Bifunctional three-way DNA junction-based strand displacement recycling for amplifiable electrochemical bivariate biosensing

It’s intriguing to utilize the branched arms of three-way DNA junction (3WDJ) for modifying specific recognizing and/or sensing elements of multivariate analytes. Herein, by using two targeting DNA segments (T and T*) specific to SARS-CoV-2 as analyte models, an electrochemical bivariate biosensor was created based on a functional 3WDJ including two-NH2-labeled recognizable probes (RP and RP*) and an assistant probe (AP), while its two branched arms hybridized with four helping DNA blockers. In the electrode surface electrodeposited in HAuCl4, the 3WDJ was stably immobilized via Au-N bonds to specifically recognize and bind T and T*, with which two modified signaling probes by electroactive methylene blue (SP-MB) and ferrocene (SP*-Fc) were introduced to initiate two strand displacement reactions. Resultantly, SP-MB and SP*-Fc were guided to be complementarily hybridized in two arms of 3WDJ, replacing T and T * to execute two individual repeatable recycling for signal amplification. Thus, MB and Fc were oriented proximal to the modified electrode surface for significantly increased electrochemical current signals, respectively dependent on T and T*. With the branched arms of rapidly assembled 3WDJ, the discernible detection of bivariate targets was achievable, showing superb simplification, high sensitivity, and potentially more accurate electrochemical assay of multivariate targets. •A unique 3WDJ is assembled to guide the creation of an electrochemical bivariate biosensor.•The branched arms of 3WDJ are conjugated with specific recognizing elements of bivariate analytes.•Two targets initiate two strand displacement reactions for recycling signal amplification.•Two electroactive electron tags proximal to electrode surface produce discernible current signals.•The electrochemical biosensor was highly sensitive to bivariate targets down to femtomole.