Clinicians in low-resource settings face several challenges when using nucleic acid amplification to diagnose disease at the point of care, because conventional methods have high costs per sample, require trained technicians, and require the use of expensive equipment which relies on grid-supplied electricity. The first step of diagnosis using nucleic acid amplification is release of amplifiable DNA from the cell. The AudioLyse is an inexpensive lysis device, which is powered by any audio device (such as a cell phone or any small mp3 player, both of which only require charging before reuse). Lysis takes just 10 minutes in a “set it and forget it” format. I show that the AudioLyse is capable of lysing methicillin-sensitive Staphylococcus aureus (MSSA). I compared the lysis of MSSA using the AudioLyse method, to lysis with the Bead Beater, a standard lysis technique. I found the AudioLyse effectively released amplifiable DNA from MSSA cells. I also tested the effects of glycerol and simulated nasal matrix, or SNM, on the lysis method’s ability to release amplifiable DNA. I found that high levels of simulated nasal matrix decreased the efficiency of amplifiable DNA release when both the bead beater and AudioLyse were used. However, we expect nasal swabs will be AudioLyse compatible, after dilution in lysis buffer. I found that the AudioLyse doesn’t perform well in releasing amplifiable DNA in viscous solutions. Glycerol was added to samples to test how the AudioLyse’s efficiency was affected. The AudioLyse displayed a statistically significant decrease in amplifiable DNA released in a 50% glycerol solution, releasing just 7.8% of the amplifiable DNA the bead beater was able to release. As of now, the AudioLyse is an efficient device in terms of releasing sufficient amplifiable DNA for disease detection, but remains limited by the viscosity of samples. Future work will include testing the AudioLyse on other genera of bacteria.

Arrhythmias kill 300,000 people annually and effective cardiac drugs are imperative in their management. However, heavy reliance on in vivo testing results in low throughput and ineffective preclinical prediction of human response. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are a promising cell source for highly predictive, high-throughput in vitro testing. Unfortunately, culture conditions in current hPSC-CM assays leave hPSC-CM tissues immature, making them ineffective at modeling human myocardial drug responses. Tissue culture conditions that mimic the myocardial microenvironment promote the maturation of hPSC-CMs with higher efficiency than non-mimetic conditions. Taking advantage of this, our lab's NanoMEA system successfully melds biomimetically nanopatterned substrates with microelectrode arrays (MEAs) that perform high-throughput electrophysiological analyses of in vitro tissues. However, greater maturation efficiency is necessary to improve NanoMEA screening throughput. Electrically conductive environments promote hPSC-CM maturation, and numerous electrically conductive scaffolds exist. Yet, overlaying MEA electrodes with these substrates results in noisy signals that bar automatic processing. NanoMEA nanopatterns are made of Nafion, a nanoporous polymer that does not negatively impact the recordings from underlying MEAs. Since Nafion exhibits robust self-assembly with electrically conductive reduced Graphene-Oxide (rGO) molecules, we hypothesize that Nafion-reduced Graphene-Oxide (NrGO) nanopatterns will convey the benefits of conducting surfaces without impacting signal clarity. To investigate this, NrGO-nanopatterns are fabricated and characterized using Raman Spectroscopy, transmission electron microscopy, scanning electron microscopy, and conductive atomic force microscopy. Then, hPSC-CM monolayers are cultured on NrGO nanopatterns of varying rGO content and differences in morphological and transcriptional maturation markers are assessed. Promising NrGO nanopatterns are then integrated into a multiwell MEA platform to examine their effects on signal clarity and electrophysiological hPSC-CM maturation. If effective, we anticipate that this enhancement to the NanoMEA system will enable faster development of safer drugs, reducing animal model reliance, and improving the treatment of arrhythmias.

The study of neonatal neuroanatomy is a complex field because of the rapid changes occurring continuously. The physical development of tissues is critical to understanding the cognitive development of behavior and learning in the neonate. These structures can be visualized and analyzed with Magnetic Resonance Imaging (MRI). The hippocampal structure in particular is vital to memory consolidation and is therefore key to understanding how newborns build upon acquired knowledge and how this affects their behavior as they grow. This strucure has been studied at many different ages with parallel work analyzing how changes in anatomy result in observable cognitive differences and developments. This study in particular has a rare cohort of scans that were taken shortly after birth. Prematurity is an especially important factor to consider here as it affects physical and behavioral growth. Here we looked at 42 premature MRI subjects and used these to build a temporal model of brain development. Our primary aims of the study are to: (1) develop a protocol for labeling the hippocampus in premature neonate scans, (2) create a growth over time model of the structure in order to automatically segment a larger number of scans and (3) observe how volume varies with behavior as projected by Bayley's Scale of Infant and Toddler Development. So far aim 1 has been achieved and current work is increasing the previous atlas size of 32 scans to 42 in order to improve automatic segmentation. The overarching goal of premature neonatal MRI segmentation is to provide further data for early diagnosis of behavior abnormalities that could develop later in the subject. Results here could produce a meaningful indicator of cognitive development through MRI analysis as well as assist in improving the accuracy of MRI automatic segmentation algorithms. 

Premature births, or births before 37 gestational weeks, are associated with structural and functional abnormalities in the brain. Magnetic Resonance Imaging (MRI) is used to examine brain injury in neonates. Previous work demonstrated correlations between neonatal cortical lobes volumes from MRI and scores on the Bayley Scales of Infant and Toddler Development-Third Edition (Bayley-III), which measures cognitive, motor, and language skills of infants. The occipital lobe is important to diagnosis of visual and neurodevelopmental delays. In this work, I examined the relationship between occipital lobe subdivision volumes and Bayley-III scores. In order to do this, I designed a parcellation or labeling protocol that divided the occipital lobe into two regions based on the calcarine sulcus, the cuneus region and the lingual region. I applied the protocol to the 32 MRI training dataset. Using this parcellation protocol, I created a spatio-temporal atlas of brain parcels, which is a time and space model of brain tissue and parcel probability estimates. This atlas was used to validate our protocol using a leave-one-out validation and to automatically parcellate the larger dataset using EM or Expectation-Maximization labeling. The dataset contained 270 T1-weighted MRI scans from premature neonates who took the Bayley-III at 18 months. Once the larger dataset was labeled, I used multi-variate linear regression, normalizing for head size, to examine the relationship between occipital lobe gray matter volumes and scores on the Bayley-III and analyzed the growth pattern of the regions of the occipital lobe. Both regions showed symmetric and exponential growth patterns. Statistically significant correlations between the lingual region of the occipital lobe and Bayley-III scores were found. This suggests that regional occipital lobe volumes are associated with developmental outcomes in premature neonates.

The need for effective, on-demand contraceptives and protection against sexually transmitted infections is constantly increasing in our ever-growing society. We plan to contribute to this need by utilizing the barrier and drug eluting properties of a non-woven matrix of electrospun nanofibers made from the hydrophobic polymers polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA). Our experiments will demonstrate that nanofiber technology can be effective as a barrier contraceptive. This fibrous network will serve as a platform to sustain the release of the hormonal contraceptive Levonorgestrel (LNG), providing a chemical barrier against unintended pregnancy. We quantify in-vitro via optical microscopy how effectively the fiber inhibits penetration by observing its interaction with sperm. To optimize the physical barrier properties of the fibers we investigate sperm penetration as a function or fiber thickness and pore size in order to obtain an optimized barrier contraceptive of the thinnest fiber and highest protection. We then look at how well the fibers sustain the release of LNG in-vitro such that the contraceptive is eluted over a span of 7 days. We will also investigate materials that can be encapsulated within the fiber barrier to expand and block the cervical os (cervix opening) for ensured protection against sperm. This system will swell to many times its original size instantaneously, providing immediate protection for the user upon insertion. Beyond contraception, we plan to further understand the capability of these materials to impede the penetration of HIV. By utilizing drug-eluting nanofibers we can produce a platform that acts not only as a contraceptive but also as a means of preventing the spread of HIV.

Current synthesis for thermoresponsive magnetic nanoparticles such as iron oxide nanoparticles coated with poly(N-isopropylacrylamide), pNIPAm, usually requires extensive surface functionalization or high temperature procedure, and the yield is very limited. Here we present a simple and reproducible approach for the synthesis of thermosensitive iron oxide nanoparticles, via coprecipitation of Fe2+ and Fe3+ ions from basic aqueous solutions at room temperature in the presence of block copolymer, poly(N-isopropylacrylamide)-block-poly(acrylic acid) with a dodecyl tail following the acrylic acid block. The polymer facilitates iron oxide nanoparticle synthesis by forming micelles via hydrophobic dodecyl tail assembly and coordinating iron oxide formation via complexation with the poly(acrylic acid) segment. The block copolymers were synthesized through reversible addition-fragmentation chain transfer polymerization. Proton NMR characterization shows incorporation of 11 acrylic acid monomeric units to pNIPAm macro chain transfer reagent with 150 degree of polymerization. The new synthesis approach is rapid, taking one hour, and can be easily purified via size exclusion chromatography. The resulting magnetic nanoparticles’ surfaces display pNIPAm, which enables particle aggregation when the solution temperature is above 34°C. The particle aggregates exhibit high magnetophoretic mobility, which results in rapid and efficient magnetic separation, ≥95% in few minutes. The particle’s hydrodynamic diameter is ca. 30 nm, characterized using dynamic light scattering. This simple method overcomes the problem of using high temperatures while maintaining reproducibility, which could make it useful for larger scale synthesis.

