Biology subjects

Olanrewaju, A. O.

Publications and source records attributed to Olanrewaju, A. O..

9 recordsLinked to original sources

bioRxiv2026

Modeling and validation of parallel co-flows layer widths in open-capillary trigger valve systems

Control of fluids is a hallmark of microfluidic systems and fundamental for the successful application of microfluidic devices. Trigger valves use geometric features to autonomously control the release of fluids in microfluidic devices. Our previous work has adapted geometries used in closed trigger valve systems to enable use in open systems, allowing for open microfluidic devices with up to three trigger valves. Here, we focus on the parallel co-flows produced by sequential release of trigger valves and present a model that predicts their layer widths as a function of the geometric characteristics of the different side channels of each trigger valve. We show layered co-flows with widths as low as 50 microns. Additionally, we expand the use of trigger valves in open microfluidic devices by incorporating 1) varied step heights, 2) devices with up to seven trigger valves, and 3) use of varied fluids and plastics. To validate the implementation and use of these trigger valves in open systems, we have developed a theoretical framework to compare predicted outcomes (i.e., fluid travel distance, velocity, and layering width) with our experimental values. This theoretical work offers applications in various fields, including hydrogel patterning for 3D cell culture, organ-on-a-chip models, at-home sample preparation, and autonomous microfluidic systems for biosensing.

bioengineering↗

bioRxiv2025

An Optimized Product-Enhanced Reverse Transcriptase Assay for Sensitive and Quantitative Detection of HIV Viral Load and Phenotypic Drug Resistance

The World Health Organization (WHO) recommends Human Immunodeficiency Virus (HIV) drug resistance testing (DRT), but current tests are too complex and expensive for routine use. Genotypic DRT is challenging to interpret because of the growing list of mutations responsible for HIV drug resistance. Although phenotypic DRT is simpler to interpret, it requires slow and labor-intensive viral culture. Phenotypic tests that measure the activity of isolated HIV enzymes are faster and less labor-intensive, but none have yet met the 2023 WHO Target Product Profile (TPP) for HIV DRT. Here we present an optimized Product-Enhanced Reverse Transcriptase (PERT) assay for sensitive and quantitative detection of HIV viral load and drug resistance. PERT combines complementary DNA (cDNA) synthesis by HIV-Reverse Transcriptase (HIV-RT) with cDNA amplification and detection by quantitative PCR (qPCR). We established sensitive detection down to 10 HIV-RT molecules in a 25 {micro}L sample corresponding to a viral load of [~]10 HIV RNA copies/mL. We demonstrated the assays feasibility for phenotypic DRT using lamivudine-5-triphosphate (3TC-TP)--to which the M184V mutation confers high-level resistance--and quantified 3TC-TP inhibition using the difference in cDNA produced between drug and no-drug conditions. We surpassed WHO minimal analytical sensitivity requirements (20% low-abundance variant detection) by differentiating 10% M184V HIV-RT fractions in heterogenous mixtures (1,000 total HIV-RT, viral load [~]2,500 copies RNA/mL). Finally, we showed that PERT could detect resistance to existing and emerging RT inhibitors including tenofovir-diphosphate (TFV-DP), doravirine (DOR), and islatravir (ISL)-TP. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/691922v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@afec9borg.highwire.dtl.DTLVardef@1d6c6eeorg.highwire.dtl.DTLVardef@19e4082org.highwire.dtl.DTLVardef@9d49e6_HPS_FORMAT_FIGEXP M_FIG C_FIG The Product-Enhanced Reverse Transcriptase (PERT) assay measures HIV-RT activity by its ability to synthesize complementary DNA, providing a phenotypic measurement of HIV viral load (by quantity of HIV-RT enzyme) and susceptibility to RT inhibitors (by level of inhibition of HIV-RT activity). IMPORTANCEAlthough antiretroviral therapy can effectively treat and prevent HIV infection, treatment efficacy and global control of the HIV epidemic are threatened by rising rates of HIV drug resistance. Inexpensive and decentralized HIV DRT could facilitate surveillance efforts to understand the prevalence of HIV drug resistance in local and global contexts. This need is especially timely and pressing considering anticipated increased rates of HIV acquisition and drug resistance due to the reductions in global HIV services driven by recent funding cuts. Our optimized PERT assay for simultaneous viral load and phenotypic drug resistance quantification is fast ([~]2 hours), sensitive, and accurate. We can also leverage existing RT-qPCR instruments used for viral load measurement to significantly improve access to HIV drug resistance monitoring and tailored regimen selection.

bioengineering↗

bioRxiv2025

REverse-transcriptase ACTivity with CRISPR (REACTR) Assay for Ganciclovir Triphosphate Monitoring

