Hybridization chain reaction and CRISPR/Cas12a-integrated biosensor for precise Ago2 detection

Area of Science:

• Biomedical Engineering
• Molecular Biology
• Biotechnology

Background:

• Argonaute 2 (Ago2) is a critical enzyme in RNA interference (RNAi) pathway, vital for gene regulation.
• Dysregulation of Ago2 is implicated in various diseases, including cancer and viral infections, necessitating sensitive detection methods.
• Existing methods for Ago2 detection may lack the required sensitivity or specificity for comprehensive analysis.

Purpose of the Study:

• To develop and validate a highly sensitive and specific electrochemiluminescence (ECL) biosensor for Argonaute 2 (Ago2) activity.
• To leverage a multi-stage signal amplification strategy combining hybridization chain reaction (HCR) and CRISPR/Cas12a systems.
• To establish a robust platform for accurate biomarker detection applicable to clinical diagnostics and disease monitoring.

Main Methods:

• Designed a biosensor integrating hybridization chain reaction (HCR) for signal amplification.
• Incorporated the CRISPR/Cas12a system to further enhance signal through collateral cleavage activity.
• Utilized electrochemiluminescence (ECL) detection with a ruthenium-based luminophore for sensitive readout.
• Ago2-mediated cleavage of hairpin RNA initiated the HCR amplification cascade.

Main Results:

• Achieved an exceptional limit of detection (LOD) of 0.126 aM for Ago2 activity.
• Demonstrated high specificity, distinguishing Ago2 from other Argonaute proteins.
• Confirmed excellent reproducibility and stability, with 94% signal retention after two weeks.
• Validated real-world applicability by accurately detecting Ago2 in spiked cell lysates with high recovery rates.

Conclusions:

• The developed HCR-CRISPR/Cas12a-ECL biosensor offers a powerful platform for sensitive Ago2 detection.
• This innovative biosensor represents a significant advancement for next-generation diagnostic tools.
• The platform shows great potential for clinical diagnostics, disease monitoring, and therapeutic evaluation due to its sensitivity and adaptability.