Birth asphyxia is the inability of a newborn to begin and maintain breathing. Twenty-three percent of neonatal deaths globally are caused by birth asphyxia. Birth asphyxia results in a neurological injury called hypoxic ischemic encephalopathy (HIE). Rapid HIE screening within six hours after birth is crucial to identify neonates at risk. Unfortunately, the diagnostic equipment is impractical for low resource settings because it is costly ($20/test and $5,000 for equipment) and requires technical staff, that are in short supply, to operate. We hypothesize that a cost-effective device can be developed for HIE analysis. pHast Cam quickly screens for birth asphyxia and HIE in infants via a paper-based blood pH sensor. The device combines an inexpensive pH sensitive dye, a smartphone camera, and a fixture that controls the imaging environment to quickly identify acidosis from samples. A low-cost paper-based strip is made with a water-soluble resin doped with a pH-sensitive dye, bromothymol blue (BTB), and a membrane to filter out red blood cells. The fixture removes lighting variation. The smartphone camera records the pH indicator image, and an algorithm captures, reduces noise, and accesses color change. pHast Cam incorporates four features: 1) accurate assessment of acidity within 0.05 pH units, 2) require only a few microliters of sample, 3) use electrical hardware and software only from the smartphone, and 4) affordability. At this stage, we have achieved a regressive linear model that predicts buffered solution acidity (y=-589.32x+4684.05 R2=0.9857), with 95% confidence interval of 0.04 pH units. In the future, we will transition from measuring buffered solutions to blood-plasma. Ultimately, we expect pHastCam to screen for birth asphyxia, and other acid-base disorders, by quantifying plasma pH in neonates so that timely therapeutic interventions and plans to address long-term complications may occur.

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 37˚C, but we decreased the reaction time to 15 minutes and removed the need for an external heating source by incubating at room temperature (25˚C). 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 < 90˚) 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

Therapeutic Drug Monitoring (TDM) serves a critical role in optimizing the effectiveness and safety of dapivirine (DPV) vaginal rings as an HIV prevention tool, ultimately leading to improved health outcomes for individuals at risk of HIV infection. Liquid chromatography-tandem mass spectrometry (LC/MS) is a commonly used method for HIV TDM, however, its limited availability leads to delayed results, high cost, and limited utility in clinical practice. The REverse TRanscrIptase Termination (RESTRICT) assay is a rapid and inexpensive test for TDM. DPV has low solubility in aqueous solutions due to its hydrophobic properties. Organic solvents like isopropyl alcohol (IPA), acetonitrile (ACN), and dimethylsulfoxide (DMSO) are typically used for extracting DPV from returned vaginal rings. However, organic solvents often interfere with the performance of enzymatic assays like RESTRICT. In this study, we investigated the compatibility of organic solvents used for DPV extraction with the RESTRICT assays. We tested mixtures of IPA, ACN, and DMSO at varying concentrations of solvent in water. After performing a preliminary experiment with 25%, 50%, and 75% concentrations of IPA in water using RESTRICT, we found similar fluorescence readouts between the different concentrations indicating that IPA and RESTRICT are compatible. The solvent that results in the smallest decrease in signal intensity compared to solvent-free assays will be selected. In the future, we will extract DPV from new and returned vaginal rings and measure drug levels using the RESTRICT assay benchmarking against a conventional laboratory technique like Raman spectrometry. This work represents a first step towards developing a user-friendly test for measuring DPV levels at the point of need.

There is a strong need for rapid and inexpensive tests for monitoring human immunodeficiency virus (HIV) drugs to prevent treatment failure. The REverse TranscrIptase Chain Termination (RESTRICT) Assay provides fast and inexpensive measurement of HIV reverse transcriptase (RT) inhibitors by measuring the extent of DNA synthesis with the HIV RT enzyme in the presence of a patient’s blood. RESTRICT uses an intercalating dye, PicoGreen, to provide fluorescence as a function of the amount of DNA synthesized. However, PicoGreen interrupts DNA synthesis and binds to any double-stranded DNA (dsDNA) present in the sample including background DNA in blood samples, precluding real-time measurements and potentially decreasing the accuracy of results. This project uses RESTRICT in conjunction with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) complexes and reporters to accurately measure RESTRICT results in real-time. Fluorescence is produced by reporters which are made up of a quencher and a fluorophore. When the CRISPR complex detects a specific complementary sequence formed from the single-stranded DNA (ssDNA) template, the Cas enzyme is activated and cleaves the fluorescent reporter. Once cleaved, the fluorophore is released from the quencher thus generating fluorescence. Because the CRISPR complex binds only to specific segments of DNA produced by RESTRICT, we can obtain more specific results without interference from background DNA. In addition, CRISPR complexes and reporters do not interfere with DNA synthesis and can be added at the same time as other RESTRICT reagents, enabling real-time monitoring of RESTRICT reactions. Using a portable fluorescence reader (T8-ISO, Axxin Pty Ltd.), positive control reactions containing RT enzyme provided at least 5X more fluorescence than negative controls without RT. Future work will focus on measuring clinically relevant levels of HIV drugs in aqueous buffers using RESTRICT with CRISPR readout.