A low-background and wash-free signal amplification F-CRISPR biosensor for sensitive quantitative and visible qualitative detection of Salmonella Typhimurium

Abstract

In traditional CRISPR-based biosensors, the cleavage-induced signal generation is insufficient because only a signals is generated at a CRISPR-induced cleavage. Herein, we developed an improved CRISPR/Cas12a-based biosensor with an enlarged signal generation which integrated the hybridization chain reaction (HCR) and low-background Förster Resonance Energy Transfer (FRET) signal output mode. The HCR with nucleic acid self-assembly capability was used as a signal carrier to load more signaling molecules. To get the best signal amplification, three different fluorescence signal output modes (fluorescence recovery, FRET and low-background FRET) generated by two fluoresceins, FAM and Cy5, were fully investigated and compared. The results indicated that the low-background FRET signal output mode with the strictest signal generation conditions yielded the highest signal-to-noise ratio (S/N) (19.17) and the most obvious fluorescence color change (from red to yellow). In optimal conditions, the proposed biosensor was successfully applied for Salmonella Typhimurium (S. Typhimurium) detection with 6 h (including 4 h for sample pre-treatment) from the initial target processing to the final detection result. The qualitative sensitivity, reliant on color changes, was 103 CFU/mL. The quantitative sensitivity, calculated by the fluorescence value, were 1.62 × 101 CFU/mL, 3.72 × 102 CFU/mL, and 8.71 × 102 CFU/mL in buffer solution, S. Typhimurium-spiked milk samples, and S.Typhimurium-spiked chicken samples, respectively. The excellent detection performance of the proposed biosensor endowed its great application potential in food and environment safety monitoring.