A triple-amplified electrochemical-photoelectrochemical dual-mode biosensing platform for viral gene assay based on catalytic hairpin assembly, CRISPR-Cas12a and liposome-entrapped bifunctional methylene blue

Taking SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene as the model due to the great destructiveness of COVID-19 in the past years, a triple-amplified electrochemical-photoelectrochemical (EC-PEC) dual-mode biosensing platform was constructed for viral gene assay based on catalytic hairpin assembly (CHA), CRISPR-Cas12a and liposome-entrapped bifunctional methylene blue. Firstly, the SARS-CoV-2 RdRp gene triggered CHA nonenzymatic amplification process to produce large amounts of double-stranded DNA (dsDNA) that could activate CRISPR-Cas12a trans-cleavage ability. Subsequently, the activated CRISPR-Cas12a trans-cleaved nearby ssDNA carrying methylene blue (MB)-encapsulated liposome sphere (MELS) and streptavidin-modified magnetic bead (SMB), and lots of MELS were released. After magnetic separation, MELS was lysed by Triton X-100, releasing a large quantity of bifunctional MB molecules that not only generate an obvious EC signal originated from the electro-oxidation of MB, but also effectively induce the photocurrent-polarity-switching of the Sb2S3 NRs-modified photoelectrode from cathodic photocurrent to anodic photocurrent and generate a large anodic photocurrent. Based on the combination of the above triple amplification processes with MB-induced photocurrent-polarity-switching and EC-PEC dual-mode sensing strategies, the SARS CoV-2 RdRp gene was sensitively, selectively and accurately assayed with a linear response range of 0.1 fM – 10 nM (1 aM – 10 nM) and a detection limit of 69.6 aM (0.11 aM) for EC (PEC) method. The proposed EC-PEC dual-mode biosensor is also easily extended to the analysis of other viral nucleic acid by changing the related sequences of H1 and H2 DNAs, providing a new way in the early diagnosis and epidemic control of diseases caused by viruses.