Engineering a CRISPR-Mediated Dual Signal Amplification-Based Biosensor for miRNA Determination

MicroRNAs, pivotal regulators of gene expression and physiology, serve as reliable biomarkers for early cancer diagnosis and therapy. As one of the earliest discovered miRNAs in the human genome, miRNA-21 provides critical information for early cancer diagnosis, drug therapy, and prognosis. In this work, we harness CRISPR as a bridge to integrate target-induced self-priming hairpin isothermal amplification (SIAM) with terminal transferase (TdT) polymerization labeling, constructing a facile, straightforward electrochemical biosensor for sensitive miRNA-21 detection. Unlike conventional single-strand template-based exponential amplification (EXPAR), the SIAM hairpin undergoes target triggered intramolecular conformational change, initiating extension and strand displacement reactions that suppress nonspecific dimer formation and lower background current. Notably, the assay requires only a single probe, enabling unidirectional signal amplification while nonspecific reactions caused by system complexity. The generated SIAM products activate the Cas12a/crRNA complex to trans-cleave PO₄^3- modified single-stranded DNAs (ssDNAs); the resulting 3' hydroxyl ssDNAs are subsequently labeled by TdT, with the assistance of SA-HRP catalyzing hydrogen peroxide, achieving robust signal amplification. Under optimized conditions, the cathodic current exhibits a logarithmic relationship with miRNA concentrations from 20 fM to 5.0 × 10⁸ fM, with a detection limit of 9.2 fM. The biosensor successfully quantified miRNA-21 in commercial serum samples and biological lysates, demonstrating its potential for cancer diagnostics and therapy.