An AND-Logic Gate-Based Biosensor for Simultaneous Detection of SARS-CoV-2 Nucleic Acids and Nucleocapsid Proteins

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Biosensor Technology

Background:

• Nucleic acids and proteins are crucial biomarkers for disease diagnosis.
• Conventional single-analyte detection methods are prone to false positives, impacting reliability.
• Current methods face challenges in simultaneous detection of multiple biomarkers like viral RNA and proteins.

Purpose of the Study:

• To develop a novel biosensor for simultaneous detection of SARS-CoV-2 RNA and nucleocapsid protein.
• To improve diagnostic reliability by minimizing false positives through an AND-logic gate approach.
• To create a flexible assay design for diverse target detection.

Main Methods:

• Developed a one-pot isothermal amplification and CRISPR-Cas cooperative system (OIACS).
• Utilized antibody-mediated target binding with blocker release for flexible target recognition.
• Generated a universal Cas12a-targetable DNA barcode via strand displacement isothermal amplification for signal amplification.

Main Results:

• The OIACS functions as an AND-logic gate biosensor for simultaneous detection.
• Achieved reliable detection of SARS-CoV-2 virus-like particles at 5000 copies/mL.
• Determined a limit of detection as low as 1698 copies/mL.

Conclusions:

• The OIACS offers a robust and reliable method for dual-biomarker detection.
• Demonstrated practical utility and potential for clinical diagnosis of SARS-CoV-2.
• The antibody-mediated recognition and DNA barcode amplification enhance assay performance.