Dual-Mode RPA/CRISPR-Cas12a Biosensor Based on Silica and Magnetic Hybrid Nanobeads for Rapid Detection of Campylobacter jejuni.

Abstract

In this study, we developed a biosensor that makes use of recombinase polymerase amplification (RPA) along with a CRISPR/Cas12a system integrated with silica nanobeads and a magnetic nanoparticle nanohybrid complex that displayed peroxidase-mimicking properties. This nanohybrid nanozyme (NZ) integration with the CRISPR/Cas system allowed dual-mode fluorometric and colorimetric responses . The nanohybrid NZ was a conjugated ssDNA quencher probe sequence with inherent fluorometric properties. In the presence of target RPA amplicons, the CRISPR/Cas12a system gets activated, cleaving the probe sequence attached to the NZ complex and leading to fluorescence signal generation. Post-CRISPR/Cas12a assay, the presence of the NZ in the reaction mixture, after being cleaved away from the probe sequence, gave a colourimetric response directly proportional to the target DNA concentration, as the ssDNA probe sequence no longer hindered its catalytic activity. Therefore, the dual-mode detection using the CRISPR/Cas12a-based fluorometric response and NZ-based colorimetric detection conferred high sensitivity and selectivity toward Campylobacter detection. The developed sensor could detect the pathogenic DNA at concentrations as low as 0.98 pg/μL and 0.96 pg/μL via fluorescence and absorbance spectroscopy, respectively. In addition, our method was also tested in raw food analysis and showed good recovery.