Quencher-free CRISPR-based molecular detection using an amphiphilic DNA fluorescence probe

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Nucleic Acid Detection

Background:

• Nucleic acid detection is vital for clinical diagnostics.
• Existing methods often rely on complex fluorophore-quencher systems.
• There is a need for simpler, sensitive, and specific detection methods.

Purpose of the Study:

• To develop a novel, quencher-free CRISPR-based nucleic acid detection system.
• To utilize an amphiphilic DNA fluorescence probe for enhanced detection.
• To enable visual quantification of target nucleic acids using a paper-based microfluidic device.

Main Methods:

• Designed an amphiphilic DNA probe with a hydrophobic Cy5 fluorophore head and hydrophilic ssDNA tail.
• Integrated the probe with a CRISPR-Cas12a system and a paper-based microfluidic device.
• Assessed system performance using human papillomavirus-16 (HPV-16) DNA and clinical samples.

Main Results:

• The system demonstrated quencher-free, CRISPR-based detection of nucleic acids.
• Fluorescence diffusion distance correlated inversely with target nucleic acid concentration for visual quantification.
• Clinical validation with swab samples showed comparable results to PCR.

Conclusions:

• The developed system offers a simple, affordable, and sensitive method for nucleic acid quantification.
• This technology has potential for diagnosing HPV-associated cancers and other infectious diseases.
• The quencher-free approach simplifies CRISPR-based detection platforms.