Label-free split G-quadruplex-based CRISPR/Cas12a biosensor for fluorescent detection of Listeria monocytogenes

Rapid and sensitive nucleic acid detection is of immense value and broad application prospects in fields such as food safety monitoring and disease diagnosis. Conventional fluorescently labeled probes, while widely used in fluorescent biosensors for nucleic acid detection, suffer from inherent limitations including high background signal, photobleaching, and cost inefficiency. Meanwhile, G-quadruplex (G4) structures present inherent issues of insufficient design flexibility and weak output fluorescent signal strength. Herein, we innovatively developed a fluorescent biosensor integrating split G4 with the recombinase polymerase amplification (RPA)-CRISPR system for the label-free, rapid, and highly sensitive detection of Listeria monocytogenes . Through RPA of target DNA, exponential amplification of the target DNA sequence was achieved, which subsequently activated the trans -cleavage activity of Cas12a. This non-specifically cleaved the linker strand, inhibiting the assembly of split G4 and resulting in proportional fluorescence attenuation corresponding to the input DNA target. Under the optimized conditions, the detection sensitivity of this method for L. monocytogenes reached 1.9 × 10 1  CFU/mL, which is two orders of magnitude higher than that of the conventional fluorescent probe methods. Furthermore, this method exhibited excellent specificity in distinguishing L. monocytogenes from other common pathogens, with successful application in natural sample analyses. Therefore, we have established a split G4-based RPA-CRISPR/Cas12a method, which provides a label-free, highly sensitive and specific analytical tool for the detection of foodborne pathogens, offering significant reference value for practical applications.