Integrated Optothermal Paperfluidic Platform for Single-Step, Model-Driven CRISPR-Cas12a Diagnostics of Mycobacterium...

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Point-of-Care Testing

Background:

• CRISPR-Cas12a diagnostics show promise for pathogen detection but face challenges in point-of-care applications.
• Complex reagent handling, thermal control, and equipment size hinder field deployment.

Purpose of the Study:

• To develop an integrated opto-thermal paperfluidic platform for autonomous, single-step CRISPR-Cas12a detection of Mycobacterium tuberculosis (MTB).
• To enable rapid, sensitive, and specific detection of MTB at the point-of-care, particularly in resource-limited environments.

Main Methods:

• Optimized platform architecture using thermal transfer printing for robust hydrophobic barriers on cellulose.
• Developed a hybrid mathematical model for an "intrinsic timer" to synchronize capillary flow with CRISPR kinetics.
• Engineered a 3D-printed module for autonomous thermal regulation (39 °C) and fluorescence imaging.
• Utilized a red-shifted ROX-quencher reporter probe and sucrose-based lyoprotection for reagent stability and reduced autofluorescence.

Main Results:

• Achieved a limit of detection of 0.0335 ng/μL for MTB with prestored reagents.
• Demonstrated autonomous operation with a single sample-loading step and results in 15 minutes.
• The integrated platform overcomes limitations of complex reagent handling and bulky equipment.

Conclusions:

• The developed opto-thermal paperfluidic platform provides a practical, low-cost solution for decentralized MTB surveillance.
• This technology enhances the translation of CRISPR-Cas12a diagnostics to point-of-care settings.
• Enables rapid and sensitive detection of Mycobacterium tuberculosis in resource-limited areas.