Dual-labeling ratiometric electrochemical strategy initiated with ISDPR for accurate screening MecA gene

An outstanding dual-labeling ratiometric electrochemical biosensor based on isothermal strand displacement polymerization reaction (ISDPR) for highly sensitive and selective detection of mecA gene has been proposed. Concretely, in the presence of mecA gene, the addition of methylene blue (MB)-labeled primer and polymerase induced recycling amplification to change the structure of the ferrocene (Fc)-labeled hairpin probe, thereby releasing abundant target gene to realize the signal amplification and dual-signal output. Through this process, the electrochemical responses of Fc (I_Fc) and MB (I_MB) were both substantially reduced and increased proportionally, ensuring that the value of I_MB/I_Fc can accurately reflect the true detection level of mecA gene. Benefiting from the "signal-on/off" strategy, the fabricated biosensor exhibited outstanding sequence specificity to discriminate mismatched mecA gene, which verified to be 2.72 times that of single-label detection for perfect match/single base mismatch (PM/MM) discrimination ratio. This strategy effectively integrated the advantages of signal amplification and ratiometric modes, making the biosensor exhibit a broad working range with 10 fM - 3000 pM and a limit of detection (LOD) with 3.33 fM (S/N = 3). Moreover, the proposed biosensor has good feasibility for mecA gene determination in water samples due to acceptable recoveries (95-115%) and repeatability relative standard deviations (RSD) value of 4%. This will provide a powerful sensing platform for improving accuracy and decreasing background signal of sensor for ARGs screening in environmental monitoring.