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

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Axl receptor tyrosine kinase is implicated in cancer and inflammation.
• Sensitive Axl detection is vital for accurate diagnosis and personalized treatment.
• Current detection methods may lack sufficient sensitivity for clinical applications.

Purpose of the Study:

• To develop a highly sensitive and specific fluorescence probe for Axl detection.
• To utilize a CHA-CRISPR/Cas13 system for signal amplification.
• To validate the probe's performance in biological samples and cell imaging.

Main Methods:

• A fluorescence probe was constructed using Axl-specific aptamers and a CHA-CRISPR/Cas13 system.
• The system relies on aptamer-mediated displacement and cyclic RNA release to activate Cas13.
• CRISPR/Cas13 cleaves a reporter, restoring FAM fluorescence proportional to Axl concentration.

Main Results:

• The probe demonstrated a linear relationship between fluorescence and Axl concentration (3.33-667 pM).
• Achieved an ultrahigh sensitivity with a detection limit of 0.84 pM.
• Successfully detected Axl in human serum, identifying higher levels in cervical cancer patients.
• Visualized Axl in tumor cells, correlating with serum detection findings.

Conclusions:

• The developed CHA-CRISPR/Cas13 fluorescence probe offers a sensitive and specific method for Axl detection.
• This technology provides valuable support for tumor diagnosis and prognosis.
• Potential applications include detecting circulating tumor cells in blood via cell imaging.