Oxygen transportation in red blood cells occurs via a protein called hemoglobin. When hemoglobin is either absent, flawed, or impaired, the transportation of oxygen is hindered and this is known as anemia. Due to the prevalence and dangers of anemia, our goal is to effectively measure the concentration of hemoglobin in small volumes of a patient’s blood in order to diagnose critical levels of anemia. In order to do this, we take advantage of Beer-Lambert’s Law – a physical law that states concentration scales linearly with absorbance (a measure of how much light a molecule can absorb at a given wavelength). With the use of a microfluidic device, whole blood flows through an 8 mm x 250 μm x 50 μm channel and is illuminated by a white LED. A portion of the light is then absorbed by hemoglobin and continues at a reduced intensity to a green filter to focus our measurements to the visible green spectra, because it is known that hemoglobin absorbs the most light at about 540 nm, which is in this region. We demonstrate that our devices can determine hemoglobin concentration effectively with less than 40 μL of blood and at a low cost. The immediate impact of hemoglobin measurements of small volumes is its use towards children. The long-term impact is a diagnostic point of care device to solve global needs.

The majority of the population living with HIV resides in African countries where the cost-effectiveness and ease of implementation of the technology is imperative. Our goal is to develop an assay that eliminates the need for expensive nucleic acid sequencing technology, to test and diagnose drug resistance by detecting variation in nucleic acid sequence of the virus. One of the missing pieces that will allow us to reach this overarching goal is to design a method for isolating the viral particles in a manner appropriate for point-of-care application. Our approach, which will contribute to the assay being developed at the same time, is to use protein G magnetic beads, which are affinity molecules, to isolate the HIV virus from plasma sample. The protein G on the magnetic beads interacts with the coat antibodies on the viral particles and facilitate their removal from plasma. We show that the protein G magnetic beads have comparable efficiency at isolating the viral particles compared to the gold standard, Qiagen extraction, without the use of strong chemical reagents and complicated instruments. Yet, further work is needed to demonstrate that the viral RNA can be extracted starting from clinical plasma specimens with a protocol applicable in point-of-care settings defined by limited use of complicated insturments and cost effectiveness of the solution. 

While, according to the World Health Organization, the successful application of antiretroviral therapy reduced global deaths from HIV/AIDS by 24% between 2005 and 2011, a corresponding rise in viral resistance to these drugs has been measured. This rise is of particular concern in low-resource settings that are collectively burdened with over two-thirds of worldwide HIV infections while lacking access to robust diagnostic assays. In response to this need, the Lutz Lab, in collaboration with the Frenkel Lab at Seattle Children’s Research Institute and the Lai Lab at UW BIOE, aims to engineer a low-cost paper-based oligonucleotide ligation assay (OLA) for drug-resistant HIV. In order to lower the chance of user errors and reduce diagnostic turnaround time, it is desirable to be able to detect a set of six point mutations indicative of drug-resistance to first-line antiretroviral treatment in a single device. However, with current techniques we are limited to being able to test for one point mutation per device. To address this challenge, we have investigated a DNA hybridization method of OLA probe capture. Using this method, each point mutation is assigned a specific DNA “barcode” that could be used to generate spatially distinct capture lines in a diagnostic device. We have designed a set of six engineered barcodes that display highly specific and sensitive binding to their engineered complements. Furthermore, the performance of this DNA barcode system has been compared to barcodes constructed from non-natural nucleic acid analogs in order to develop a method that optimizes sensitivity and specificity of probe capture. The completion of this project brings the OLA one step closer to being able to make a previously expensive, technology intensive, and operationally complicated test, accessible to those in low-resource settings.

Due to the rise of pathogen genetic knowledge and implementation methods of diagnoses pathways for patients, an increase in access to speedy and efficacious therapies is needed. The development of inexpensive, high-performance point-of-care (POC) tests does not only improve healthcare in low resource settings, but also moves the diagnoses out of hospitals and into homes and primary care offices. Paper-based microfluidic devices help both identify and screen for pathogens by allowing for more rapid care and treatment. The multiplexed, autonomous, disposable nucleic acid amplification test devices fabricated in the Yager Laboratory performs sequential functions - sample preparation, nucleic acid amplification, and lateral flow detection – for target identification. My project aims to bridge the last two segments into a single unit by introducing a method of inhibitory competition that permits for measurable, fluorescent-based analysis of isothermal DNA amplification from dry reagents in porous media. To prove this concept, a simplified experimental model of a multi-region, paper-based nucleic acid amplification test was designed, and new interference amplification mixtures developed. The isothermal strand displacement amplification (iSDA) technique utilized in these devices incorporates a fluorescent hybridization probe that allows for the detection of the increase in amplicons using an optical detection method. By incorporating a dilution of comparable, secondary strand of DNA as an internal control, a competition for reagents is established which creates different chemical sensitivities in the amplification zones. As a result, target DNA from the pathogen is amplified against a competitive threshold in a range. In the long run, fluorescence imaging of all regions can yield a quantitative measure of the concentration of pathogens in a sample. Overall, this project tests the fundamental groundwork for a new family of optical-based, quantitative devices by testing the use of fluorescent detection for POC tests, and investigating the principle of inhibitory competition.

Macrophages are cells of the immune system that play key roles in surveillance, destruction of pathogens, and tissue repair. In the context of cancer, macrophages can infiltrate solid tumors and adopt an anti-inflammatory profile resembling that of the “M2” macrophage subclass. These M2-like tumor-associated macrophages (TAMs) promote tumor growth and metastasis by suppressing the immune response. Therefore, selective destruction of TAMs using a biomolecule that targets and binds to TAMs may have therapeutic benefit in cancer. We have previously identified a novel peptide, M2pep, that selectively binds to M2 macrophages over other immune system cells. In other experiments, a specific pattern of amino acids, named the M2pep motif, was observed to be critical for peptide binding to TAMs. This project seeks to improve M2pep binding and selectivity to TAMs by constructing and screening a library of peptides containing the M2pep motif for binding to M2 macrophages. The library was constructed by genetically engineering bacteriophages to display all possible peptides containing the M2pep motif. This was achieved by: (1) designing DNA sequences encoding for peptides with certain amino acid positions “fixed” with the M2pep motif, and the other positions randomized; (2) inserting these sequences into bacteriophage DNA; and (3) incorporating the engineered DNA into bacteria to generate bacteriophages that display the peptide library on their outer surface. Then, the peptide library was screened for binding to M2 macrophages, and the peptides that bound were sequenced. Several candidates were evaluated for M2 macrophage-binding and compared with M2pep to identify sequences with improved binding and selectivity. This method of peptide library construction and screening using a bacteriophage platform can be a powerful tool for future drug development, and enable further refinement of binding molecules with established binding motifs.

Macrophages are broadly classified as pro-inflammatory M1 and anti-inflammatory M2 macrophages. The majority of tumor-associated macrophages (TAMs) in tumors are known to express M2 phenotypes (M2-TAMs) which secrete cytokines and growth factors to promote cancer cell proliferation, tumor angiogenesis, and metastasis. Hence, depletion of the M2-TAMs is a promising adjuvant therapy to the current cancer treatments. Our lab has previously developed M2 macrophage-binding peptide (M2pep) which can bind to both M2 macrophages and M2-TAMs. However, in vivo targeted delivery of cytotoxic drugs to M2-TAMs using M2pep as a targeting ligand was limited by poor serum stability of M2pep. Previously, we engineered and tested different linear variants of M2pep; however, none of these was able to improve serum stability while retaining M2 macrophage-binding activity. In this study, we investigated cyclization of M2pep as a strategy to improve its serum stability since cyclic peptides are usually more serum resistant than their linear counterparts due to their higher structural rigidity. To synthesize a cyclic M2pep variant, the original M2pep was synthesized with two flanking cysteines on each terminus. These cysteines were then oxidized to form a disulfide bond yielding cyclic M2pep. The serum stability of cyclic M2pep was evaluated by incubating the peptide in serum and withdrawing aliquots of the serum at various time points for analysis by mass spectrometry. Compared to linear M2pep which was rapidly degraded in serum within 4 hours, the cyclic variant was detected even after incubation in serum for more than 24 hours. In conclusion, we have developed an optimized cyclic M2pep which exhibits enhanced serum stability while retaining M2-TAM binding activity. Our peptide can be utilized as a more effective ligand for targeted delivery of cytotoxic drugs to M2-TAMs. Future development of M2pep-based TAM-depleting therapeutics would serve as an effective adjuvant platform to potentiate current cancer therapies.

Gene delivery has successfully been accomplished with the use of polycations, both in vivo and in vitro. However, a major problem in this category is the inherent cytotoxicity associated with a high density of positive charges. Polymers that can be degraded within the cells are typically better tolerated compared to their non-degradable counterparts. This research studies the use of disulfide bonds to reversibly connect cationic polymer moieties to a stable polymer backbone. Reducible and non-reducible, statistical co-polymers of 2-aminoethyl methacrylate (AEMA) and oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA) were synthesized and evaluated for a comb-like, backbone chain. These monomers have been chosen due to their low cytotoxicity as well as their high degree of polymerization control in reversible addition-fragmentation chain transfer (RAFT) polymerization. Backbones containing 50 and 100 monomers per polymer have been successfully synthesized. The current phase of the project is to functionalize AEMA with a unique biomolecule called dibromomaelimide that contains two points for positively charged moieties. This final polymer will be used to transfect cells with genetic material to test its feasibility for gene delivery. Success of this project will create a well-defined polymer containing positively charged cations that are easily dispersed upon internalization by cells.

