Allosteric Switching-Triggered Electrocatalyst Recruitment Enables Indicator-Free, Milliampere-Level Hydrogen Evolution Readout for MicroRNA-21 Detection

Abstract Electrochemical nucleic acid biosensors typically rely on redox labels or exogenous mediators to generate analytical signals, which can complicate assay design and constrain signal magnitude. Herein, we report an enzyme-free and indicator-free electrochemical biosensor for microRNA-21 based on allosteric switching-triggered electrocatalyst recruitment and hydrogen evolution reaction (HER) readout. The sensing interface comprises a hairpin probe DNA that undergoes target-induced allosteric switching upon hybridization with microRNA-21, thereby exposing a streptavidin-binding domain for recruitment of the HER electrocatalyst FLN-Pt@SA. This target-triggered interfacial assembly converts molecular recognition into a catalyst-amplified HER response, using H2O as an abundant electrochemical substrate rather than conventional redox indicators or mediators. The resulting biosensor generates a stable milliampere-level signal and enables quantitative detection of microRNA-21 over a wide concentration range from 100 aM to 100 nM, with a detection limit of 34 aM. The biosensor also exhibits good selectivity against non-target and mismatched miRNA sequences, as well as satisfactory reproducibility, stability, and recovery in serum samples. This work establishes an enzyme-free, indicator-free HER-based signal-transduction strategy that couples nucleic acid recognition with electrocatalytic amplification for electrochemical biosensing.