The effective treatment of individuals with HIV relies on maintaining therapeutic drug concentrations, necessitating accurate measurement of antiretroviral (ARV) drug levels. Current methods, such as liquid chromatography tandem mass spectrometry (LC-MS/MS), are limited by cost and accessibility. Our research addresses this gap by developing the INTEGRase activITY (INTEGRITY) assay for measuring integrase strand transfer inhibitors (INSTIs), a leading class of ARV drugs. This 2-step assay quantifies INSTIs using a DNA strand transfer reaction and quantitative polymerase chain reaction (qPCR). The presence of INSTI drugs disrupts the strand transfer reaction, inhibiting full-length target DNA formation, which is then measured through real-time qPCR. My work focused on optimizing the limit of detection of INTEGRITY by altering the strand transfer reaction conditions and protocol. Specifically, I conducted experiments altering INSTI drug concentrations and optimizing pre-incubation times of integrase with the drug to enhance the LOD. I observed that preliminary incubation of integrase and INSTI drugs for 5 minutes at 37 degrees Celsius improved the LOD of INTEGRITY by an order of magnitude. The simplicity of the INTEGRITY assay, utilizing standard laboratory equipment, holds immense promise for broadening access to routine clinic-based ARV drug level monitoring. This advancement has the potential to significantly enhance HIV care on a global scale by offering a cost-effective and accessible solution for monitoring therapeutic drug concentrations.

Lithium-ion batteries (LIBs) are vital energy storage devices for electric vehicles (EVs). Conventionally, LIBs have planar electrodes that present trade-offs between energy and power (charge/discharge speed) due to ion diffusion limitations. EVs require a high energy battery to enable long mileage ranges while also being able to charge quickly (< 15 minutes). 3D battery electrodes can potentially overcome this trade-off, achieving both high energy and power by leveraging 3D structures that create fast ion transport pathways. However, a scalable manufacturing process for 3D electrodes is needed. We are investigating processes for this, and we need a method to characterize our 3D electrodes. There is no method to automatically quantify the features within these 3D structures, which is required for rapid, high quality analysis. By accurately measuring 3D electrode feature sizes, correlations between features and optimal battery performance can be determined. We hypothesize that fabricating fine 3D features (order of 10s of microns) will improve battery performance. To address this need, I have developed an image processing script that characterizes 3D electrode samples. I investigate how threshold values improve accuracy in comparison to manual measurements and am able to achieve < 10% error. I also connect the code’s feature size measurements to our manufacturing process operating conditions to inform how manufacturing conditions can be altered to precisely control feature sizes, which impact battery performance. We expect that higher operating frequencies for our manufacturing process will result in our target fine feature 3D electrodes, achieving high-performance Lithium-ion batteries. This material is based upon work supported by the U.S. Department of Energy’s Office of Energy Efficiency and Renewable Energy (EERE) under the Advanced Manufacturing Office (AMO) Award Number DE-EE0010226. The views expressed herein do not necessarily represent the views of the U.S. Department of Energy or the United States Government.
 

With recent advances in wearable technologies, there is a growing demand for high power density batteries with more complex geometries. However, conventional battery manufacturing processes such as blade casting are incapable of producing the desired complex form factors. As an alternative manufacturing method, researchers are using additive manufacturing (AM), which allows for the rapid and efficient production of complexly shaped lithium-ion batteries (LIBs). A commonly used type of AM is direct-ink write (DIW) printing, a manufacturing technique where material is extruded through a syringe using displacement-controlled mechanisms. However, DIW typically lags from when pressure is applied to the syringe to when the battery material gets dispensed onto the surface. This lag can result in the printing of inaccurate features that negatively impact battery performance or even cause device failure. To account for this lag, we created a software module using the programming language C#, allowing users to print with micron-level precision. The module was integrated into the Rhino and Grasshopper platforms, a commercial computer-aided design (CAD) software package, enabling direct application into CAD models. The module can accept a list of curves as an input and will output a transformed list of curves that are more accurate to the CAD design. This module eases the challenge of printing material at the micron level, however, further research must be conducted to implement this module into lithium-ion batteries AM.