Cancer cells can grow into life-threatening tumors and metastases partly because of their ability to evade the immune system. Immunotherapies combat cancer by boosting the natural immune response towards cancer cells. Immunotherapies are often more successful than standard treatments like chemotherapy because they are more targeted and have longer lasting effects with milder side-effects. A key component of immunotherapy is the identification of ligands that selectively target immune cells for drug delivery or immunomodulation. Peptide ligands are of special interest because they are inexpensive to synthesize and can be identified by a library screening technique called phage display. The purpose of this project is to identify peptide ligands to address two needs in cancer immunotherapy. The first need is to enable selective depletion of M2 tumor-associated macrophages, which promote tumor growth and suppress the adaptive immune response. We successfully identified 6 peptide ligands that preferentially bind murine M2 macrophages over M1 macrophages by using phage display and Illumina next-generation sequencing. The second need is to develop technologies for T-cell based immunotherapies. T-cells are an effective target for immunotherapies because they kill pathogens and regulate short-term and long-term immune responses. The identification of inexpensive peptide ligands to specific surface receptors on T-cells will be useful in increasing accessibility and efficacy of these treatments. We applied the successful next-generation sequencing methods from M2 macrophage ligand identification to target the CD28 and CD3e surface receptors on T-cells.

Melioidosis, a disease caused by Burkholderia pseudomallei, is the third most fatal infectious disease in Thailand and causes morbidity and mortality in many other Southeast Asian countries. Due to fears that this bacterium could be weaponized, the US government lists this bacterium as a tier 1 select agent. Current treatments involve weeks of IV-delivered antibiotics and months of pills to prevent reoccurrence of the infection. Despite the availability of these treatments mortality can be as high as 40% in some locations. This situation calls for a more effective means of treatment to reduce loss of life, treatment time, and cost involved in fighting melioidosis. In order to develop a more efficient treatment, I created a novel delivery system to deliver large doses of antibiotics and, potentially, prophylactically load cells with therapeutic to prevent infection. I formulated nanoparticles using reversible addition-fragmentation chain transfer (RAFT) polymerization and microemulsion. The particles were first formulated using an inert monomer to test uniformity of the constructs and optimize the microemulsion procedure. Once this procedure was optimized, I added a prodrug monomer containing ciprofloxacin to the construct and the new constructs were characterized for size, shape, and the kinetics of prodrug degradation to its active form. Cytotoxicity to mammalian cells was tested with MTS assays using RAW 264.7 cells. Planktonic assays were also performed to test the efficacy of this novel delivery system to combat Burkholderia as compared to free ciprofloxacin.

Heart failure is one of the leading causes of morbidity and mortality worldwide. Heart failure (HF) is the inability of the heart to keep up with its workload, and at least half of HF patients suffer from decreased ventricular contractility or systolic dysfunction. Improving cardiomyocyte contraction is a potential therapy for diminished heart systolic function, but there is no current effective therapy that directly improves cardiomyocyte contraction. Previous studies from the Regnier Lab have shown that heart muscle exhibits a significant increase in contractility and force when naturally occurring 2-deoxyadenosine triphosphate (dATP) is used in place of adenosine triphosphate (ATP) as the substrate for contraction. Intracellular dATP levels can be elevated by the overexpression of the enzyme ribonucleotide reductase (RNR). In order to assess the efficacy of this alternative myosin binding substrate as a potential therapy, it is necessary to design a reliable and accurate method to quantify the dATP levels in cardiac tissue and/or cultured cells. The goal of this work was to design a protocol for tissue sample preparation to run using High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) for quantitative assessment of nucleotides. A standard curve was produced to allow for absolute quantification of the nucleotides. Finally, these methods were implemented to study the cardiac levels of nucleotides in a transgenic mouse that overexpressed the enzyme attributed for de novo dATP synthesis on dATP levels. Correlation of dATP levels and cardiac function can provide validation of the influence of elevated dATP to improve cardiac muscle contraction for use in studies of animal models of heart failure.

In diagnostics, the ability to detect small quantities of molecules with high sensitivity and specificity is crucial for a quick and accurate diagnosis of the presence of target antigens. The ELISA assay, which uses fluorescence to detect the presence of target molecules, is a standard diagnostic and research method for antibody-antigen detection. However, current ELISA methods do not have fast binding kinetics at low target antigen concentrations, requiring several hours for complete results. Therefore, there is a significant need for development of a simple and inexpensive method to improve the molecular binding kinetics in ELISA assays. My hypothesis was that rotational stimulation during enzyme incubation would greatly increase the sensitivity of the assay in a much shorter amount of time. To test this hypothesis, I compared the measured fluorescence of two different types of incubations – the traditional static incubation and a rotational incubation method. I measured the fluorescence generated by each set of assays at varying enzyme concentrations to determine the limit of detection, the minimum concentration that can be detected in a sample, for each method. I also analyzed the binding kinetics of each method using time-based data of each method. It is expected that the rotational method increases the sensitivity of the assay using half the concentration of target molecules compared to that of the static method. It also significantly decreases the time required to reach a binding equilibrium state by several hours. Promising results would suggest that rotational stimulation could significantly improve the molecular binding kinetics in diagnostic assays and could be implemented in future protocols to produce a more efficient diagnostic assay procedure.

Current drug delivery methods lack the ability to effectively deliver a drug to a targeted region and fail to escape from the endosome. The ability to gain greater control over a drug’s effective delivery would lead to an enhanced efficacy of existing drugs, decrease the amount administration sessions, and reduce the number of side effects. Nanotechnology is a means to address these issues as the unique quantum properties witnessed on this scale allow for enhanced targeting and greater control over the delivery of the targeted drug. However, biocompatibility and toxicity are concerns with many developments within nanotechnology. To address these concerns, we have designed a siRNA-aptamer chimera based on protein biosynthesized calcium phosphate (CaP) nanoparticles. This was done due to CaP’s well-known biocompatibility and biodegradability as the calcium phosphate dissolves within the endosome, therefore releasing the siRNA into the cytoplasm of the cell. Within this project, we have targeted cancer cells as a means to quantify the efficacy of our nanoparticles. Through engineering a dual-functional protein expressed in E.coli, we were capable of 1) biosynthesizing CaP nanoparticles utilizing the protein’s affinity to calcium phosphate, and 2) binding siRNA onto the designed protein for delivery, which is utilized to inhibit the translation of the targeted cancer cells. The biomineralized nanoparticles were characterized via DLS and TEM to reveal that we had synthesized uniform 100nm nanoparticles, a promising result which supports our goal of designing nanoparticles capable of establishing greater control over drug delivery. The completion of this project would entail the successful design of biocompatible nanoparticles that could be effectively used to silence cancer cells. As the protein’s affinity for a substrate can be manipulated, these siRNA-aptamer chimeras could see use with other drugs as well; effectively leading to a powerful and versatile drug delivery system.

A cell’s outer membrane displays a broad range of proteins and other biomolecules that play an essential role in mediating interactions between the cell and its environment. Some cell surface molecules, known as antigens, can be used by clinicians and researchers to identify specific cells for use in diagnostics and targeted therapies. Cell identification based on surface antigens is known as phenotyping, which is typically performed in the clinical laboratory using a flow cytometer. In flow cytometry, cells are labeled with fluorescently-labeled antibodies that bind to a specific antigen on the cell surface in order to identify the cell’s phenotype. Conventional platforms for flow cytometry are costly, and fluorescence-based phenotyping involves tedious labeling steps and advanced detection capabilities. For laboratory facilities without the resources or need for a flow cytometry platform, a method for simple, specific, and direct cell characterization is desirable. The Ratner Lab uses silicon photonic technology in biosensing applications to detect when mass binds to a sensing surface. In this project, the sensing region of the silicon photonic chip was coated with antibodies to target a specific antigen displayed by a cell. A silicon photonic sensor was functionalized to phenotype prostate carcinoma cell lines for prostate-specific membrane antigen (PSMA). The cells were fragmented to yield membrane microparticles that could be flowed across the antibody-coated photonic sensor in order to characterize the cells for the presence or absence of the cancer marker. This study employed model cell lines as a proof-of-concept for simple, label-free cell phenotyping, departing from traditional fluorescence-based methods for cellular analysis.

A barrier to quality healthcare in low-resource, rural areas is the lack of surgical care, and current anesthetic delivery devices either are expensive or require resources such as electricity. The Bioengineers Without Borders have proposed a low-cost, point-of-care anesthetic vaporizer, and seek a tool to assist in designing the device. To this end, a finite element model was constructed to predict the performance of a device in different operating conditions. We have imported the prototype’s geometry into finite element multiphysics software and added a domain representing the liquid anesthetic. Conservation equations and boundary conditions for mass, momentum, and heat transfer were applied to the domains of the device and numerically solved over clinically relevant time periods. Physical properties of the liquid halothane, stainless steel, and air were approximated using literature values of similar compounds. The model is currently being validated by comparing the vaporizer’s benchtop measurements of concentration and temperature to the computational model’s prediction. Computational results indicate that a vaporizer with no external heat transfer will experience a significant temperature and concentration drop, which in turn lowers the amount of anesthetic vaporized. When validated, the model will prove useful to engineers as proposed modification may be tested computationally before production, allowing for design refinement and less chance of device failure.

