High-Sensitivity Enzyme-Free Fluorescence Probe Based on CRISPR/Cas13 and the Isothermal Amplification Strategy for...

Axl is an important receptor tyrosine protein kinase that plays a key role in the development and progression of various diseases, such as cancer and inflammation. Developing a highly sensitive Axl detection method can help improve accuracy, better address-specific clinical needs, and guide personalized treatment. In this study, a CHA-CRISPR/Cas13 fluorescence probe was established using Axl-specific aptamers as a mediator to displace the polynucleotide chain (TA). Through TA construction, an entropy-driven nucleotide catalytic hairpin assembly system was created to cyclically release RNA that activates clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13 activity, triggering its cleavage activity. The activated CRISPR/Cas13 system cleaves the reporter labeled with BHQ1 and FAM at both ends, leading to the recovery of FAM fluorescence. Based on the optimization design using the free energy (△G) and secondary structure software simulation results of the nucleic acid sequence, the fluorescence intensity of the probe is proportional to the concentration of Axl. Results showed a good linear relationship between fluorescence intensity increment and log C_Axl (C_Axl in the range of 3.33-667 pM, r = 0.9907). The probe exhibited ultrahigh sensitivity with a detection limit of 0.84 pM. It was successfully applied in the detection of human serum samples, showing a higher Axl level in cervical cancer patients compared to breast cancer patients. The probe was also successfully applied in the imaging of various tumor cells, consistent with serum detection results. In conclusion, this probe represents an effective new method for detecting Axl, demonstrating outstanding specificity and sensitivity. It provides technological support for tumor diagnosis and shows the potential for detecting circulating tumor cells in blood through cell imaging.