A novel label-free universal biosensing platform based on CRISPR/Cas12a for biomarker detection

Area of Science:

• Biotechnology
• Molecular Biology
• Nanotechnology

Background:

• Developing universal, sensitive, and cost-effective biosensors for disease biomarker detection remains a significant challenge in clinical diagnostics.
• Existing methods often require complex labeling or suffer from high background noise, limiting their clinical utility.

Purpose of the Study:

• To develop a programmable and universal biosensing platform for sensitive and specific detection of proteins, small molecules, and nucleic acids.
• To integrate the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system with a fluorescent label-free approach for enhanced signal amplification.

Main Methods:

• Utilized an exonuclease III-mediated target cycle to release a universal trigger chain, activating CRISPR/Cas12a enzyme activity.
• Employed single-stranded DNA-templated silver nanoclusters (ssDNA-Ag NCs) as a fluorescent reporter probe, with hydrolysis leading to fluorescence quenching.
• Demonstrated flexibility by modifying DNA hairpin sequences for recognition of diverse targets like proteins, small molecules, and microRNAs.

Main Results:

• Achieved sensitive and specific detection of various disease-related biomarkers without the need for pre-labeling or specific reporter molecules.
• The label-free system exhibited minimal background signal, leading to a significant decrease in fluorescence intensity upon target detection.
• Successfully detected protein, small molecule, and microRNA targets in biological samples using a single, adaptable platform.

Conclusions:

• The developed CRISPR/Cas12a-based fluorescent label-free biosensing platform offers a sensitive, specific, and versatile tool for biomarker detection.
• This innovative approach provides a low-cost, rapid, and simple method for clinical diagnosis, addressing limitations of current technologies.
• The programmable nature of the system presents a powerful new strategy for developing advanced clinical diagnostic tools.