In the United States, kidney failure (end stage renal disease/ ESRD) affects more than 871,000 people and costs over 40 billion dollars for treatment. The majority of these ESRD patients can sustain their lives via dialysis, which requires blood access. Synthetic vascular grafts are one way of creating the access, but they oftentimes fail within the first two years, in part due to the lack of endothelial ingrowth into the vessel walls. Our lab is developing a synthetic vascular graft that rivals the performance of native blood vessels by promoting complete healing of native vessels (e.g. endothelial cells) into the graft. This pro-healing effect is made possible by a precision-engineered microporous structure. However, due to this device’s porous properties, a sealant is required to prevent bleeding (hemorrhage). In order to simultaneously prevent hemorrhages and promote endothelial in-growth during the healing process, I have engineered a series of cross-linked gelatin with tunable biodegradability, mechanical properties, and low toxicity. In particular, cross-linkers genipin and EDC/NHS were explored. The shear strength of the hydrogel was examined by coating the luminal surface of the graft with the gelatin, incorporating this model into a flow regime, and analyzing the resulting surface effects with Scanning Electron Microscopy (SEM). Toxicity was analyzed through mice fibroblast seeding on a cross-linked hydrogel, and biodegradability was estimated by soaking the gelatin in a polyphenylene sulfide (PPS) buffer solution (with a physiological pH of 7.4) and measuring the resulting Young’s modulus with a universal testing machine (UTM). While lower crosslinking densities have rendered a more rapid remodeling by the body, higher crosslinking densities tend to display more durable mechanical properties; the crosslinking density was optimized based on these trends, such that the gelatin can effectively prevent initial bleeding while maintaining the biodegradability to allow the native blood vessel to heal into.

Current chemotherapies exploit the larger pore size of tumor vasculature to reach cancer cells. However, this rudimentary form of targeting offers low selectivity and carries the risk of severe systemic toxicity. In addition, chemotherapy distribution to tumors is limited by the large distance between blood vessels in tumors, increased interstitial fluid pressure, and physiological resistance by the extracellular matrix (ECM). With the goal of addressing these limitations, researchers have developed drug-bearing polymers of various sizes and architectures that selectively target tumor cells. The Pun lab previously produced a novel drug delivery polymer, the sunflower polymer, that can be utilized for anticancer drug-delivery. The sunflower polymer is a macrocyclic brush polymer that can be functionalized with a folic acid targeting ligand and an anticancer drug, doxorubicin, that is released in the acidic tumor microenvironment. In order to maximize the potential of anti-cancer drug-delivery polymers such as this, the impact of polymer size and architecture on tumor penetration must be better understood. This is because the efficacy of any anticancer drug, regardless of its mechanism of action, is limited by its ability to navigate the tumor microenvironment and reach all viable cells in a tumor. This project seeks to discover how polymer size and architecture impacts tumor penetration by using a 3D in vitro perfusion chip model of the tumor microenvironment. The model consists of microfluidic channels seeded with KB carcinoma cells and ECM, and has been optimized using model polymers of polyethylene glycol (PEG) and dextran. Tumor penetration is studied by perfusing fluorescently labelled polymers of various morphologies through the seeded microfluidic channels. The information obtained from this project could potentially be used to optimize the size and architecture of drug-delivery polymers for tumor penetration. In turn, these optimizations could present a significant contribution to the development of anticancer drug-delivery polymers.

Highly controllable adhesives refers to adhesives that can be activated to attach strongly when needed but deactivated for easy and non-damaging detachment. There currently is a need to develop highly controllable adhesives that work optimally in wet environments, like those found inside the human body. Single fimH proteins, found at the tip of E. coli type 1 fimbria, form bonds with mannose that can be activated to bind strongly when pulled at high speeds, but are not activated under low speeds and as a result bind weakly. We hypothesize that thin-films composed of E. coli type 1 fimbriae can act as a nanoscale locking seatbelt, where they lock in at high speeds but not at low speeds, and that we can use mechanical force to control the adhesive behavior. Using atomic force microscopy (AFM) we tested the adhesive behavior between fimbriae thin-films and mannose covered surfaces. We observed that the force required to break the surfaces apart, as well as the number of activated bonds formed, increased the faster the surfaces were pulled away from each other. Also, by using the AFM to preload the bonds with high force, we observed that we could increase the number of activated bonds by a near 4 fold, as opposed to pulls with no preload. Inactivation after preload was also observed by allowing enough time for bonds to inactivate under no force, which decreased the bonds activated to only a 2 fold increase. These results show that these adhesive thin-films are capable of acting like a nanoscale locking seatbelt and can be controlled through mechanical force. An adhesive that can display these properties can have the potential to be incorporated into devices like pill endoscopy, giving an increased ability of controlled precise movement in fields like medical micro-robotics.

Optical coherence tomography (OCT) is an non-invasive imaging technique that is able to produce micrometer resolution images of human tissue. Building upon this imaging technique, the Wang lab has developed novel OCT based techniques to image the microvasculature of the human eye. With these high resolution OCT images, it would be possible to provide quantitiatve measurements of ocular features that can assess eye diseases non-invasively. These measurements would include blood flow rate, vessel density, vessel tortuosity, fractal dimensions, etc. The goal in this research is to determine which of these physical parameters in the eye can provide objective measures that correlate well with the severity of diabetic retinopathy (DR), the leading cause of blindness worldwide. Specifically, this research focused on five quantifiable metrics - vessel diameter, vessel area density, vessel skeleton density, vessel perimeter index, and vessel complexity index - and their correlations with the progression of the disease. The main hypothesis was that at least one of these parameters would show significant correlation with DR progression.Each of these metrics were quantified from OCT images of patients with DR using semi-automated computer software. Then, statistical analysis was done to calculate the correlations of each metrics with disease progression. Finally, an evaluation was performed to see which of these objective parameters can serve as sensitive biomarkers, possessing the potential to be used in diagnosing or monitoring the therapeutic treatments of diabetic retinopathy. 

 Light Chain Amyloidosis (AL) is a disease defined by the deposition of aggregated immunoglobulin light chains in the form of fibrils in several organs. While these fibrils are insoluble, the soluble oligomers that precede them in the pathway involving unfolding and then aggregation of the protein are toxic to the cell. A novel secondary structure, alpha-sheet, was hypothesized to be the cause of toxicity. This structure is rarely seen in normal proteins, but it is populated in all peptides and proteins involved in amyloid diseases studied so far in our lab. In addition, the correlation between thermal stability and fibril formation has led to the study of the impact of amino acid mutations on the unfolding pathway. As is the case for most amyloid diseases, Light Chain Amyloidosis is incurable and fatal . The determination of specific patterns and mutations that destabilize the protein and lead to increased toxicity is important to better understand how to intervene. To this end, we are comparing two patient-derived light chains, one non-amyloidogenic and the other amyloidogenic. By analyzing their differences, we can determine regions of the protein that contribute to aggregation. We performed molecular dynamics simulations of each protein and analyzed their unfolding pathways and characterized the structure of the amyloidogenic interemediate associated with toxicity. The docking of pre-existing amyloid inhibitors developed in the lab to the resulting structures was done to determine a lead compound. Through rational and evolutionary design, several iterations were done on the lead compound and an inhibitor with an overall better binding score was developed.

Amyloid diseases like Alzheimer’s disease, type 2 diabetes, and Parkinson’s disease represent some of the most pressing concerns facing our healthcare system today. Human consequences aside, these disorders place an enormous economic burden on society; Alzheimer’s disease care in the United States alone costs upwards of $226 billion annually, and these expenses will continue to rise as the population ages. As such, substantial effort has been invested in the development of novel compounds that would halt or reverse the effects of these diseases. Our group’s previous research suggests that amyloid diseases, though caused by the selective dysfunction of different and unrelated proteins, share a common mechanism of pathogenicity, wherein misfolded proteins aggregate into toxic oligomers typified by novel α-sheet secondary structure. Synthetic peptides designed to adopt a structure complementary to this motif inhibit in vitro aggregation of several different amyloid systems. In this study, we show that the inhibitory effects of these molecules also extend into the realm of simple biological models, in this case inhibition of toxicity in neuroblastoma cells. We co-incubated IMR-32 cells with Aβ42 peptide (whose aggregation is associated with Alzheimer’s disease) and our synthetic α-sheet compounds for periods of twenty-four to forty-eight hours, then assessed cytotoxicity by a MTT cell viability assay. When compared with neat Aβ42, the designs were able to rescue cells from necrosis, significantly lessening detectable toxicity. While preliminary, these results suggest that our peptides may have promise as potential therapeutic agents for treating a broad spectrum of amyloid diseases.

Thrombotic microangiopathy is characterized by endothelial activation and microvascular thrombosis, and is associated with a group of life-threatening disorders, such arteriothrombosis, sepsis, and other small vascular diseases. With systemic inflammation, the arteriolar and capillary vessels are often found to be obstructed by platelet rich thrombi, leading to tissue ischemia and organ failure. Understanding the mechanism behind endothelial activation and induced thrombi formation during inflammation and vessel injury has important clinical implications that can lead to therapeutic development. Here, I developed a novel in vitro microvascular platform that mimics the in vivo arteriole architecture and function. This platform is generated based on a biocompatible polymer, polydimethylsiloxane, using injection molding. The device is lined with an extracellular matrix (ECM) using type I collagen for structural support. Human coronary artery smooth muscle cells (hCASMCs) are seeded within the bulk collagen to allow spontaneous reorganization and migration. A channel is created within the ECM using needle-based subtraction molding and is then seeded with human umbilical vein endothelial cells (HUVECs) to form an endothelium. Optimization was performed to provide consistent cell seeding of HUVECs within the channel, to allow cellular reorganization and alignment, and to form a functional small-diameter arteriole. We observed reorganization and alignment of both HUVECs and hCASMCs after 3 days of culture under continual flow. In addition, luminal patency was maintained with proper hCASMC contractile function. On-going studies include development of functional assays to recapitulate the inflammatory response and thrombi formation in arterioles. The study is expected to not only further elucidate the fundamental mechanism of arteriothrombosis, but to also provide a base platform to develop preventative or therapeutic medicine for those affected by these pathological phenomena.

