesize the fluorescent DNA-templated CuNCs. The corresponding fluorescence signal is related to the amount of ALP conjugated on antibody, and the fluorescence intensity can be used to detect target antigen through an ELISA platform. Human immunoglobulin G (IgG) is taken as the model antigen for the proof-of-concept, and human IgG could be detected in 0.05 - 12 ng/mL range. The detection limit was 7 pg/mL, which is much lower than those in most of the previously reported ELISA. This simple turn-on fluorescent ELISA platform provides a cost-effective approach for sensitive detection of target proteins.