Gold Nanoparticle-Labeled CRISPR-Cas13a Assay for the Sensitive Solid-State Nanopore Molecular Counting

Area of Science:

• Biotechnology
• Nanotechnology
• Molecular Diagnostics

Background:

• CRISPR-Cas13a systems offer precise RNA targeting capabilities.
• Nanopore sensing provides a platform for label-free electrical detection of biomolecules.
• Existing nucleic acid detection methods can be complex and require specialized equipment.

Purpose of the Study:

• To develop a sensitive and quantitative nucleic acid assay using gold nanoparticles (AuNPs) and CRISPR-Cas13a.
• To enable rapid, portable detection of RNA biomarkers via solid-state nanopore sensing.
• To achieve high sensitivity and a wide dynamic range for RNA quantification.

Main Methods:

• A CRISPR-Cas13a assay was developed, labeling RNA targets with AuNPs via non-covalent conjugation.
• Upon CRISPR activation, AuNPs were liberated, isolated, and detected by a solid-state nanopore sensor.
• Detection was based on monitoring changes in ionic current caused by AuNP translocation, with each event enumerated.

Main Results:

• Achieved a sensitive detection limit of 50 fM for SARS-CoV-2 RNA segments without amplification.
• Demonstrated a broad dynamic range of six orders of magnitude for quantitative RNA sensing.
• The assay operates at physiological temperature and utilizes a smartphone-sized nanopore reader.

Conclusions:

• The developed AuNP-labeled CRISPR-Cas13a assay provides a sensitive, quantitative, and simplified method for RNA detection.
• The system's portability and ease of use make it suitable for rapid biomarker detection in diverse settings.
• This technology has potential applications in both clinical diagnostics and resource-limited environments.