Soft electronics are small, flexible devices that have potential in next-generation applications such as wearable electronics, robotics and biomedical devices. Additive manufacturing (AM) has been demonstrated for fabrication of electronic circuitry and insulation, but some electronic components cannot currently be made with AM. These components are typically placed onto devices by hand. We aim to develop an AM process that uses a single motion platform to switch between printing circuitry and placing electrical components without the need for manual intervention. In the pursuit of full automation, we designed and built a pick and place (PnP) tool that is compatible with the Jubilee open-source 3D printing platform, and developed software to create integrated toolpaths. The Jubilee is the ideal platform for testing multi-material 3D printing in conjunction with PnP because of its ability to switch between tool heads mid-print. Our workflow for AM of soft electronics includes 3D printing of electrical circuitry via a material extruder tool, part placement with the PnP tool, and automated switching between the tools. We have programmed software that takes user input of starting and ending component location and creates machine code. The machine code tells the Jubilee where to go and when to activate or deactivate the vacuum system. We validate the functionality of our PnP tool and software by placing components in designed patterns. We then incorporate our PnP tools into the AM process to demonstrate fully automated fabrication of a simple circuit design. Integration of our PnP tool into the Jubilee open-source platform enables circuit printing and part mounting to be executed without manual intervention and is a step towards complete automation of AM for soft electronics.
 

As the world’s reliance on Lithium-ion batteries increases for technologies like electric vehicles, we need to improve battery performance. Traditional Lithium-ion batteries are composed of planar electrodes whose thickness can be optimized for energy capacity or charge rate (power). Thinner electrodes have a faster charge/discharge rate but low energy capacity while thicker electrodes have slow charge rates but higher energy capacity. Three-dimensional (3D) electrode structures that deviate from traditional planar electrodes can mitigate these trade-offs by allowing for fast ion transport while still maintaining a high ion quantity. One structure of interest due to its theoretical performance improvements shown in literature is a line patterned electrode. Line patterned electrodes have material and structural design features that greatly impact battery performance; it is therefore important to have methods to characterize and quantify features prior to battery testing. To rapidly and accurately analyze 3D line electrode feature sizes, we have developed an image processing code that analyzes cross-sectioned images of 3D line electrodes made from battery materials, enabling automated quantification of features such as line width, spacing and height. Cross-sectioned images are converted to black and white, which can then be processed by a function to detect the line edges and calculate the feature sizes. The results of our image processing code were compared to manual measurements to quantify accuracy. We draw connections between 3D line patterned electrode features and Lithium-ion battery performance to demonstrate how 3D electrode structures can be tuned to improve performance. This material is based upon work supported by the U.S. Department of Energy’s Office of Energy Efficiency and Renewable Energy (EERE) under the Advanced Manufacturing Office (AMO) Award Number DE-EE0009112. The views expressed herein do not necessarily represent the views of the U.S. Department of Energy or the United States Government.

Repeat thrombotic events have been associated with increased levels of Von Willebrand Factor (VWF) in patients prescribed dual antiplatelet therapies (DAPT), medications designed to prevent thrombosis. VWF is a protein that regulates platelet adhesion during hemostasis, allowing platelets to aggregate at sites of vascular injury. A microfluidic device containing a rigid block that simulates vascular injury and a flexible post to measure platelet contractile force through its displacement will be used to form shear-induced thrombi. I will determine how VWF affects platelet activation by testing whole blood samples and samples doped with DAPT (0.3 mM ASA and 10 μΜ 2-MeSAMP), 50 μg/ML VWF, or both VWF and DAPT. Platelet activation is measured through the area of platelet-plug, intracellular calcium levels, and platelet-plug contractile force. Preliminary experiments have shown that high VWF levels produced the largest platelet plugs whereas adding DAPT led to opposite effects. My results present that adding both VWF and DAPT to whole blood leads to similar platelet activation and larger platelet plug size as whole blood doped solely with VWF. The data obtained from this research can provide new insights into the improvement of therapeutic agents that aim to target VWF’s interaction with platelets and ultimately prevent repeat thrombotic events.

Heart disease takes an estimated 17.9 million lives each year, highlighting the pressing demand for cost-effective treatments. Melusin, a chaperone protein in the heart, holds potential as a target for heart failure therapeutics. Previous studies done in wild-type (WT) and melusin knockout (MelKO) mice discovered the absence of melusin was associated with a hypertrophic response indicative of heart failure. I plan to investigate the biomechanical role of melusin in humans using human-engineered heart tissues (EHTs) created from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that lack melusin and their isogenic controls. EHTs are a 3D in vitro model of the human heart, ideal for studying the role of melusin in humans. I hypothesize that WT EHTs subjected to mechanical stress will outperform the MelKO EHTs. The EHTs are suspended between one flexible and one rigid silicone post. The EHT displaces the flexible post as it contracts, from which the displacement can be measured to calculate various auxotonic properties of the tissue. To induce mechanical stress on the tissues, I use a brace to restrict the movement of the flexible post. I am using histology to determine if there are any morphological differences between tissue types resulting from the brace. Thus far, I have cast WT and MelKO EHTs and completed twitch force measurements two and three weeks post-casting. Overall, I found the MelKO EHTs demonstrated lower contractile force than the WT EHTs. I plan to cast and collect more EHT data with and without braces in order to provide insight into the role of melusin in humans. Furthering our understanding of the heart’s mechanotransduction properties using EHTs is important in expanding our knowledge about the various pathologies of the heart. Ultimately, studying the pressure overload pathways involving melusin can lead to the development of future therapies for cardiovascular disease.