BackgroundCytomegalovirus (CMV) can cause severe disease and death in infants and immunocompromised people. Although effective at treating CMV, the first-line drug - ganciclovir (GCV) - has high rates of pharmacokinetic variability which leads to significant rates of underexposure or toxicity. Intracellular concentrations of ganciclovir triphosphate (GCV-TP) - GCVs active anabolite - are associated with dose-dependent neutropenia and could enable dose individualization to improve treatment efficacy and reduce adverse effects. However, GCV-TP is currently measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) which is impractical for routine use, especially in resource-limited settings, because of its high cost, labor-intensiveness, and need for specialized equipment. To address this gap, we adapted the REverse transcriptase ACTivity with CRISPR (REACTR) assay to measure GCV-TP. MethodsWe leveraged our earlier work using REACTR to measure reverse transcriptase (RT) inhibitors used to treat and prevent human immunodeficiency virus (HIV) because GCV-TP serendipitously also inhibits HIV RT. We designed custom DNA templates, primers, and CRISPR complexes to accurately measure GCV-TP spiked into buffer and blood. We evaluated the assays analytical performance with 40 dried blood spots from infants with congenital CMV. ResultsREACTR reproducibly measured clinically relevant GCV-TP concentrations using a simple workflow and equipment that are readily available in many clinical laboratories. REACTR measurements of clinical samples correlated with LC-MS/MS GCV-TP measurements (r = -0.7891; p<0.0001). ConclusionsThis study highlights the potential of REACTR as a rapid and accessible alternative to LC-MS/MS for therapeutic drug monitoring of GCV.

bioengineering↗

bioRxiv2025

To the homeRNAmax: Developing an Improved Blood Self-Collection and Stabilization Platform for Remote Transcriptomic Studies

Shifting human subjects research from research sites to participants homes removes barriers to participation, including transportation and scheduling difficulties. Previously, we developed homeRNA, a kit for immediate stabilization of RNA in self-collected blood using a custom-engineered tube containing RNA stabilizer fluid. The stabilized RNA is extracted and used for downstream gene expression analysis. Here, we introduce homeRNAmax, which improves our original design by interfacing with a commercially available blood collection tube (BD Microtainer), allowing homeRNAmax to be used with any blood collection method that uses this tube and doubling the possible sample volume that can be collected and stabilized compared to the original homeRNA. Through a pilot study (n=19 participants), we show that homeRNAmax (with the Tasso+ blood collection device) produces RNA samples of sufficient quality (mean RIN=7.8) and yield (mean yield=1.93 g) for downstream analysis and can reach participants across the United States, who generally (n=17/19) found the homeRNAmax kit easy to use. A key aspect of the homeRNA and homeRNAmax platforms is a fluidic feature that prevents the RNA stabilizer from spilling, however our previous work had not yet fully characterized the mechanism of this feature. Here, we developed a theoretical model of the spill-resistant feature. In brief, fluid in the tube is suspended due to a balance of pressures; an increase in air volume within the tube reduces the air pressure above the fluid, creating a small vacuum, and preventing fluid leakage. Overall, we show that homeRNAmax is a user-friendly, effective tool for remote blood RNA stabilization.

bioengineering↗

bioRxiv2025

CandyCollect Open to Closed (O2C) Microfluidic System for Rapid and User-Centric Detection of Group A Streptococcus

We created CandyCollect, a lollipop-inspired, child-friendly saliva sampling device that provides enhanced comfort compared to common throat and mouth swabs. The device has an open microchannel and a functionalized surface that effectively captures and stores the saliva and pathogens. We recently demonstrated the ability to combine the CandyCollect with rapid antigen detection tests (RADTs) for fast and accessible detection of group A streptococcus (GAS). However, the current procedure necessitates manual steps to elute and transfer captured antigens from the lollipop to RADTs, which relies on the user to follow complex instructions, increasing the likelihood of error. In this work, we developed the CandyCollect Open to Closed (O2C) microfluidic system to automate antigen elution and streamline detection with RADTs. The O2C system securely seals the open microchannels of the CandyCollect device to create a continuous closed channel for seamless delivery of elution reagents to release antigens at the press of a button for subsequent lateral flow detection. Our system leverages and combines the unique benefits of both open channel and closed channel microfluidics into one platform, further enhancing the potential of both methods. We performed experiments with GAS bacteria spiked into saliva and compared the manual and O2C elution methods. Both the manual and O2C methods showed clear test lines on the RADT strips at clinically relevant GAS concentrations (between 5 x105 and 109 CFU/mL). The O2C provided comparable or better results than the manual procedure in a more convenient form factor. The O2C platform has the potential to enable user-friendly screening for respiratory pathogens by minimally trained users in decentralized settings.

bioengineering↗

bioRxiv2024

Rapid enzymatic assay for antiretroviral drug monitoring using CRISPR-Cas12a enabled readout

Maintaining efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse-transcriptase ACTivity-crispR (REACTR) assay for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTRs sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.

bioengineering↗

bioRxiv2024

REverse transcriptase ACTivity (REACT) assay for point-of-care measurement of established and emerging antiretrovirals for HIV treatment and prevention

