Enzyme-assisted catalytic hairpin assembly based aggregation-induced emission assay for trace miRNA-196a in serum

MicroRNA-196a (miRNA-196a) is a clinically significant biomarker for the early diagnosis of pancreatic cancer. However, its trace level in serum necessitates highly sensitive and robust detection methods. Combining the merits of the enzyme-free CHA DNA circuits and the nucleotide enzymes, an enzyme-assisted CHA (eaCHA) strategy is proposed and verified in an AIE biosensing platform targeting miRNA-196a to further boost amplification of CHA. The target (miRNA-196a) initiated an Nb·BbvCI assisted CHA, the products of which drove the assembly of two TPE-labeled probes (Probe 1 and Probe 2), resulting in a significant "turn on" of AIE signals. To further enhance sensitivity, exonuclease III was employed to assist the TS1 recycling from blunt-ended duplexes, enabling cascaded signal amplification. The biosensor exhibited a wide linear range from 1 pM to 50 nM with a detection limit of 120 fM. Generally, the assay demonstrated high selectivity, excellent reproducibility (RSD < 3%), and excellent performance in clinical sample assay, indicating its potential capability for early pancreatic cancer screening.