Direct quantitative analysis of HCV RNA by atomic force microscopy without labeling or amplification

Area of Science:

• Biophysics
• Nanotechnology
• Molecular Diagnostics

Background:

• Hepatitis C Virus (HCV) poses a significant global health challenge.
• Accurate and sensitive detection of HCV RNA is crucial for diagnosis and treatment monitoring.
• Current methods often require amplification or reverse transcription, adding complexity and time.

Purpose of the Study:

• To develop a direct, label-free method for quantitative detection of HCV RNA using atomic force microscopy (AFM).
• To establish a sensitive detection platform for viral RNA at low concentrations.

Main Methods:

• Utilized force-based AFM for direct detection and quantification of HCV RNA.
• Designed specific DNA capture and detection probes immobilized on a substrate and AFM probe, respectively.
• Employed dendron self-assembly for controlled DNA probe spacing and generated adhesion force maps.

Main Results:

• Specific unbinding events showed a mean force of 32 ± 5 pN.
• Hydrodynamic distance of captured RNA was measured to be 30-60 nm.
• A linear relationship was observed between cluster number and HCV RNA concentration (0.76 fM to 6.0 fM).

Conclusions:

• AFM enables direct, quantitative analysis of HCV RNA without amplification.
• The developed method demonstrates high sensitivity, with potential for detecting few-copy numbers of HCV RNA.
• This approach offers a promising alternative for rapid and sensitive viral RNA diagnostics.