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.