Integrated Optothermal Paperfluidic Platform for Single-Step, Model-Driven CRISPR-Cas12a Diagnostics of <i>Mycobacterium tuberculosis</i>

CRISPR-Cas12a molecular diagnostics offer high sensitivity and specificity for pathogen detection, yet their translation to point-of-care (POC) settings is often hindered by complex reagent handling, precise thermal requirements, and reliance on bulky laboratory equipment. In this study, we present an integrated opto-thermal paperfluidic platform engineered for the autonomous, single-step CRISPR-Cas12a detection of Mycobacterium tuberculosis (MTB). The platform’s architecture was optimized by comparing laser and thermal transfer printing, identifying the latter as the superior method for creating robust, leak-proof hydrophobic barriers while preserving the structural integrity of the cellulose substrate. To achieve autonomous operation, we developed a hybrid mathematical model─incorporating boundary resistance and thermal evaporation─to design an “intrinsic timer” that aligns capillary flow front progression with the 15 min CRISPR reaction kinetics. Thermal regulation and signal readout are managed by a custom-engineered, 3D-printed hardware module that provides closed-loop 39 °C incubation and high-contrast fluorescence imaging. By utilizing a red-shifted ROX–quencher reporter probe to overcome paper autofluorescence and sucrose-based lyoprotection for reagent stabilization, the platform achieved a limit of detection of 0.0335 ng/μL with prestored reagents. Our integrated detection system requires only a single sample-loading step and provides results in 15 min, offering a practical, low-cost solution for decentralized tuberculosis surveillance in resource-limited environments.