Solution-phase nucleic acid reaction weaves interfacial barriers on unmodified electrodes: Just-in-time generation of sensor interface for convenient and highly sensitive bioassays

Electrochemical bioassays that rely on sensor interfaces based on immobilized DNA probes often encounter challenges such as complex fabrication processes and limited binding efficiency. In this study, we developed a novel electrochemical bioassay that bypasses the need for probe immobilization by employing a solution-phase nucleic acid reaction to create interfacial barriers on unmodified electrodes, enabling rapid, just-in-time sensor interface formation. Specifically, a 3 '-phosphorylated recognition probe was used to identify the target microRNA-21 (miR-21), followed by target recycling facilitated by duplex-specific nuclease (DSN), which resulted in extensive hydrolysis of the recognition probe into DNA fragments with 3 '-hydroxyl ends. These fragments were then extended by terminal deoxynucleotidyl transferase (TdT) to form long poly(A) tails. The extended products hybridized with a thiolated assembly probe rich in thymine bases and subsequently assembled on the unmodified gold electrode (AuE) surface, creating a "barrier effect" that hindered the adsorption of streptavidin-HRP (SA-HRP) on the AuE, generating a detectable electrochemical signal. This method demonstrated excellent analytical performance, with a linear detection range from 10 fM to 10 nM and a detection limit as low as 4.3 fM. Moreover, the assay was successfully applied to detect miR-21 in real biological samples, including cell lines and bladder urothelial carcinoma surgical resection specimens, showing strong concordance with RT-qPCR results. The developed method offers a new approach for establishing electrochemical bioassays without the need for pre-immobilization of probes and with minimal reagent use, presenting a promising tool for clinical diagnostics and cancer research.