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.

One third of adults from ages 20 to 44 have untreated dental caries, typically treated through irreversible fillings and root canals (Centers for Disease Control and Prevention, 2012). Current technologies require professional consultation for caries detection – an inconvenient and costly process. Our objective is creating an optical system of detecting early caries formation to be performed at home on a weekly basis via spectral imaging of the plaque on teeth. The bacterial growth in plaque and demineralization of underlying enamel is detected by optically monitoring the fluorescent byproduct of bacterial metabolism, porphyrin. By imaging teeth in blue reflectance light and red fluorescence signal, concentration and growth of plaque deposits are measured using target versus background (T/B) pixel peak values (PPV) and T/B pixel count (PC) of PPV. Repeated measurements are plotted for plaque deposits and trends. Statistical analysis and comparison of T/B ratios and PCs trending can identify the most efficient measurement for locating plaque deposits most susceptible to caries development, prompting more efficient preventative care and triggering a dental examination. This new concept was simulated in vitro using a dental training model of teeth, measured applications of red stain, image stitching software (123D, Autodesk), image analysis software (ImageJ), and a mobile phone. Red stain was applied in increasing concentration and volume to both occlusal and interproximal crevasses that would most likely be missed by brushing and flossing. At each time period, a series of 2D images were acquired to reconstruct 3D models of simulated bacterial growth and were processed using ImageJ for T/B ratios of PPVs and PCs, which were plotted against each time period. T/B ratios of PPVs produced the most consistent measure of trending of the simulated growth, R^2 values 0.77, with statistically significant differences in variance with PC ratios (f-test).

This project aims to develop a better model for testing gene therapy vectors in vitro. Gene therapy uses a modified virus to insert a functional copy of a gene into a patient’s cells, but testing new viral vectors on 2D cell culture does not accurately predict success in vivo. To address this need, I have designed a 3D-printed microfluidic device for optimized viral vector testing. Cells are cultured in 3D in one channel, while media and viral vector load can be easily adjusted in a connected channel through a gravity perfusion system. The device is currently undergoing tests for cell viability and demonstrating its utility for gene therapy applications. The primary advantage of this device is that cells cultured in 3D mature more similarly to true skeletal muscle and thus are better models for predicting the success of a viral vector in a patient. The small size and fine tune control over the cell’s environment also save time and money during testing, allowing the maximum number of options to be explored. A new, physiologically relevant model for testing viral vectors would expedite gene therapy research to help bring treatments faster to patients in need of gene replacement therapy.

Approximately half a million mortalities occur in the United States due to cardiac arrest alone; only a few survive from the initial shock. To improve these chances, first responders must resuscitate patients before their arrival to the hospital for proper medical treatment. Unfortunately, none of the devices on the market can be deployed to guide resuscitation by generating useful hemodynamic information to the responder. Animal studies further simulate that obtaining hemodynamic data during medical procedures such as CPR provides optimum patient outcomes. We have created a non-invasive ultrasound-controlled system that measures flow patterns of oxygenated blood within the ascending carotid artery, as a means to guide resuscitation during cardiac arrest. We designed and tested our ultrasound transducers on a water-submerged string phantom that mimics the flow properties of blood. The transducer propagates a pressure wave towards the moving string, and then records the reflected and frequency shifted wave resulting from the Doppler effect. Once the data has been collected in MATLAB, we produce a graphical user interface (GUI) containing a real-time pulsed Doppler spectrogram displaying the blood flow speeds, which are proportional to the Doppler-shifted frequencies. Our team collected data from multiple swine in-vivo to capture the flow rate in the carotid artery. We analyzed and compared the baseline, trauma, and resuscitation measurements to significant parameters such as heart rate and mean arterial pressure. Successful analysis of those pig studies will motivate retrospective human trials that, in turn, should provide sufficient results to motivate commercialization of this technology.