Advanced Electrochemical Biosensing toward <i>Staphylococcus aureus</i> Based on the RPA-CRISPR/Cas12a System and Conductive Nanocomposite

<i>Staphylococcus aureus</i> (<i>S. aureus</i>) is a prevalent foodborne pathogen that poses significant challenges to food safety. Herein, a sensitive and specific electrochemical biosensor based on RPA-CRISPR/Cas12a is developed for evaluating <i>S. aureus</i>. In the presence of <i>S. aureus</i>, the extracted target DNA fragments are efficiently amplified by recombinase polymerase amplification (RPA). The designed crRNA, binding to Cas12a, effectively recognizes the target fragment cleaving hpDNA. The signal molecule of hpDNA is cleaved from the sensing interface, resulting in a reduction of current response. Under optimal experimental conditions, the developed electrochemical biosensor exhibits remarkable sensitivity in detecting <i>S. aureus</i>. The linear range for quantifying <i>S. aureus</i> in pure culture is 1.04 × 10¹-1.04 × 10⁸ CFU/mL, with a detection limit as low as 3 CFU/mL. In addition, the biosensor enables the accurate and sensitive detection of <i>S. aureus</i> in milk within a linear range of 1.07 × 10¹-1.07 × 10⁷ CFU/mL. The electrochemical biosensor enhances anti-interference capability owing to the specific amplification of RPA primers and the single-base recognition ability of crRNA. The RPA-CRISPR/Cas12a biosensor exhibits exceptional anti-interference capability, precision, and sensitivity, thereby establishing a robust foundation for real-time monitoring of microbial contamination.