Ultrasensitive “On–Off” Ratiometric Fluorescence Biosensor Based on RPA-CRISPR/Cas12a for Detection of Staphylococcus aureus

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Abstract
Staphylococcus aureus (S. aureus) is a major pathogenic bacterium responsible for bacterial foodborne diseases, making its rapid, specific, and accurate detection crucial. In this study, we develop a ratiometric biosensor based on the recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats/CRISPR associated protein 12a (RPA-CRISPR/Cas12a) system and Eu-metal-organic framework (Eu-MOF) fluorescent nanomaterials for the high-sensitivity detection of S. aureus, combining with RPA for efficient isothermal amplification, this sensor enhances specificity and sensitivity by utilizing the target activation of CRISPR/Cas12a. The Eu-MOF serves a dual function, providing stable red fluorescence as a reference signal and adsorbing FAM-labeled probes for fluorescence quenching, forming a dual-signal system that significantly reduces background interference. This ratiometric design enables accurate and quantitative detection over a wide range (7.9 × 100 to 7.9 × 108 CFU/mL) with a low detection limit of 3 CFU/mL. Overall, with these merits of simplicity, rapid response, high sensitivity, and specificity, this dual-signal biosensor offers a promising method for accurately evaluating S. aureus contamination in food under complex substrate conditions.
Funding Information
  • Natural Science Foundation of Jiangsu Province (BK20241924)
  • Key Research and Development Program of Zhenjiang City (NY2023002, SH2024010)
  • Open Project Program of State Key Laboratory of Dairy Biotechnology (SKLDB2024-009)
  • Agricultural science and technology independent innovation project of Jiangsu Province (CX(23)3041)

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