Heart disease is the leading cause of death in the United States. Due to the inability of cardiac tissue to self-heal, extreme cases necessitate heart transplants and most patients do not fully recover. A promising novel approach to create engineered heart tissue focuses on 3D extrusion-based bioprinting of stem cell-derived cardiomyocytes. Mature iPSC derived cardiomyocytes, the cells responsible for the contraction of the heart, do not proliferate. Therefore, the printed tissue construct must be created with the final desired cell-density de novo in order to mimic native cardiac tissue. Researchers need a viable method for extruding high cell-density bioinks to form functional constructs. In my research project, I am working  to generate 3D bioprinted cardiac tissues with high cell-density to measure both electrophysiological characteristics and contractile force output. The use of cell-only bioinks is atypical, and there is limited research in the literature documenting its properties. I measured the acute change in viability of NIH 3T3 cells extruded through a needle to investigate the effect of the needle’s hydrodynamic forces on the cells at high density. The data shows that cells extruded at high density maintain a high viability, and we observed strong structural cohesion in the extruded filaments. I optimized the extrusion parameters, needle diameter and flow rate, to create long-lasting filaments, and constructs remained intact over a 5 day observation period. However, we found they fail easily with agitation. Further work is needed to optimize bioink and conduct further studies using flexible posts to measure contractile force output and calcium imaging to determine the electrophysiological characteristics of our cardiac constructs. Quantifying these properties is critical to ensuring that constructs recapitulate the characteristics of native cardiac tissue. This research may aid in the development of engineered cardiac tissue for transplantation and drug discovery.

Every forty seconds in the United States, someone suffers from a heart attack or stroke. Heart attack and stroke can be caused by the blockage of blood vessels by small, transient emboli, or clots. The risk of these attacks increases with age, and there are sex and ethnic based inequalities in the prevalence of these thrombotic events. These emboli can form through various pathways. One factor that can affect platelet aggregation is Von Willebrand Factor, which acts as a scaffold for platelets to bind to, creating a stable clot. In our microfluidic devices, I have observed the detachment of small emboli from large platelet aggregations, which I believe can model transient thrombotic embolisms in the body. Von Willebrand Factor is of specific interest to me, as I have observed that higher levels result in larger platelet aggregations, and therefore more emboli detachment events. This project investigated the question: What is the composition of these detached emboli and what part does Von Willebrand Factor play in their makeup? I hypothesized that these emboli will have high Von Willebrand Factor content and a core of activated platelets surrounded by inactivated platelets, which is what allows for the emboli’s detachment and transient nature. I employed flow cytometry, which causes specific components stained by fluorescent antibodies like PAC-1 and P-selectin to light up. This allows me to determine the composition of the emboli in terms of activated platelets and Von Willebrand factor. If my hypothesis that the emboli have high Von Willebrand Factor content is correct, this could have significant implications for treatments for heart attack and stroke, as current antiplatelet therapies do not target Von Willebrand Factor. If we can create more efficient treatments by knowing what the treatments should be targeting, there are countless people whose health can be positively impacted.

Open-top light sheet (OTLS) microscopy enables the volumetric imaging of large tissue specimens for research and potential clinical assays. No destructive sectioning is required, allowing the tissue to be used for standard downstream assays (e.g. H&E and molecular analyses) after the 3D pathology process is completed. While our typical 3D pathology workflow uses many of the same reagents as standard formalin-fixed paraffin-embedded (FFPE) histology, including xylene and ethanol along with a food-grade cinnamon oil (ethyl cinnamate), we would like to show that our processes do not negatively impact the quality of molecular biomarkers in valuable archived clinical specimens (FFPE). In previous research, we qualitatively demonstrated that tissue morphology and immunohistochemistry markers were unchanged before and after our 3D pathology workflow. Here, we aim to quantitatively assess the effects of our processing methods on FFPE breast carcinoma tissues using standard ER and HER2 immunohistochemistry (IHC) and HER2 fluorescent in situ hybridization (FISH) analyses, as well as nucleic acid integrity metrics (RIN scores, RNA bulk yield, signal quality). We hypothesize that the molecular characteristics of our processed specimens are statistically equivalent to those of unprocessed specimens. To demonstrate this, I have obtained two adjacent 3-mm diameter punch biopsies from 26 archived (FFPE) breast specimens. For each specimen, one sample will undergo our standard 3D pathology workflow. I will then submit both samples to pathology labs for quantitative comparison of standard clinical biomarkers (e.g. HER2 and ER expression) and nucleic acid integrity metrics. We will demonstrate that our lab’s 3D pathology protocols do not negatively impact tissues in terms of molecular characteristics, which will be important for clinicians to allow our nondestructive 3D pathology methods to be performed on valuable archived tissue specimens, and for our methods to more-easily translate into standard clinical practice.

