Localized Cas12a-based cascade amplification for sensitive and robust detection of APE1

Area of Science:

• Biochemistry
• Molecular Biology
• Nanotechnology

Background:

• Apurinic/apyrimidinic endonuclease 1 (APE1) is crucial for DNA repair and overexpressed in cancers, making it a potential diagnostic biomarker.
• Detecting low APE1 levels in early cancer stages remains challenging due to sensitivity limitations in current methods.

Purpose of the Study:

• To develop a highly sensitive and stable method for detecting APE1, addressing the limitations of existing diagnostic tools.
• To introduce a novel localized Cas12a-based cascade amplification (LCas12a-CA) strategy for enhanced biosensing.

Main Methods:

• Confinement of terminal deoxynucleotidyl transferase and crRNA/Cas12a complex onto gold nanoparticles (AuNPs) to create LCas12a-CA.
• Utilizing APE1's enzymatic activity to initiate a cascade reaction involving TdT and Cas12a.
• Employing Cas12a's trans-cleavage activity on reporters for fluorescence signal generation.

Main Results:

• The LCas12a-CA method achieved a detection limit of 2.51 × 10^-6 U/mL, over 60 times lower than free Cas12a-CA.
• The localized enzyme confinement enhanced enzyme stability and reaction rates via substrate channeling.
• The method demonstrated robustness in extreme conditions and effectiveness in detecting APE1 in clinical samples.

Conclusions:

• The LCas12a-CA platform offers significantly improved sensitivity and stability for APE1 detection.
• This novel biosensing approach holds promise for early cancer diagnosis and prognosis.
• The localized enzyme strategy provides a robust platform for various diagnostic applications.