High-Performance Ratiometric Electrochemical Method Based on the Combination of Signal Probe and Inner Reference Probe in One Hairpin-Structured DNA.

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 ((IFc-sP/IMB-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.

Integrated signal probe based aptasensor for dual-analyte detection.

For the multi-analyte detection, although the sensitivity has commonly met the practical requirements, the reliability, reproducibility and stability need to be further improved. In this work, two different aptamer probes labeled with redox tags were used as signal probe1 (sP1) and signal probe2 (sP2), which were integrated into one unity DNA architecture to develop the integrated signal probe (ISP). Comparing with the conventional independent signal probes for the simultaneous multi-analyte detection, the proposed ISP was more reproducible and accurate. This can be due to that ISP in one DNA structure can ensure the completely same modification condition and an equal stoichiometric ratio between sP1 and sP2, and furthermore the cross interference between sP1 and sP2 can be successfully prevented by regulating the complementary position of sP1 and sP2. The ISP-based assay system would be a great progress for the dual-analyte detection. Combining with gold nanoparticles (AuNPs) signal amplification, the ISP/AuNPs-based aptasensor for the sensitive dual-analyte detection was explored. Based on DNA structural switching induced by targets binding to aptamer, the simultaneous dual-analyte detection was simply achieved by monitoring the electrochemical responses of methylene blue (MB) and ferrocene (Fc) This proposed detection system possesses such advantages as simplicity in design, easy operation, good reproducibility and accuracy, high sensitivity and selectivity, which indicates the excellent application of this aptasensor in the field of clinical diagnosis or other molecular sensors.