Unlocking Ultrasensitive MicroRNA-21 Signals with a Dual-Output Strand Displacement Biosensor

The efficient and quantitative detection of miRNA-21 plays a critical role in early cancer diagnosis, as well as in the screening and treatment of early-stage infectious diseases. In this study, we developed an electrochemical biosensor for the detection of miRNA-21 in human serum samples. The sensor is constructed based on carboxyl-modified cobalt-doped nitrogen-doped carbon (Co-NC-COOH), a dual-output toehold-mediated strand displacement (TMSD) reaction, and gold nanoparticles (Au NPs) to achieve electrocatalytic signal amplification toward acetaminophen (AP). Co-NC-COOH possesses abundant active and catalytic sites, along with a porous surface morphology, which facilitates electron transfer and enhances the loading capacity of the capture probe (CP). In the presence of miRNA-21, the dual-output TMSD reaction is initiated, resulting in the release of a large amount of product strand (P). Subsequently, P hybridizes with the CP immobilized on the electrode surface, enabling the attachment of Au NPs to the electrode and thereby achieving significant electrochemical signal amplification. The results demonstrate that the proposed biosensor exhibits excellent analytical performance, including a wide linear detection range (0.1 fM-50 nM), an ultralow detection limit (63 aM), and satisfactory spike recovery rates (95.18%-103.59%). Moreover, the biosensor shows good stability and high specificity, providing a reliable platform for the precise quantification of miRNA-21 in human serum.