Flying insect robots (FIRs), owing to their minuscule weight and size, offer unparalleled advantages in terms of material cost and scalability. However, their size introduces control hurdles, notably high-speed dynamics, restricted power, and payload capacities. While there have been notable advancements in developing lightweight sensors, often drawing inspiration from biological systems, the challenge remains in executing controlled flight without external feedback. We introduce Tiny Sense, a novel avionics system tailored for FIRs, encompassing an integrated sensor package — an inertial measurement unit, a pressure sensor, and an optical flow sensor. Coupled with a Kalman Filter (KF), this system weighs a mere 78.4 mg, drawing 15 mW of power. This is lighter and more power-efficient than previous sensor suites of the same capabilities. Our system uses a global-shutter camera as an optical flow sensor to collect pixel intensities for accurate optical flow calculations at 100 Hz. We collected raw data from the Tiny Sense by attaching it to a Crazyflie quadcopter and tested the KF by comparing its results to the measurements from the Crazyflie. We will continue to integrate the Tiny Sense with sub-gram FIRs and are currently working on mounting it to a 74-mg RoboFly. Our sensor suite allows even smaller FIRs to be able to achieve autonomous control.

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

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.

Tissues like the meniscus, a wedge-shaped pad of connective tissue found in the knee, are fibrous and have complex architecture that regenerates poorly and undergoes active mechanical stimulation which modifies cell signaling and tissue health. In vitro models are beneficial for characterizing these interactions as they create a controlled environment where single variables can be altered. We previously used the J1 Mechanoculture bioreactor to apply strain on a fibrous polymer scaffold laden with primary meniscal cells and observed nonsignificant variances between testing groups with mock injury vs. no injury. Applied strain was modeled after physiological strain levels, ~10%. Based on the minimal changes in cell behavior observed in mock injury samples, it is likely that the mock injuries in conjunction with the applied strain did not induce comparable plastic deformation to that experienced post injury within the native meniscus. We hypothesize that increasing strain and applied force to achieve plastic deformation within the electrospun samples will create a fibrotic and apoptotic response like that in vivo. Ongoing work is analyzing how the bioreactor will interact with unaligned electrospun polymer samples with no cells present. This will demonstrate the optimal parameters to instigate a significant material response. By inducing significant changes to scaffold material properties and underlying structure, it is more likely cells with demonstrate fibrotic and apoptotic responses in vitro mimicking immediate cell reactions to meniscal injuries in vivo. This response will be assessed by assaying for fibrosis through αSMA activation and apoptosis by caspase-3 activation. On the conclusion of this study, we expect that greater applied stress and associated strain will cause more plastic deformation within the polymer scaffold. This can be applied to an in vitro meniscus injury model to better understand the response of primary meniscal cells to stress in an environment with disrupted mechanics.

It is well documented that women are predisposed to various musculoskeletal injuries, including hip labrum, Anterior Cruciate Ligament (ACL), and meniscus tears. Literature has detailed the regenerative potential of human mesenchymal stromal cells (hMSCs), and clinical trials have confirmed their medicinal application within tissue repair and healing, particularly in the musculoskeletal system. hMSCs are commonly used in the field of tissue engineering due to their immunomodulatory capabilities, differentiation capacity, and autograft availability. Across the lifespan, cells in male and female bodies experience varying levels of estrogen exposure, which elicits different responses. During in vitro cell culture, cells are typically exposed to sources of exogenous estrogens, including phenol red and fetal bovine serum (FBS). Although these additives are commonplace in the practice of cell culture, little research has been done to understand the impact of these additives on hMSCs in in vitro cultures, especially among female hMSCs. To investigate these cellular responses, we studied the impact of these estrogen-mimetic media components on cell proliferation and metabolism in vitro for hMSCs derived from male and female donors. Specifically, we investigated phenol red, which is shown to behave as an estrogen, and FBS, which naturally contains 17β-estradiol (E2). We hypothesized that estrogen-mimetic compounds would be associated with an increase in cellular proliferation and metabolism in a sex-dependent manner. These results clarify the response patterns of male and female hMSCs due to exogenous estrogen exposure, improving our sex-specific understanding of their potency for in vitro studies and regenerative medicine applications.