Multiple self-cleaning paper-based electrochemical ratiometric biosensor based on the inner reference probe and exonuclease III-assisted signal amplification strategy

Rapid and accurate detection of nucleic acids plays a major role in biological research and clinical diagnostics. Here, a 3D multiple self-cleaning electrochemical ratiometric microfluidic paper-based analytical device (SER-mu PAD) has been constructed for manganese super oxide dismutase (MnSOD) gene detection on the basis of the inner reference probe and exonuclease III (Exo LLD-assisted analytes recycling amplification method. To simplify manual operations, a multipath self-cleaning tab that could manipulate fluid transport was introduced into the paper-based device, realizing time-programmable self-cleaning of the electrode. For achieving sensitive detection of MnSOD gene, the methylene blue (MB)-modified capture probe (CP) as the inner reference element was first self-assembled on triangular Au nanosheets modified paper working electrode to provide a built-in correction and improve the detection accuracy. When MnSOD gene existed, it hybridized with the hairpin-structured signal probe, triggering the cyclic amplification with the assistance of Exo III selective digestion to engender numerous residual DNA labeled with ferrocene (Fc) that could be captured on electrode surface by CPs. Hence, the Fc tags were close to the electrode surface, resulting in the oxidation peak current of Fc (I-FC) increase, while that of MB (I-MB) was constant on account of the unchanged distance between the MB tags and the electrode. The value of I-FC/I-MB was linear with MnSOD gene concentration from 10 nM to 1200 nM, and the detection limit was 3.91 nM. This strategy provides an accurate, robust, and sensitive method for nucleic acids detection and shows great potential in the construction of portable devices.