CRISPR/Cas12a-powered evanescent wave fluorescence nanobiosensing platform for nucleic acid amplification-free detection of Staphylococcus aureus with multiple signal enhancements

Although CRISPR-based biosensors for pathogenic detection are highly specific, they not sensitive enough and nucleic acid amplification is generally required to improve their sensitivity. However, this allows only binary operations and significantly limits practical applications. Here, a CRISPR/Cas12a-powered Evanescent wAve fluorescence nanobiosensing plaTform (CREAT) was developed for ultrasensitive nucleic acid amplification-free quantitative detection of pathogens with multiple signal enhancements. In addition to collateral cleavage amplification of the CRISPR/Cas12a system, we constructed nanophotonic structure-based evanescent wave fluorescence enhancement, Mg²⁺ or DNA-mediated fluorescence enhancement, and air-displacement fluorescence enhancement strategies for ultrasensitive detection of Staphylococcus aureus (S. aureus). Especially, the fluorescence signal detected by CREAT can be significantly enhanced by adding a simple air displacement step, thus improving detection sensitivity. This nanobiosensor detected real samples containing S. aureus, with a detection limit of 592 CFU/mL and 13.2 CFU/mL in 45 min and 90 min, respectively, which are comparable to those of RT-qPCR. This paves a new way for simple, rapid, sensitive, robust, and flexible on-site detection of S. aureus as well as other pathogens.