A Label-Free CRISPR/Cas12a-G4 Biosensor Integrated with FTA Card for Detection of Foodborne Pathogens

Area of Science:

• Molecular Biology
• Biotechnology
• Food Safety

Background:

• CRISPR/Cas diagnostics offer high specificity but face limitations in field use due to complex sample preparation and reliance on fluorescent probes.
• Existing methods often require specialized equipment and costly reagents, hindering accessibility in resource-limited environments.

Purpose of the Study:

• To develop a simplified, label-free, and equipment-free CRISPR/Cas12a diagnostic platform for detecting foodborne pathogens.
• To integrate efficient nucleic acid extraction with a novel signal reporting mechanism for visible fluorescence output.

Main Methods:

• Developed a system combining FTA card-based nucleic acid extraction with CRISPR/Cas12a.
• Utilized a pre-folded G-quadruplex (G4)-Thioflavin T (ThT) complex as a label-free signal reporter, leveraging G4-ThT structural binding for fluorescence.
• Tested the platform for the detection of *Escherichia coli* O157:H7 in spiked food samples.

Main Results:

• Achieved a limit of detection (LOD) of 10¹ CFU/mL for *Escherichia coli* O157:H7.
• Demonstrated 100% concordance with quantitative PCR (qPCR) and standard fluorescent probe-based CRISPR/Cas12a systems.
• The platform requires minimal equipment, including portable heating and imaging devices.

Conclusions:

• The developed label-free, equipment-free platform significantly enhances the field applicability of CRISPR/Cas diagnostics.
• This innovative system offers a cost-effective and simplified approach for detecting foodborne pathogens, particularly beneficial for resource-limited settings.
• The G4-ThT reporter system eliminates the need for fluorescently labeled probes, streamlining the diagnostic process.