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

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.