Subtherapeutic drug levels can lead to the failure of antiretroviral therapy (ART) regimens used in Human Immunodeficiency Virus (HIV) treatment and prevention. However, gold-standard HIV drug level monitoring techniques—such as mass spectrometry—require bulky and expensive instruments that are not widely accessible at the point-of-need. Our group developed the REverSe TRanscrIptase Chain Termination (RESTRICT) enzymatic assay to rapidly (30 min) and inexpensively measure tenofovir diphosphate (TFV-DP), a nucleotide analog used in >90% of oral ART regimens and in all approved prevention regimens. However, RESTRICT currently requires trained operators to perform multiple time-sensitive liquid-handling steps. To reduce user intervention and minimize the need for laboratory equipment, we harnessed 3D-printed capillaric microfluidics to self-propel liquids using only surface tension effects encoded in microchannel geometry and surface chemistry. Specifically, we translated the manual tube-based RESTRICT to an automated microfluidic protocol by using autonomous trigger valves to pre-load multiple RESTRICT assay reagents and serpentine channels to control assay timing. Currently, RESTRICT reactions are incubated for 30 minutes at 37ËšC, but we decreased the reaction time to 15 minutes and removed the need for an external heating source by incubating at room temperature (25ËšC). There was only a 15% decrease in overall signal intensity in the faster, room temperature assays, and measured readout was distinguishable between clinically relevant concentrations of TFV-DP. Our results represent a first step towards integrating RESTRICT reactions and fluorescence readout onto a rapidly fabricated microfluidic chip. We hope to achieve a device that increases the accessibility of HIV drug level monitoring at the point of need without specialized equipment or highly trained operators.

Capillary microfluidics capitalize on surface tension effects encoded in microchannel geometry and chemistry to transfer liquids without external instruments, making them a user-friendly technology for point-of-care tests. For most applications, hydrophilic surfaces (contact angle < 90Ëš) are necessary to induce surface tension driven flow. Currently, this is achieved with vacuum plasma chambers that alter surface chemistry. Unfortunately, the hydrophilic properties made with plasma processing are temporary and unstable. Alternatively, an inherently stable hydrophilic 3D-printing resin containing polyethylene glycol diacrylate (PEGDA) and acrylic acid (AA) was recently developed for capillary microfluidics. However, this hydrophilic resin has not been thoroughly validated for inexpensive (<$300) liquid crystal display (LCD) printers. Our objective is to optimize and validate 3D-printing parameters including exposure time, UV power, layer thickness, and lift/retract speed using this hydrophilic PEGDA-AA resin with three LCD 3D printers (AnyCubic Photon Mono X 6K, AnyCubic Photon Mono M5s Pro, and Phrozen Sonic Mini 8K). Validation includes measuring hydrophilic properties as well as the dimensional fidelity of the printed channels compared to the design specifications. Our proof-of-concept prints on the Mono X 6K printer had average contact angle measurements of 42.8° ± 8.77. The percent differences between designed and printed channel lengths, widths, and depths were 31.5 ± 0.23%, 28.9 ± 3.41%, and 2.40 ± 13.9% respectively. By optimizing the print parameters of cost-effective 3D printers with the inherently stable hydrophilic resin, we enable capillary microfluidic technologies for users in low income/resource settings who may not have access to vacuum plasma chambers. Future work will explore additional resin modifications to encourage applications like spatial patterning of hydrophilicity and protein immobilization in microchips. [1]V. Karamzadeh, A. S. Kashani, M. Shen, and D. Juncker, “Digital Manufacturing of Functional Ready‐to‐Use Microfluidic Systems,” Advanced Materials, vol. 35, no. 47