A naked-eye biosensing system based on one-pot RPA-CRISPR/Cas12a driver G4-hemin self-assembly for Mycobacterium tuberculosis

Under optimized conditions, the biosensing system achieved ultrasensitive naked-eye detection of MTB with a limit of detection (LOD) of 10 copies/μL, comparable to traditional four-step fluorescent assays. Clinical validation using 104 patient samples demonstrated high concordance with standard diagnostic methods. This approach combines the advantages of recombinase polymerase amplification (RPA), CRISPR/Cas12a specificity, and G4-hemin DNAzyme-based colorimetric signal amplification, enabling simple, equipment-free visual detection. Given its speed, sensitivity, and ease of use, this biosensing system holds significant promise for point-of-care MTB nucleic acid testing in resource-limited settings.