The primary function of the kidneys is to filter waste from blood, and the kidney glomeruli are the major site for filtration. The glomerulus is a blood filtration barrier with low resistance to small molecules, but is relatively impermeable to macromolecules. Each glomerulus consists of a blood capillaries network with endothelial cells on a basement membrane, the other side of which is lined with specialized epithelial cells known as podocytes. The integrity and function of the glomerular filtration barrier is critical for kidney function, and disruption of this barrier correlates with kidney disease progression, and eventually kidney failure. Current research models for kidney disease include animal models, 2D, and 3D kidney epithelial cell cultures. However, animal models suffer from high cost and are too complex for studying detailed cellular interactions, while existing in vitro models lack the architecture and components to fully reproduce in vivo conditions. We propose a 3D microphysiological system that recapitulates the endothelial-epithelial interface found in the kidney glomerulus. We have developed a method to form independent microscale channels within a collagen I substrate, separated by a very thin layer of collagen. Collagen I is a native matrix protein that supports cell growth, invasion, and exchange of cellular signals. Primary endothelial and epithelial cells has been seeded into the channels to form closed lumina, whose overlaps form the endothelial-epithelial interface. However, culture conditions remain to be optimized to support both types of cells. Structure, morphology, and function of the interface are monitored by confocal microscopy, biochemical assays, and electron microscopy. When fully developed and validated, this culture system can be used to study the transport properties and injury responses of glomerular filtration. It can also provide a platform for drug screening to identify compounds toxic to the kidney, or therapeutic compounds for kidney diseases.

Bacterial endocarditis is an infection on the inner lining of the heart. When studying bacterial adhesion, researchers often ignore that blood is pumped through the heart in a pulsatile manner. Because of this, all studies have been done with constant flow. We hypothesize that pulsatile flow conditions play a critical role in this disease. We studied how Streptococcus gordonii, a strain of bacteria that is known to cause bacterial endocarditis, bound in constant and pulsatile flow. The rate of adhesion in both constant and pulsatile flow was measured at different shear stresses. When suspended in buffer, the bacteria bound at the same rate in both flow conditions. The overall trend showed the rate of bacterial adhesion was highest at 10 dyne/cm². At the shear stresses predicted to be present within the heart, 20 – 80 dyne/cm², only about 10% of bacteria bound as compared to the peak value. In a solution with 50% buffer and 50% red blood cells (RBCs), the percentage commonly found in the blood, there was a dramatic change in the trend of bacterial adhesion in constant flow. Instead of having a peak value, the rate of adhesion increased with the level of shear stress, allowing for a much higher rate of binding in the 20 – 80 dyne/cm² range. We were unable to repeat this experiment using pulsatile flow due to limitations in our flow device but this could mean a dramatic change in the trend observed in pulsatile flow as well. We have created a new device to further investigate our hypothesis and better simulate the conditions found in the heart.

Antibodies play a critical role in our health system, adding specificity to diagnostics and therapeutics with their strong binding capabilities. Unfortunately, the binding cannot be controlled over time, introducing contamination of the target and preventing further testing with the bound sample. We propose an alternative to antibodies: a switchable binding protein. This protein has affinity for its binding target which can be modulated by environmental change without directly altering the binding site, otherwise known as allosteric regulation. This presents the potential to later change the binding target to a medically relevant antigen. A binding protein FimH, found on the end of bacteria pili, was used as the scaffold for the design because its binding depends on a significant structural change. I predict that if this protein is locked in place with a cross-linker or covalent bond, which experiences a large structural change under a simple external stimulus, FimH binding could be precisely triggered. Inserting a disulfide bond or a photoswitchable linker are two methods I am investigating with their binding triggers being dithiothreitol (DTT) and ultra-violet (UV) light, respectively. The photoswitchable linker design is ideal because it would induce reversible binding, allowing for reuse of our protein. Using past structural knowledge, multiple mutation designs are being created and their binding tested under external stimulus. The design requires mutation placed far from the binding site in order to create this allosteric switch. Initial results indicate the design with a disulfide bond between residues 112 and 189 does switch binding affinity, triggered to unbind, when DTT is added. This research also has the potential to contribute a novel approach to regulating large proteins and add specificity in diagnostics by concentrating the target.

Many biological, socioeconomic, and cultural factors increase women’s risk of acquiring HIV, especially in low-resource settings. Currently, there aren’t any options that allow women to discreetly protect themselves from HIV acquisition. To address this, we developed a topical nanofiber mesh to be inserted into the vagina to deliver antiretrovirals for HIV prevention. Benefits of this dosage form include high drug loading, tunable drug release, and imperceptibility to users while protecting them from HIV over the course of a week. We designed a “fiber-in-fiber” system: the “burst” release nanofiber delivers an initial bolus of drug, while the “sustained” release nanofiber (polyester) prolongs drug release over a week. Nanofibers are synthesized via electrospinning, which involves exposing a polymer and drug solution to an electric field to create a nanofiber mesh. After polyester fibers are electrospun, they are micronized using a food processor and electrospun directly into the burst release fibers. The final product is a burst release nanofiber mesh that contains small polyester fiber particles. Initially, the polyester fiber particle sizes were large and variable, ranging from 8 to 2000 micrometers. After optimizing the micronization process, the median particle size was reduced to 179 micrometers. We compared this to the median particle size of post-electrospinning, which was 26 micrometers. The data indicate that smaller nanofibers are more likely to be electrospun into the fiber-in-fiber mesh. Thus, minimizing particle size is essential to achieve the desired drug loading. Furthermore, loading the micronized fibers with a fluorescent dye revealed that they are evenly distributed throughout the burst release fiber mesh. Recent experiments assessed the drug release profiles of this fiber-in-fiber system, which showed that it is able to sustain drug release. Evaluating the drug release benefits of this fiber-in-fiber system will allow this system to be applied to other topic drug delivery systems.

The Joint United Nations Program on HIV/AIDS (UNAIDS) reported in 2014 that approximately 36.9 million people in the world are currently living with HIV/AIDS. Many of these individuals reside in low and middle income countries where access to STD prevention methods is limited and risk of infection is high. Electrospun nanofibers are a promising drug delivery platform in development, with the potential to provide either pericoital or sustained protection at an affordable cost. Here, we fabricate and characterize nanofibers composed of polyvinyl alcohol (PVA) and agarose (AGR), a naturally occurring polysaccharide that is both mechanically strong and biocompatible. Previous studies in our group have reported challenges in electrospinning polymer blends from AGR and PVA, likely due to the high gelling properties of agarose. Subsequently, a non-uniform distribution of fiber diameter resulted in variable mechanical properties with an undesirable burst release of hydrophilic small molecule drugs (tenofovir, TFV). In this study, the electrospinning process was improved to achieve higher AGR loading in the nanofibers. We used a melt-electrospinning technique where the polymer solution was heated at a constant temperature of approximately 60°C, which is below the glass transition temperature of PVA. Constant heating of the polymer solution reduced AGR gelation and improved fiber throughput. The melt-electrospinning process enabled fabrication of nanofibers with up to 30% AGR content. Mechanical testing on the nanofibers revealed that the Young’s Modulus was approximately 100 – 200 MPa. Drug release studies revealed that AGR loading in the nanofibers did not significantly affect the burst release of TFV from the fibers. Fiber properties indicate that the PVA-AGR system must be optimized to further validate experimental results. In general, the melt-electrospinning process was key to enable the fabrication of PVA-AGR nanofibers with increased AGR content, which allows for enhanced characterization of these fibers as an antiretroviral drug delivery platform.

Recent disease models have shown that majority of sexually active individuals will acquire sexually transmitted infections (STIs) sometime in their lives. Compartmentalization of vaginal mucosal immunity, as well as the immunosuppressive immunity exhibited by the Langerhans cells in vaginal mucosal tissue, pose challenges in developing efficacious STI vaccines. To address both of these challenges, we are developing multifunctional nanoparticles for programming ex vivo dendritic cells (DCs), which subsequently can be delivered intravaginally to stimulate Langerhans cells (LCs) in the vaginal epithelial tissue. We designed our DC transfection nanoparticles using a polycationic protein called protamine to complex antigen-encoded DNA plasmids for antigen presentation, and a double stranded RNA adjuvant, poly(I:C), for provoking DC maturation. In determining the optimal formulation, nanoparticles were formulated at four different protamine to mock DNA mass ratios (P:D ratios), 8:1, 2:1, 1:1 and 0.5:1. The nanoparticles were analyzed for their size, stability and DNA loading efficiency. Integrating results from these studies, we determined that nanoparticles with P:D ratio of 2:1 is the best candidate since it is the smallest P:D ratio that yields nanoparticles with the optimal size for DC uptake, as well as high particle stability and DNA loading efficiency. Additionally, DC transfection efficiency of the nanoparticle formulated at this ratio is also discussed. Successful development of the DC programming nanoparticles will demonstrate the feasibility of developing novel DC-based intravaginal vaccine, which will enhance vaginal mucosal immunity and can potentially be translated to efficacious vaccines against various types of STIs.

