Exploiting the size exclusion effect of protein adsorption layers for electrochemical detection of microRNA: A new mechanism for design of E-DNA sensor

The assay performance of electrochemical DNA (E-DNA) sensors is deeply influenced by the state of DNA probes immobilized on electrode. Moreover, the immobilization procedures for DNA probes are tedious and vary according to the probes and analytes. In this work, we find that the adsorption layers of bovine serum albumin (BSA) on gold electrode (AuE) possess a size exclusion effect to distinguish between single-stranded (-ss) DNA probes and the DNA fragments generated from enzymatic digestion of ssDNA probes. In detail, the BSA layers act as a gatekeeper that hinders the adsorption of a ssDNA probe on AuE but permits the DNA fragments with much smaller sizes to pass through the adsorption layers and adsorb on AuE. This finding is developed into a novel E-DNA sensor for microRNA (miRNA) detection by coupling with duplex-specific nuclease (DSN)-assisted target recycling strategy. The ssDNA probe in solution phase is enzymatically digested during the DSN-assisted target recycling process initiated by target miRNA-21, generating plenty of DNA fragments. The adsorption of these DNA fragment on BSA/AuE is permitted, which arouses electrochemical signals after binding with [Ru(NH₃)₆]³⁺ to indicate the recognition of miRNA-21. The developed E-DNA sensor possesses a wide calibration range from 0.001 to 100 pM and a low detection limit of 0.48 fM. Significantly, accurate evaluation of miRNA-21 expression levels in cancer cell lines and non-small-cell lung carcinomas (NSCLC) serum samples are successfully achieved using the developed method. This work provides a new mechanism for constructing sensitive E-DNA sensor without tedious probe immobilization procedures.