CRISPR-BASED POINT-OF-CARE HIV-1 RNA DETECTION USING A MICROFLUIDIC PLATFORM

Author

• Bushra Raahat, Southern Illinois University CarbondaleFollow

Date of Award

8-1-2026

Degree Name

Master of Science

Department

Electrical and Computer Engineering

First Advisor

Li, Hui

Abstract

Human Immunodeficiency Virus type 1 (HIV-1) remains a major global health challenge, requiring rapid and sensitive diagnostic methods for early detection and effective disease management. Conventional nucleic acid amplification tests provide high sensitivity but often require complex instrumentation, centralized laboratory facilities, and extended turnaround times, limiting their application in resource-limited and point-of-care settings. This thesis presents the development of a microfluidic platform integrated with reverse transcription recombinase polymerase amplification (RT-RPA) and CRISPR-Cas12a-based fluorescence detection for rapid and sensitive HIV-1 RNA detection. The assay was first optimized and validated in a benchtop format by evaluating RT-RPA amplification conditions and CRISPR-Cas12a trans-cleavage activity. RT-RPA parameters, including primer concentration and reaction conditions, were investigated, followed by optimization of Cas12a and guide RNAs concentrations to achieve enhanced fluorescence signal generation. The optimized RT-RPA-CRISPR assay demonstrated specific detection of HIV-1 target sequences with a limit of detection of 10 copies/µL. Assay specificity was evaluated against non-target bacterial DNA, demonstrating selective amplification and detection of HIV-1 sequences. A multilayer microfluidic device containing different reaction volumes, including nanoliter-scale wells, was fabricated using SU-8 photolithography and PDMS-based molding techniques. The device design enabled compartmentalization of individual reactions and provided a platform for digital fluorescence-based detection. Fluid loading behavior, surface treatment conditions, and plasma bonding parameters were investigated to improve well filling and assay compatibility. The optimized device was integrated with the RT-RPA-CRISPR assay and evaluated using fluorescence microscopy for on-chip detection. The developed microfluidic CRISPR-based platform provides a compact and sensitive approach for HIV-1 RNA detection and demonstrates potential for future point-of-care diagnostic applications. Further improvements, including integration of sample preparation and direct detection from clinical samples, could enable development of a complete sample-to answer HIV-1 diagnostic system suitable for decentralized healthcare settings.