Dual-mode sensing of Mycobacterium tuberculosis with DNA-functionalized gold nanoparticles and asymmetric RPA-triggered PAM-free CRISPR system

Mycobacterium tuberculosis (MTB) causes the ancient infectious disease of tuberculosis, and its rapid, specific, and sensitive detection has long been a challenging endeavor. Herein, we propose integrating DNA-functionalized gold nanoparticles and asymmetric RPA-triggered PAM-free CRISPR for sensing of MTB. Upon activating the CRISPR/Cas12a complex by specific single-stranded products of asymmetric RPA and subsequent DNA degradation between AuNP and fluorophore, metal-enhanced fluorescence occurred with visible color changes. Therefore, this method has a dual signal output mode for point-of-care pathogen detection. Significantly, the entire detection process occurs at a constant temperature within a single tube, thereby minimizing the risk of aerosol contamination. In the experiment, the detection limit for MTB is remarkably low at 1 copy μL−1, and importantly, this method exhibits no cross-reactivity with other common respiratory pathogens. Notably, we observed a 100 % concordance rate with clinical qPCR results in 32 sputum samples, underscoring the reliability and accuracy of this method. This biosensing platform not only effectively overcomes the limitations posed by PAM requirements but also holds great promise for point-of-care applications. •The asymmetric RPA-assisted CRISPR system is PAM-free, eliminating the limitations of PAM sites.•Functionalized gold nanoparticles enables both fluorescence enhancement and visual sensing.•Dual-mode sensing of Mycobacterium tuberculosis holds promise for POC applications, thus increasing its practical relevance.