Advanced Electrochemical\nBiosensing toward <i>Staphylococcus aureus</i> Based on\nthe RPA-CRISPR/Cas12a System\nand Conductive Nanocomposite

Staphylococcus aureus (S. aureus) is a prevalent foodborne pathogen that\nposes significant challenges to food safety. Herein, a sensitive and\nspecific electrochemical biosensor based on RPA-CRISPR/Cas12a is developed\nfor evaluating S. aureus. In the presence\nof S. aureus, the extracted target\nDNA fragments are efficiently amplified by recombinase polymerase\namplification (RPA). The designed crRNA, binding to Cas12a, effectively\nrecognizes the target fragment cleaving hpDNA. The signal molecule\nof hpDNA is cleaved from the sensing interface, resulting in a reduction\nof current response. Under optimal experimental conditions, the developed\nelectrochemical biosensor exhibits remarkable sensitivity in detecting S. aureus. The linear range for quantifying S. aureus in pure culture is 1.04 × 10<sup>1</sup>–1.04 × 10<sup>8</sup> CFU/mL, with a detection limit\nas low as 3 CFU/mL. In addition, the biosensor enables the accurate\nand sensitive detection of S. aureus in milk within a linear range of 1.07 × 10<sup>1</sup>–1.07\n× 10<sup>7</sup> CFU/mL. The electrochemical biosensor enhances\nanti-interference capability owing to the specific amplification of\nRPA primers and the single-base recognition ability of crRNA. The\nRPA-CRISPR/Cas12a biosensor exhibits exceptional anti-interference\ncapability, precision, and sensitivity, thereby establishing a robust\nfoundation for real-time monitoring of microbial contamination.