A Near-Infrared Light-Driven Photoelectrochemical Biosensor Based on CRISPR/Cas12a for Highly Sensitive Detection of Pseudomonas fluorescens in Dairy Products

Pseudomonas fluorescens ( P. fluorescens ) is a psychrotrophic bacterium causing dairy spoilage. Plate count and PCR methods, as conventional detection standards, are either time-consuming or dependent on expensive instruments, restricting widespread application. Herein, a near-infrared (NIR)-driven photoelectrochemical (PEC) biosensor based on CRISPR/Cas12a was constructed by using SH-ssDNA/AuNPs/AgBiS 2 as the working electrode. AgBiS 2 possesses broad-spectrum absorption and an appropriate bandgap for efficient NIR utilization. In the presence of P. fluorescens, the CRISPR/Cas12a system was activated by the LAMP amplification products of the target DNA to perform the trans-cleavage activity toward the SH-ssDNA modified on the AuNPs/AgBiS 2 electrode surface. This cleavage released SH-ssDNA from the electrode, reducing steric hindrance and accelerating interfacial electron transfer, thereby enhancing the PEC signal. The constructed biosensor achieved sensitive detection of P. fluorescens with a limit of detection (LOD) of 1 CFU/mL in the range of 10 1 –10 8 CFU/mL, offering a reliable strategy for detecting the psychrotrophic bacteria in dairy products.