High-Performance Ratiometric Electrochemical Method Based on the Combination of Signal Probe and Inner Reference...

In this work, the dual signal-tagged hairpin structured DNA (dhDNA)-based ratiometric probe was developed by the combination of ferrocene-labeled signal probe (Fc-sP) and methylene blue-modified inner reference probe (MB-rP) in one hairpin-structured DNA. On the basis of this, a high-performance ratiometric electrochemical method was proposed for biomarker detection. In contrast to the conventional ratiometric electrochemical probe, this dhDNA ratiometric probe integrated sP and rP into one structure, which ensured the completely same modification condition and the interdependence of sP and rP on one sensing interface. As a result, the dhDNA ratiometric probe possesses a stronger ability to eliminate the disturbance of environmental change, which was proven by the fact that the changes of the surface roughness and pH value had no significant effects on the reproducibility and stability of the sensor. Moreover, in the proposed strategy, the initial ratio responses of Fc-sP to MB-rP ((I_Fc-sP/I_MB-rP)⁰) are controllable and can be kept constant at 1:1, which is favorable for the increase in signal-to-noise ratio and sensitivity. When the sequence of Fc-sP is designed as the aptamer of mucin 1 (MUC1), the dhDNA ratiometric sensor with signal amplification of Au nanoparticles becomes feasible for the sensitive detection of MUC1 by one-step incubation procedure. Compared with the conventional ratiometric sensor, the proposed dhDNA sensor has higher reproducibility, accuracy, stability, sensitivity, and simplicity, which are significant for the development of the sensor in various fields for practical applications.