Francisella tularensis and Burkholderia pseudomallei, the agents of tularemia and melioidosis, respectively, have been identified by the Centers for Disease Control and Prevention (CDC) as bioterrorism agents that could lead to mass casualties and severe threat to public health. These pathogens are highly infectious and aerosolizable intracellular alveolar pathogens that can cause fatal respiratory tract infections. The intracellular compartmentalization of these pathogenic organisms within alveolar macrophages is a significant barrier to bacterial clearance and contributes to their associated morbidity and mortality. Currently, there is no effective treatment to eradicate melioidosis and tularemia other than prolonged antibiotic therapy and even with several months of intensive antibiotic treatment, complete clearance of these pathogens is not guaranteed. Therefore, there is an urgent clinical need to develop new therapeutic drug nanocarriers that can deliver antibiotics intracellularly to alveolar macrophages to increase treatment efficacy of tularemia and melioidosis. The goal of this project is to design biodegradable and nontoxic polymeric nanoparticles with various drug release rates and drug loading densities to overcome pathogens’ drug resistance mechanisms. The nanoparticle scaffolds will be synthesized using Polysorbate 80 via Thiol-ene and Thiol-Michael “click” reactions to maintain biocompatibility while incorporating functional groups that are amenable to chemical modification for enhanced drug conjugation. Once the nanoparticle scaffolds are characterized, they will be conjugated to antibiotics such as ciprofloxacin via hydrolytically degradable ester bonds. Drug release profiles will be characterized in serum containing media via reverse phase HPLC to evaluate their efficacies. Once ciprofloxacin is conjugated to the nanoparticle scaffolds, in vitro studies will be conducted to evaluate the polymeric nanoparticles’ therapeutic efficacy and toxicity in co-culture macrophage models of bacterial infection. Development of these polymeric nanoparticles will lead to rapid clearance of tularemia and melioidosis with shorter antibiotic administration while reducing the chances of relapse and antimicrobial resistance.

Point-of-care diagnostic tests allow clinicians or care providers to make rapid, informed treatment decisions in settings where patient follow-up is unlikely or not an option (i.e. rural regions in developing countries). The Anisotropic Ratchet Conveyors (ARC) platform developed in our laboratory can transport small, individual droplets with a passive surface pattern and orthogonal vibration. In this work, a novel ARC system is characterized for future use in automated point-of-care diagnostic devices. Effects of rung radius on droplet transport were characterized with respect to the acceleration amplitude of the vibration required to initiate transport. The acceleration amplitude was significantly lower on the 1000 µm radius and 1500 µm radius rungs compared to the 500 µm radius rungs and slightly lower on the 1000 µm radius rungs compared to the 1500 µm radius rungs. Interestingly, the radius of the rungs was also observed to influence the natural frequency of the transported droplets. The results of this work will be applied to optimize droplet transport across functionalized detection regions on chip. Thus, ongoing work aims to functionalize the silicon dioxide rungs for immunosensing. In order to demonstrate proof-of-concept immunosensing in droplets, antibodies (e.g. antibodies targeting human immunoglobulin gamma - anti-IgG) will be bound to the silicon dioxide rungs using a linking molecule (e.g. silane-PEG-NHS or APTES) via micro contact printing, a common technique used to transfer proteins to a surface with elastomeric stamps. This process will be optimized to control the density of bound antibodies. Fluorescent microscopy will be used to verify and characterize the capture of fluorescently tagged target molecules (e.g. FITC-IgG) suspended in droplets transported on ARC tracks. Experiments will also be performed to characterize how antibody capture affects the velocity droplets transported on rungs of each radius group. The ability to capture target molecules suspended in droplets will present the potential of using ARC devices as a platform for rapid, efficient healthcare diagnostics.

In 2012, the World Health Organization (WHO) estimated that in low resource countries 4 in 10 deaths among children under the age of 15 were associated with infectious disease. Point-of-care diagnostic devices that can quickly and effectively identify the source of the infection will drastically improve patient treatment times increasing rates of survival. We therefore investigated whether traditional RNA handling techniques used in a laboratory setting are necessary for these point-of-care devices, by assessing the stability of RNA under various conditions. Using respiratory syncytial virus (RSV) as an example, we discovered that RSV can be thermally lysed without significantly degrading the integrity of the RNA. In addition, the use of RNase inhibitors or tRNA does not have a statistically significant effect on sample integrity. A comparison between RSV RNA derived directly from patient nasal swabs, with swabs containing purified RSV RNA, shows that RSV virions protect their RNA prior to lysis. Amplification by qRT-PCR, indicates that RSV RNA is directly amplifiable suggesting that RNases are inactivated at 95°C, and do not regain activity prior to amplification. Our data provides the necessary framework for performing point-of-care testing without the need for traditional RNA handling procedures. The results have important implications since we expect that the research concerning RNA handling and integrity will translate to other RNA based viruses which include Ebola haemorrhagic fever, influenza, hepatitis C, West Nile fever, polio, and measles. With recognition of these capabilities, we can implement minimal RNA sample processing into point-of-care devices.

Cardiovascular disease is the leading cause of death worldwide due largely to the inability of the heart to respond effectively to injury through tissue regeneration. Coronary artery narrowing initiates cessation of arterial blood supply to the heart generating an ischemic injury, characterized by low oxygen and nutrient supply and the accumulation of metabolic waste. Currently, research approaches to study ischemia rely on high-cost and low-throughput in vivo models. As such, there is a need for an in vitro platform for mimicking ischemia/reperfusion injury in a three dimensional cellular environment. The research and design pursued here addresses this deficit in modeling capabilities by developing an in vitro ischemic injury and reperfusion model utilizing engineered cardiac micro tissues. Cardiomyocyte microtissue particles (MTPs) are formed through centrifugation of a single cell suspension of stem cell derived cardiomyocytes without scaffold materials into polydimethylsiloxane micro-wells. Ischemic injury is induced in MTPs by exposing tissue cultures to mineral oil layers on top of small volumes of spent culture media to restrict oxygen supply and metabolic waste exchange. This simulated injury results in an observable reduction in live cells as evidenced by a decreased live cell fluorescence signal associated with duration of injury from a calcein and EthD-1 live/dead stain. Optimization of this staining protocol for characterizing the extent of the induced injury in an adapted plate reader format is currently underway. Thus far, percent of live cells has been shown to positively correlate with normalized calcein fluorescence intensity and current efforts are directed towards increased efficacy of EthD-1 dead cell marking and sufficient tissue infiltration of the stain. Implementation of this protocol will create a baseline metric for monitoring the state of the engineered cardiac tissue during injury and reperfusion and provide a foundation for development of an accurate and cost effective cardiac ischemia injury model.

Point of care diagnostics devices are aimed at addressing health disparities in low resource settings. They must be cheap, easy to use and offer quick results. Nucleic acids (DNA, RNA) can be used to identify infectious pathogens, and employ molecular diagnostic tools such as PCR to do so. PCR is a process which utilizes DNA Denaturation, where the hydrogen bonds that form between DNA and complementary strands break under various temperatures, forming to single strands of DNA. The melt temperature, the point where half of a given solution is single stranded DNA, and half of the solution is double stranded DNA, can be calculated by using fundamental thermodynamic equations. Our goal is to create a lab teaching module in which students can perform experiments measuring the melt temperature of various pieces of DNA with its complementary strand. Learning Objectives were produced, and a lab was designed in order to fulfill each objective. The module consists of a prelab portion, in which students review the basic concepts behind thermodynamics, read and analyze scientific papers, and calculate melt temperatures (by using thermodynamic values as well as industry standard software). The actual lab portion consists of an experiment which tests the effect of DNA length on melt temperatures. Students will then interpret and analyze the melt curves generated in lab. Lastly, during the post lab portion students will brainstorm future experiments and solidify their understanding of how thermodynamic tools can be used to measure various nucleic acid processes, namely denaturation.

M. tuberculosis (TB) infects one third of the global population and causes 1.5 million deaths annually. Most of the disease burden occurs in low-income countries. Although TB is curable, the lack of proper diagnostic tools in developing areas restricts treatment and propagates the spread of this highly contagious airborne disease. Currently, the gold standard for pulmonary TB detection is a sputum culture, which is not only time intensive, invasive, and requires a clinical setting with laboratory analysis, but also increases risk of transmission and is difficult to obtain. In collaboration with Dr. James Lai of UW Bioengineering and Dr. David Horne of UW Global Health, we are developing a noninvasive and highly sensitive TB test for point-of-care settings. We propose a sequence specific capture modality of TB transrenal DNA (trDNA) in easily collected urine samples. Recent literature has shown the ability to detect TB trDNA in patient urine samples, but the current methods target long trDNA fragments which are unlikely to pass through the renal system and lack both sensitivity and specificity. As a result from specific DNA capture and targeting short DNA fragments, the proposed protocol allows detection of TB trDNA fragments from urine at a thousand-fold lower concentration.

Vessels of different organs have heterogeneous permeability to serve distinct physiological functions. Differences in basal vascular permeability may result from heterogeneity in the endothelial cells that line the vessel walls, which are influenced by many factors—including differences in the complexity of cell-cell junctions and the expression of various diaphragms that span the cell body. In addition, many diseased states are often associated with elevated levels of permeability, including viral hemorrhagic fever and tumor-induced angiogenesis. To understand such fundamental heterogeneity of the blood vessels and further investigate the change of vascular structure and function in disease scenarios, we characterized the permeability of endothelial cells harvested from different areas of the body. Human primary cells—including endothelial cells from various organs and epithelial cells from the proximal tubules of the kidney (PTECs)—were seeded on gelatin-coated transwells. We measured transendothelial electrical resistance (TEER) using a Millicell ERS Voltohmmeter, and determined solute permeability by tracking the perfusion of fluorescent dextran across the cell monolayer over time. Immunostaining was performed to observe monolayer formation, while transmission electron microscopy (TEM) was utilized to observe ultrastructure. Preliminary TEM images demonstrate distinct morphological differences between endothelial cell types, specifically in the complexity of cell-cell junctions. Investigations into the impact of these morphological differences on vascular permeability are ongoing, and a definitive conclusion will be made upon the study’s completion.

