Antiretroviral therapy (ART) prevents the progression of human immunodeficiency virus (HIV) by suppressing viral load, limiting transmission. Of the ~20 million people receiving ART, 30-40% do not maintain adequate medication adherence, resulting in treatment failure and drug resistance. Monitoring HIV medication adherence improves the efficacy of ART but requires bulky and expensive instruments that are not widely accessible at the point-of-need (e.g. doctor’s office or patient’s home), so a rapid and accessible diagnostic alternative is necessary. Our group developed the REverSe TRanscrIptase Chain Termination (RESTRICT) enzymatic assay to provide rapid and inexpensive measurement of HIV drug adherence by measuring antiretroviral drug activity indicated by fluorescence. However, one current limitation of RESTRICT is the need for trained operators to complete multiple precisely timed steps required in the enzymatic activity assay. We aim to create a 3D-printed microfluidic device that will automate the liquid handling steps required for RESTRICT via precisely tuned capillary action for rapid and user-friendly measurement of antiretroviral drugs. To that end, we first demonstrated a proof of concept by creating a microchip with a controlled 15 minute liquid delivery time and consistent liquid extraction. Through optimization of 3D-printing methods, channel geometry, and surface treatment, we created a microchip designed to deliver liquid in 14.64 minutes that ran experimentally in 19.12 ± 2.33 minutes. In the future, we will demonstrate the feasibility of RESTRICT run on-chip and fluorescence measured off-chip by testing clinically-relevant drug concentrations using the controlled liquid delivery time and liquid extraction methods developed. By creating an automated and rapidly fabricated microfluidic chip for therapeutic drug monitoring, we hope to achieve a hands-off device that removes external manipulation to increase the accessibility of RESTRICT-on-a-chip for point-of-need settings without specialized equipment or highly trained operators.

Capillary microfluidics devices automate point-of-care diagnostic assays because of their instrument-free operation, small size, and low material cost. However, capillary microfluidics currently require expensive fabrication instruments limiting rapid prototyping and deployment in low-resource settings. State-of-the-art capillary microfluidics are fabricated by using digital light project stereolithography (DLP-SLA) 3D-printers that offer high resolution (40 µm) but are expensive ($10,000 – $20,000). Liquid crystal display (LCD) SLA printers have recently emerged with comparable resolution and much lower cost ($300 – $1000). However, our initial experiments with LCD-SLA printers exhibited defects and post-processing issues. Our goal is to optimize the fabrication process of capillary microfluidics using an inexpensive LCD-SLA printer (Anycubic Photon Mono 6K, ~$400) and calibrate performance relative to a DLP-SLA printer (CADWorks Pr 4K, ~$15,000). By varying printing parameters including UV power, exposure time, layer height, retraction speed, and resin choice, we found optimal conditions that produced microfluidic channels with comparable dimensions on the Anycubic and CADWorks printers (i.e. coefficient of variation <20%). With printer settings of 40% UV Power, 1.8s exposure time, 20 µm layer height, and 0.1 mm/s retraction speed with CADWorks clear resin, the Anycubic 3D printer produced microchannels down to 100 μm, the smallest feature size we achieved with the CADWorks printer. Optimizing 3D-printing of capillary microfluidics using inexpensive LCD-SLA printers like the Anycubic has the potential to enable rapid prototyping of point-of-care diagnostics in low-resource settings. Next steps include printing more complex microfluidic components including domino valves and trigger valves.