Enhanced CRISPR/Cas12a\nFluorimetry via a DNAzyme-Embedded\nFramework Nucleic Acid Substrate

CRISPR/Cas12a fluorimetry has been extensively developed\nin the\nbiosensing arena, on account of its high selectivity, simplicity,\nand rapidness. However, typical CRISPR/Cas12a fluorimetry suffers\nfrom low sensitivity due to the limited trans-cleavage efficiency\nof Cas12a, necessitating the integration of other preamplification\ntechniques. Herein, we develop an enhanced CRISPR/Cas12a fluorimetry\nvia a DNAzyme-embedded framework nucleic acid (FNAzyme) substrate,\nwhich was designed by embedding four CLICK-17 DNAzymes into a rigid\ntetrahedral scaffold. FNAzyme can not only enhance the trans-cleavage\nefficiency of CRISPR/Cas12a by facilitating the exposure of trans-substrate\nto Cas12a but also result in an exceptionally high signal-to-noise\nratio by mediating enzymatic click reaction. Combined with a functional\nnucleic acid recognition module, this method can profile methicillin-resistant Staphylococcus aureus as low as 18 CFU/mL, whose\nsensitivity is approximately 54-fold higher than that of TaqMan probe-mediated\nCRISPR/Cas12a fluorimetry. Meanwhile, the method exhibited satisfactory\nrecoveries in food matrices ranging from 80% to 101%. The DNA extraction-\nand preamplification-free detection format as well as the potent detection\nperformance highlight its tremendous potential as a next-generation\nanalysis tool.