Hydrostatic pressure is important to many vascular processes, including endothelial activation (inflammation), vasodilation, vasoconstriction and angiogenesis (branching of new blood vessels from existing ones). The problem of pressure regulation in in vitro studies is usually solved by syringe pump based systems. Harvard Apparatus provides a catalogue of “Constant Pressure Syringe Pumps,” the cheapest of which costs $4500. This is prohibitively expensive for many applications. Thus, there is a need to create an affordable device that can reliably regulate hydrostatic pressure. I present a syringe pump based system that maintains liquid pressure within a flow chamber at a fraction of the cost of commercially available devices. My pump was designed from open-source and off the shelf components, including a pressure transducer and Arduino Microcontroller, at a total cost of ~$350. I have tested the pump’s ability to maintain constant liquid pressure across a high-impedance microfluidic system and have shown that it maintains pressure at a user-defined set point between 0 and 70 cmH2O of gauge pressure. This device illustrates the power of open-source software and hardware, 3D-printing and rapid prototyping to allow labs to design customized hardware to meet specific research needs at a reasonable price. Once completed, I will use it to study the pressure dependence of angiogenesis and vasculogenesis (formation of new blood vessels by endothelial cells embedded in interstitium). We will vary hydrostatic pressure to optimize and document blood vessel network formation. This is relevant to studying embryonic development and tumor vascularization and to engineering grafts to repair the damage from myocardial infarction (heart attack). Other possible applications include eliminating the confounding variable of pressure variation in Doppler echocardiography or in studies of endothelial cell response to variation in microvessel geometries.

Extracellular matrices are more than structural scaffolds used merely to provide mechanical support to cell and tissue organization—rather, they provide physical and biochemical signals to change the cell structure and function. Here, my project is to investigate on the diffusion properties of human kidney extracellular matrix (kECM) and their impact on proximal tubular epithelial cells (PTECs) in an engineered 3D physiological system. Outcomes of the project include: 1) diffusion coefficients of dextran of different sizes in the matrices made from pure type I collagen, pure kECM, and mixtures of type I collagen and kECM (3:1, 1:1, and 1:3). 2) A standard protocol and an assistive Matlab program for diffusion measurement and vessel perfusion experiments. 3) characterization of structure, morphology and transporters (Immunostaining) of proximal tubular epithelial cells inside the flow-derived microphysiological system. I expect the study to provide supportive understanding of cell-matrix interactions and facilitate the further development of the bioengineered human kidney microphysiological system to study kidney function and response to drugs and toxins.

In developed regions, laboratory-based molecular assays are routinely performed to enable early diagnosis and improve patient outcomes. However, several key technology gaps prevent the widespread use of molecular assays in low-resource areas. For instance, the lack of trained laboratory personnel in resource-poor settings can limit their capacity to perform labor-intensive tests. Additionally, many commonly used enzymes (e.g. polymerase and ligase) require costly cold chain storage. To simplify molecular assays for use in sub-optimal laboratories, we are developing a dried reagent platform for drying, storing, and dispensing test reagents. Our current work is focused on optimizing the platform for PCR and ligation, which are key steps in the Oligonucleotide Ligation Assay (OLA)-based HIV drug-resistance test being developed by the Frenkel lab (Seattle Children’s Research Institute) and the Lai and Lutz labs (UW Bioengineering). Multiple formulas containing preservatives up to 10% w/v were evaluated based on their assay compatibility and enzyme-stabilizing effect during freeze-drying. We found that the presence of trehalose and poly(ethylene glycol) in the dried reagent mixtures best maintained assay performance for both PCR and ligation. Preliminary data also showed that ligation performance of the new formula with the dried reagent platform was comparable to the performance of freshly prepared mixtures and that the dried reagents remained stable after 28-days of ambient storage (<15% relative humidity, 20-25°C). Long-term storage studies with the preserved PCR mixture are still in progress but we have demonstrated that lyophilized mixtures stored overnight produce amplified products in the same range as those from freshly prepared PCR mix. Future work will aim to achieve dried reagent stability up to 3-months and incorporate the platform into the first prototype of our drug-resistance test. Ultimately, the platform will reduce user manipulation and assay preparation time for the OLA and could be expanded to simplify other complex molecular assays.

The plasma protein von Willebrand Factor (VWF) is secreted by vascular endothelial cells. VWF’s primary function is to recruit platelets to the site of blood vessel injury. The interaction between VWF and platelets happens through binding between the VWF A1 domain and the platelet surface receptor, glycoprotein Ibα (GpIbα). VWF requires shear to be activated. Recently, it has been shown that oxidizing agents increase the platelet-binding activity of VWF, although the exact molecular mechanism is not understood. To explore whether neighboring domains of A1 play a role in the oxidant-induced activation of VWF, we used a flow chamber microscopy assay. Two experimental constructs of VWF, one containing the A1 domain and the neighboring A2 and A3 domains, the second containing A1 and the neighboring D`D3 domain were expressed and bound to separate plates. One control construct containing only the A1 domain was also expressed and bound to plates. Then GpIbα coated beads were washed at different shears over plates that had either been pre-oxidized or not. The movement of beads was recorded. Subsequently, rolling velocities and total number of beads were measured and analyzed using MATLAB. The results showed that, at high shear, both the A1A2A3 and the D`D3A1 constructs were more active in binding to beads when oxidized compared to the unoxidized controls. The control construct did not show a difference in binding to beads when oxidized compared to unoxidized controls. These findings suggest that the A2, A3 and D`D3 domains could have an inhibitory function on the A1 domain and that oxidation removes this inhibition resulting in an increased binding activity of VWF to platelets. Understanding how oxidation alters the function of VWF domains is necessary to explain the increased risk of thrombosis in patients suffering of chronic or acute inflammation.

HIV remains a major global health problem due to high transmission levels and emergence of drug resistant strains. According to WHO, in 2014, sub-Saharan Africa had 25 million people living with HIV and 70% of new HIV infections globally. A high number of circulating drug resistant HIV remains undetected due to lack of affordable HIV genotyping methods. The lab based oligonucleotide ligation assay (OLA) is a highly sensitive method for detecting HIV drug resistance as an alternative to the expensive and complex complete-genome sequencing, but its reliance on moderately expensive lab equipment prevents its universal access.  Our lab, in collaboration with the Frenkel Lab (Seattle Children’s Research Institute) and the Lai Lab (UW BioE), focuses on developing a simplified OLA-based HIV drug resistance test. The ligation step of the OLA relies on the specificity of DNA ligase to target single nucleotide polymorphisms (SNP’s) that can confer HIV drug resistance. However, ligation currently requires a thermal cycler to cycle between 95 °C to de-hybridize target DNA and 37 °C for annealing of oligonucleotide probes to target DNA. Eliminating the temperature cycling component of ligation would be a key advantage because thermal cyclers are not widely available in low resource settings. We measured ligation at intervals from 25 °C to 70 °C and were able to pinpoint an optimal temperature range for isothermal ligation. We plan to further increase ligation yield, and therefore detection signal, by optimizing the concentrations of salts and cofactors, adjusting probe lengths, and modifying probe sequences.  With the development of isothermal ligation, the OLA will be further simplified for potential widespread use in low resource settings. This will allow HIV infected individuals to be accurately diagnosed and receive the proper line of HIV drugs, controlling transmission rates and reducing the evolution of drug resistant HIV within entire populations. 

In the United States, kidney cancer has consistently been in the top 10 most prevalent cancers with 62,700 new cases and 14,240 fatalities each year. Renal cell carcinoma (RCC) derives from the epithelium of the kidney’s proximal convoluted tubule, and comprises the overwhelming majority of all kidney cancers. A critical step in the formation and spread of all cancers, including RCC, is angiogenesis: the formation of new blood vessels. Cancerous cells secrete numerous signals to recruit endothelial cells to form new blood vessels; these vessels provide primary tumors with oxygen, nutrients, and a path to metastasize throughout the body. While cell and animal models have been utilized to study angiogenesis and test anti-angiogenesis drugs in RCC, successes fail to translate in clinical human studies. Because of the added complexity of the human body, a better model to represent human RCC is required to study angiogenesis in vitro. In order to address this need, we are developing a tissue engineered vascularized RCC microphysiological 3D system. Microfluidic devices will be utilized to recapitulate the angiogenic interaction between blood vessels and RCC cells. Sprouting from the endothelial-lined channel, simulating kidney microvasculature, toward collagen suspended RCC cells will be analyzed to determine the efficacy of our device. In addition, we have unprecedented access to normal and cancerous tissue sample pairs from the same kidney cancer patient at the UW Medical Center. Cultures from the tissue sample pairs will be utilized to validate the device and display differences in angiogenesis between RCC and normal kidney tissue. Tissue pairs were also utilized in qRT-PCR to determine up regulated pro-angiogenesis genes in RCC. Commercial drugs along with those currently in development or clinical trials will be administered to our device to determine their efficacy in preventing angiogenesis in RCC.

According to the World Health Organization, more than 100 million people annually drop into poverty because of health care related costs. Point-of-care (POC) diagnostic testing works to alleviate this issue. Point-of-care diagnostics is testing that delivers cheap, accurate, and timely diagnosis in the hospital, the field, the office, or the home. In this way, POC testing can provide vital health information in a way that reduces the burden of health care globally. The lab of Dr. Paul Yager has developed a POC diagnostic device known as the multiplexed autonomous disposable nucleic acid amplification test, or MAD NAAT, which returns results in less than one hour. This device uses a two-dimensional paper network (2DPN) to automate the fluidic steps of a nucleic acid-based diagnostic assay, which detects the presence of one or more pathogens (e.g. MRSA, RSV) in a patient’s test sample. The downstream region of the 2DPN uses nitrocellulose strips to support a visual, colorimetric detection system. The detection system produces a series of visible red lines on the nitrocellulose strip that indicate the result of the test and associated controls. My research is focused on the optimization of the detection system, specifically: increasing the visible intensity of these red lines without increasing device cost. I have investigated the effects of varying the geometry of the 2DPN through the detection region. The goal of this research is to obtain the most intense test line signal with the least amount of expensive reagents, at the lowest possible pathogen count, in the least amount of time. I have also used MATLAB to implement patient sample analyzations for the MAD NAAT device. These efforts support further development of an affordable POC device, which will improve patient health outcomes in communities that lack proper healthcare.

