Self-assembled bifunctional nanoflower-enabled CRISPR/Cas biosensing platform for dual-readout detection of Salmonella enterica

Sensitive detection and point-of-care test of bacterial pathogens is of great significance in safeguarding the public health worldwide. Inspired by the characteristics of horseradish peroxidase (HRP), we synthesized a hybrid nanoflower with peroxidase-like activity via a three-component self-assembled strategy. Interestingly, the prepared nanozyme not only could act as an alternative to HRP for colorimetric biosensing, but also function as a unique signal probe that could be recognized by a pregnancy test strip. By combining the bifunctional properties of hybrid nanoflower, isothermal amplification of LAMP, and the specific recognition and non-specific cleavage properties of CRISPR/Cas12a system, the dual-readout CRISPR/Cas12a biosensor was developed for sensitive and rapid detection of Salmonella enterica . Moreover, this platform in the detection of Salmonella enterica had limits of detection of 1 cfu/mL (colorimetric assay) in the linear range of 10 1 –10 8 cfu/mL and 10 2 cfu/mL (lateral flow assay) in the linear range of 10 2 –10 8 cfu/mL, respectively. Furthermore, the developed biosensor exhibited good recoveries in the spiked samples (lake water and milk) with varying concentrations of Salmonella enterica . This work provides new insights for the design of multifunctional nanozyme and the development of innovative dual-readout CRISPR/Cas system-based biosensing platform for the detection of pathogens. Environmental implication The presence of bacterial pathogens is pervasive in the environment and poses a significant threat to public health safety. To prevent foodborne disease outbreaks, it is crucial to develop sensitive and reliable pathogen detection strategies for rapid and on-site detection of pathogens in both environmental and food samples. Herein, we developed a novel dual-readout CRISPR/Cas biosensing platform based on nanozyme with bifunctional properties and loop-mediated isothermal amplification (LAMP) for highly sensitive and on-site detection of Salmonella enterica in lake water and milk samples. The proposed method has good stability and high accuracy in the detection of Salmonella enterica .