CRISPR/Cas12a Protein Switch Powered Label-Free Electrochemical Biosensor for Sensitive Viral Protease Detection

Area of Science:

• Biotechnology
• Molecular Biology
• Biosensor Technology

Background:

• Viral proteases are key targets for understanding viral pathogenesis and developing antiviral therapies.
• Accurate detection of viral proteases is crucial for diagnosing infections and screening drugs.
• Existing methods may lack sensitivity or require complex procedures.

Purpose of the Study:

• To develop a sensitive, label-free electrochemical biosensor for viral protease detection.
• To integrate CRISPR/Cas protein switches with an electrochemical interface for enhanced detection.
• To demonstrate the biosensor's utility in monitoring specific viral proteases in biological samples.

Main Methods:

• Utilized protease-responsive CRISPR/Cas protein switches (CasPSs).
• Functionalized an electrochemical interface with hemin aptamers.
• Leveraged viral protease-mediated cleavage to release active Cas12a, amplifying electrochemical signals.
• Tested specificity for hepatitis C virus NS3/4A protease and enterovirus 71 3C protease.

Main Results:

• Achieved femtomolar sensitivity for hepatitis C virus NS3/4A protease detection.
• Demonstrated successful monitoring of enterovirus 71 3C protease activity in infected cell samples.
• Showcased the biosensor's adaptability to different viral proteases by modifying the CasPS module.
• Validated the label-free, electrochemical detection mechanism.

Conclusions:

• The developed CRISPR-based electrochemical biosensor offers a sensitive and specific platform for viral protease detection.
• This approach provides a valuable tool for viral infection monitoring and antiviral drug discovery.
• The integration of CRISPR biosensing with electrochemistry presents a promising strategy for future diagnostic applications.