Studies have shown that Electrical Stimulation (ES), in the form of electric or electromagnetic field can enhance wound healing by promoting cellular proliferation and expression of growth factors. We hypothesized that ES mediated by negative electric charge is predominantly effective in inducing cellular rehydration of dehydrated mammalian cells, resulting in increased viability and survival. This was tested by analyzing the effects of ES induced in a cellular model through negative electric charge build-up in a parallel plate capacitor system. The system consisted of two identical gold-plated surfaces separated by two polytetrafluoroethylene pillars. Electric charge built up on the plate was manipulated by altering the voltage of the power supply connected in turn to the gold plated surface. Experiments were conducted on the commonly used standard cellular model of mouse embryonic fibroblast NIH/3T3 cells under sterile conditions to reduce extraneous variables. The strength of ES was predetermined through a quantitative and qualitative analysis of temperature, pH, and bubble formation in different standard solutions. Cells challenged by partial dehydration with an established range of sodium chloride concentrations will be subjected to the predetermined ES and transferred to different cell culture media- Phosphate Buffered Saline (PBS) or Dulbecco's Modified Eagle Medium (DMEM). Cellular viability will be analyzed by Trypan Blue dye-exclusion method. In this way, we will determine whether negative charge promotes viability.

Water forms liquid crystal structures near hydrophilic (water-loving) surfaces such as human cells . Compounds are excluded from dissolution in the zone containing these structures, and thus this zone is known as the Exclusion Zone or EZ water. EZ is believed to contribute to cell properties and is thus important for healthy cellular function - therefore, it is essential to try and maximize EZ content as much as possible. We evaluated the EZ content of various purported therapeutic or crystallizing waters using the well-established technique of using a microscope to determine the width of the EZ around a piece of tubing. We concluded that different types of water have varying amounts of EZ, with the highest being those observed in KW and Crystal Energy Water. The effect of probiotics, helpful bacteria which reside in the intestine and have varied health benefits, was also investigated. The most clinically studied probiotic, Lactobacillus rhamnosus GG, is known to have positive effects on various body systems, including digestive and immune. This was theorized to be a result of their effects on the EZ content of body water. We investigated the effects of the probiotic by varying its concentration in DI water. It was found that EZ dramatically increased in the presence of probiotic, with a 50% increase over control observed for the recommended dose of probiotic. As a control, we conducted a heat-response study by heating a solution of the probiotic (1 mg/40 ml) to 90°C, at which temperature the bacteria would be killed. The EZ greatly declined, suggesting a large role for live probiotic in EZ formation and therefore health. This research has wide implications for health, suggesting that changing the type of water ingested and taking probiotics could greatly improve cell function and overall health.

The cultivation of tomatoes is currently a 58 billion dollar industry worldwide. Over the past few years, tomato plants have been subjected to more severe and abrupt climatic fluctuations than ever before. In-depth understanding of the tomato leaf anatomy and physiology can have significant consequences in agriculture. Fluxes (fluid flow rate) in leaf water and sugar-conducting tissues, xylem and phloem, are challenging to visualize in real time. It is important to attempt this measurement using intact leaves as excision interrupts control of normal flow paths. The possibility that vein density and flux influence photosynthetic rate is being investigated here.  Optical Coherence Tomography (OCT) is a non-invasive imaging modality that performs high-resolution cross-sectional imaging, allowing to visualize the structure and motion in biological samples in 3D.  In this study, we have used a state-of-the-art OCT system with 7 μm resolution, operating at 1300 nm wavelength, to investigate leaf structure, venation and vein flow in live tomato leaves for the first time. The low intensity imaging beam (3 mW) is in the near IR spectral region and imaging is performed at high speed (4 ms/frame) hence, does not interfere with the leaf’s natural physiological processes. We have used Rutgers’ tomatoes for this study. To improve the image contrast in the leaf veins, we are investigating using dye and lipid particles to be taken up by the leaves

Duchenne muscular dystrophy (DMD) is an incurable disease affecting approximately 1 in every 3600 males in the United States that is caused by a genetic mutation leading to the loss of dystrophin expression in muscles. Dystrophin is a protein that links the cytoskeleton of muscle cells to the extracellular matrix (ECM), and a lack of dystrophin leads to extensive damage to muscle fibers and subsequent loss of muscle function. Proposed methods of treatment, such as direct stem cell injections, have not been effective due to poor cell viability and difficulties associated with the culture of muscle stem cells in vitro. However, the development of implantable, vascularized and functionally mature skeletal muscle tissue patches may be able to address some of these issues. These tissues are made by growing primary muscle cells on biodegradable nanopatterned scaffolds that have been functionalized with sphingosine 1-phosphate (S1P), a potent angiogenic and myogenic biomolecule. The nanopatterning of these scaffolds is designed to mimic the nanotopography of native muscle ECM such that the anisotropic alignment and maturation of cultured muscle cells is induced. With this combination of topographical and biochemical cues, there was an observed increase in the formation of mature, contracting myotubes and the presence of differentiated endothelial cells. Furthermore, increased expression of myogenic and angiogenic genetic markers were found in cells cultured on substrates functionalized with S1P. Current work is focused on the validation of these results with cells featuring a knockout of the S1P receptor 1 gene. Lack of this receptor should lead to S1P-functionalized substrates having little to no effect on cell differentiation and tissue maturation, thereby illustrating the importance of S1P on the observed results. With further research and development, these tissue constructs may be used in the future to treat chronic muscle diseases such as DMD.

Shear-enhanced adhesion is a mechanism in which binding is enhanced at high flow rates. Some species of bacteria within the genus Streptococcus demonstrate shear-enhanced adhesion under high flow conditions, which are present throughout the human body. The purpose of our research is to understand and compare the effect of constant and pulsatile flow in Streptococcus gordonii, a strain of bacteria that causes infections by binding to human platelets. Our research focuses primarily on cases of infective endocarditis, a life-threatening infection of heart valves caused by various strains of bacteria. Rates of incidence in a general population has been estimated between 2 and 6 cases per 100,000 person-years. In our model system, we utilized a flow chamber to mimic pulsatile flow conditions in heart valves, which oscillate between a low and high flow, with a maximum shear stress of 3 Pa. We took videos of S. gordonii, which uses a serine rich-repeat adhesin to bind to platelets, and determined the number of bacteria that bonded to the surface under constant low, peak, average, high, and pulsatile shear stresses. We analyzed the videos to determine the conditions that enhance adhesion and compared our results under pulsatile flow to constant flow for the same range of shear stresses. Our results indicated that pulsatile conditions promote low amounts of adhesion above 1-2 Pa, demonstrating that pulsatile flow does not explain how Streptococci bind to heart valves at 2-8 Pa. These results disproved our hypothesis that pulsatile flow enhances adhesion at 2-8 Pa. Further research may look to testing the mechanism behind which Streptococci bind to heart valves in bacterial lesions exposed up to 8 Pa in the circulatory system. By understanding the adhesive properties of S. gordonii, our research may contribute to the development of anti-adhesive therapies to treat bacterial endocarditis.

Current tissue-engineered vascular grafts (TEVG) lack physiologically relevant internal structural organization which limits their mechanical properties and contractility. It has previously been shown that through the utilization of anisotropically nanopatterned scaffolds, vascular smooth muscle cells (vSMC) can be manipulated to express enhanced contractile phenotypes. We hypothesize that by using vSMCs cultured on nanopatterned surfaces we can create a TEVG, which exhibits native internal structure, that has increased mechanical properties and contains vSMCs with increased expression of contractile markers compared to a graft which lacks internal organization. In this study, we developed a flexible thermoresponsive nanofabricated substratum (fTNFS) which allows for the fabrication of multi-layered cell tubes with well defined internal structural configurations and diameters. Using the fTNFS, individual cell sheets can be stacked into a multi-layered tissue construct which can then be wrapped into a cylindrical shape. The cell tube structure can then be stabilized using a hydrogel. Here, we fabricated multi-layered vSMC tubes with structural characteristics similar to native blood vessels. The TEVGs were then immaged using immunofluorescence microscopy for expression of contractile proteins. The  vSMC tubes produced in this project maintauned internal structures analogous to native blood vessels and expressed contractile proteins.

Past studies in the Pollack Lab have discovered the existence of negatively charged solute-free "exclusion zones" adjacent to hydrophilic surfaces in aqueous solutions. One of these studies has shown that radiant energy expands exclusion zones adjacent to nonmetal surfaces based on wavelength. Another study indicated that these zones also exist adjacent to metal surfaces, but with positive charge. As a result, it is expected that radiant energy will have an opposite effect and decrease the size of the zone. My project explores the effects of radiant energy on these solute-free zones next to metals. To conduct these experiments, an aluminum sample is cleaned and placed into a customized chamber containing a diluted microsphere solution. The sample is left in the dark for 5 minutes to allow for formation of the zone. After the initial 5 minutes, the sample is subjected to the desired wavelength and intensity of light. Pictures are taken at 5 minute intervals to compare the size of the exclusion zones over 20 minutes. Initial results show that lower wavelengths have a larger effect on exclusion zones. However, varying intensities with the same wavelengths tend to produce unusual results where some intensities initially decrease the size of the zone then later causes an increase. More trials are necessary to validate these preliminary findings. Generally, the trends show that radiant energy tends to have a negative effect on these metal exclusion zones. These findings will give way for more research into areas such as electrochemical potential and energy harvesting as we discover the reasons for this behavior.