High-Sensitivity Enzyme-Free Fluorescence Probe Based\non CRISPR/Cas13 and the Isothermal Amplification Strategy for Axl\nSensing

Axl\nis an important receptor tyrosine protein kinase that plays\na key role in the development and progression of various diseases,\nsuch as cancer and inflammation. Developing a highly sensitive Axl\ndetection method can help improve accuracy, better address-specific\nclinical needs, and guide personalized treatment. In this study, a\nCHA-CRISPR/Cas13 fluorescence probe was established using Axl-specific\naptamers as a mediator to displace the polynucleotide chain (TA).\nThrough TA construction, an entropy-driven nucleotide catalytic hairpin\nassembly system was created to cyclically release RNA that activates\nclustered regularly interspaced short palindromic repeats (CRISPR)/Cas13\nactivity, triggering its cleavage activity. The activated CRISPR/Cas13\nsystem cleaves the reporter labeled with BHQ1 and FAM at both ends,\nleading to the recovery of FAM fluorescence. Based on the optimization\ndesign using the free energy (△<i>G</i>) and secondary\nstructure software simulation results of the nucleic acid sequence,\nthe fluorescence intensity of the probe is proportional to the concentration\nof Axl. Results showed a good linear relationship between fluorescence\nintensity increment and log <i>C</i><sub>Axl</sub> (<i>C</i><sub>Axl</sub> in the range of 3.33–667 pM, <i>r</i> = 0.9907). The probe exhibited ultrahigh sensitivity with\na detection limit of 0.84 pM. It was successfully applied in the detection\nof human serum samples, showing a higher Axl level in cervical cancer\npatients compared to breast cancer patients. The probe was also successfully\napplied in the imaging of various tumor cells, consistent with serum\ndetection results. In conclusion, this probe represents an effective\nnew method for detecting Axl, demonstrating outstanding specificity\nand sensitivity. It provides technological support for tumor diagnosis\nand shows the potential for detecting circulating tumor cells in blood\nthrough cell imaging.