Split G-Quadruplex-Integrated CRISPR-Cas Biosensor for One-Pot, Signal-On Visual Detection of Pathogen Nucleic Acids

Pathogen nucleic acid analysis has emerged as an indispensable component of contemporary healthcare systems, serving dual roles in personalized clinical management and population-level disease surveillance. Herein, we present a novel G-quadruplex-integrated CRISPR-Cas biosensing platform that performs in a signal-on mechanism for colorimetric detection of pathogen nucleic acids in one-pot. By harnessing the unique properties of split G4 structures, we develop a universal visual probe that generates a distinct green colorimetric signal upon target recognition, which effectively couples with both Cas12 and Cas13 systems. Using the monkeypox virus (MPXV) B7R gene and a conserved respiratory syncytial virus (RSV) sequence as model targets for Cas12a and Cas13, respectively, we establish a detection workflow combining recombinase polymerase amplification (RPA) with CRISPR-mediated cleavage, visualized through enzymatic mediated color conversion. The tube-in-tube cartridge architecture adopted in this work enables seamless integration of RPA and CRISPR-based detection within a single closed-tube system, effectively eliminating cross-contamination risks. We successfully validate the platform for detection of MPXV in environmental samples and RSV in clinical specimens, achieving a detection limit of 1 copy per test and perfect concordance with PCR methods (40/40 agreement). The colorimetric biosensing platform developed herein demonstrates rapid (<60 min) and facial performance, establishing a novel molecular diagnostic paradigm that achieves laboratory-comparable accuracy for real-time surveillance and point-of-care applications.