Target-driven triple-binder assembly of MNAzyme for amplified electrochemical immunosensing of protein biomarker.

A simple electrochemical immunosensing method is presented for highly sensitive and selective detection of protein biomarker. This method uses a newly designed assembly of Mg(2+)-dependent MNAzyme via target-driven triple-binder proximity hybridization to catalyze the cleavage of methylene blue (MB)-labeled hairpin, which leads to the departure of MB from the electrode surface and thus an amplified decrease of electrochemical signal for immunoassay of the target protein. The MNAzyme assembly is achieved by the simultaneous recognition of target protein with three DNA-labeled antibodies in the presence of Mg(2+), which greatly improves the detection sensitivity and selectivity. As a proof of concept, this strategy can detect carcinoembryonic antigen (CEA) ranging from 0.002 to 500 ng mL(-1) with a detection limit of 1.5 pg mL(-1). The whole assay including the target-driven MNAzyme formation and subsequent cleavage of hairpin can be completed with one step in 40 min. The immunosensor, prepared with a hairpin DNA, possesses good extensibility for large protein biomarkers as CEA by using corresponding antibodies, though the protein target size dependence was not investigated in this work. The proposed immunoassay method shows the advantages of easy operation, high sensitivity, wide concentration range, good selectivity, and excellent versatility, displaying potential application for protein analysis.