Synergistic cascade amplification by catalytic hairpin assembly and terminal deoxynucleotidyl transferase for ultrasensitive detection of microRNA-21.

The accurate early detection of miRNA-21 is crucial for effective cancer diagnosis and prognosis assessment. However, traditional amplification strategies are limited by spatial obstacles and have insufficient signal transmission efficiency at the electrode interface. In this study, we designed a highly sensitive electrochemical biosensor, using ordered mesoporous carbon (OMC-COOH) as the electrode substrate material, combined with the catalytic hairpin assembly (CHA) reaction and the cascade amplification effect of terminal deoxynucleotidyl transferase (TdT). Through the catalytic oxidation of acetaminophen (AP) by gold nanorods (Au NRs), the presence of miRNA-21 was converted into a measurable electrochemical signal, enabling quantitative detection. The OMC-COOH possesses abundant carboxyl functional groups and a layered porous structure, which enables the high-density immobilization of DNA probes. The conformational switch triggered by miRNA-21 initiated a tandem enzyme reaction, enabling TdT to extend a poly-T chain at the 3' end of the CHA product. Subsequently, the P chain connected to Au NRs was introduced onto the electrode substrate, thereby achieving significant electrochemical signal amplification. This electrochemical biosensor achieved a wide linear range from 0.1 fM to 10 nM, an extremely low detection limit (0.28 fM), and satisfactory spiked recovery rates (99.59%-104.81%), and was verified through consistency with qRT-PCR results in clinical samples, confirming the accuracy of this method in complex biological matrices. These characteristics highlight the great potential of this electrochemical biosensor as a reliable and robust tool for quantitative determination of miRNA-21.