Maintaining adequate levels of antiretroviral (ARV) medications is crucial for the efficacy of HIV treatment and prevention regimens. Monitoring ARV levels can predict or prevent adverse health outcomes like treatment failure or drug resistance. However, conventional ARV measurement using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is slow, expensive, and centralized delaying clinical and behavioral interventions. We previously developed a rapid enzymatic assay for measuring nucleotide reverse transcriptase inhibitors (NRTIs) - the backbone of HIV treatment and prevention regimens - based on the drugs termination of DNA synthesis by HIV reverse transcriptase (RT) enzyme. Here we expand our work to include non-nucleoside reverse transcriptase inhibitors (NNRTIs) - an ARV class used in established and emerging HIV treatment and prevention regimens. We demonstrate that the REverse Transcriptase ACTivity (REACT) assay can detect NNRTIs including medications used in oral and long-acting/extended-release HIV treatment and prevention. We demonstrate that REACT can measure NNRTIs spiked in either buffer or diluted plasma, and that fluorescence can be measured on both a traditional plate reader and an inexpensive portable reader that can be deployed in point-of-care (POC) settings. REACT measured clinically relevant concentrations of five NNRTI spiked in aqueous buffer. REACT measurements showed excellent agreement between the plate reader and the portable reader, with a high correlation in both aqueous buffer (Pearsons r=0.9807, P < 0.0001) and diluted plasma (Pearsons r = 0.9681, P < 0.0001). REACT has the potential to provide rapid measurement of NNRTIs in POC settings and may help to improve HIV treatment and prevention outcomes.

bioengineering↗

bioRxiv2023

Democratizing access to microfluidics: Rapid prototyping of capillary microfluidics with a low-cost masked stereolithography 3D printer

Microfluidics offer user-friendly liquid handling for a range of biochemical applications. 3D printing microfluidics is rapid and cost-effective compared to conventional cleanroom fabrication. Typically, microfluidics are 3D printed using digital light projection (DLP) stereolithography (SLA), but many models in use are expensive ([≥]$10,000 USD), limiting widespread use. Recent liquid crystal display (LCD) technology advancements have provided inexpensive (<$500) SLA 3D printers with sufficient pixel resolution for microfluidic applications. However, there are only a few demonstrations of microfluidic fabrication, limited validation of print fidelity, and no direct comparisons between LCD and DLP printers. We compared a 40 {micro}m pixel resolution DLP printer ([~]$18,000 USD) with a 34.4 {micro}m (<$380) LCD-SLA printer. Consistent with prior work, we observed linear trends between designed and measured channel widths [≥] 4 pixels on both printers, so we calculated accuracy above this size threshold. Using a standard IPA wash resin and optimized parameters for each printer, the average error between designed and measured widths was 2.11 {+/-} 1.26% with the DLP printer and 15.4 {+/-} 2.57% with the 34.4 {micro}m LCD printer. The average coefficient of variation [CV] was [~]2% for both printers. Printing with optimized conditions for a low-cost water wash resin designed for LCD-SLA printers resulted in an average error of 2.53 {+/-} 0.94% with the 34.4 {micro}m LCD printer and 5.35 {+/-} 4.49% with a 22 {micro}m LCD printer. We characterized additional parameters including surface roughness, channel perpendicularity, and light intensity uniformity, and as an application of LCD-printed devices, we demonstrated consistent flow rates in capillaric circuits for self-regulated and self-powered delivery of multiple liquids. In conclusion, LCD printers are an inexpensive alternative for fabricating microfluidics, with minimal differences in fidelity and accuracy compared with a 20X more expensive DLP printer.

scientific communication and education↗

bioRxiv2019

An enzymatic assay to measure long-term adherence to pre-exposure prophylaxis and antiretroviral therapy

Poor adherence to pre-exposure prophylaxis (PrEP) and antiretroviral therapy (ART) can lead to human immunodeficiency virus (HIV) acquisition and emergence of drug resistant infections, respectively. Measurement of antiviral drug levels provides objective adherence information that may help prevent adverse health outcomes. Gold standard drug-level measurement by liquid chromatography/mass spectrometry is centralized, heavily instrumented, and expensive and is thus unsuitable and unavailable for routine use in clinical settings. We developed the REverse TranscrIptase Chain Termination (RESTRICT) assay as a rapid and accessible measurement of drug levels indicative of long-term adherence to PrEP and ART. The assay uses designer single stranded DNA templates and intercalating fluorescent dyes to measure complementary DNA (cDNA) formation by reverse transcriptase in the presence of nucleotide reverse transcriptase inhibitor drugs. We developed a probabilistic model for the RESTRICT assay by calculating the likelihood of incorporation of inhibitors into cDNA as a function of the relative concentrations of inhibitors and nucleotides. We validated the model by carrying out the RESTRICT assay using aqueous solutions of tenofovir diphosphate (TFV-DP), a measure of long-term adherence to PrEP and ART. We used dilution in water as a simple sample preparation strategy to detect TFV-DP spiked into blood. The RESTRICT assay accurately distinguishes TFV-DP drug levels within the clinical range for adherence and has the potential to be a useful test to identify patients with poor adherence to ART and PrEP.

bioengineering↗