Aminoglycoside antibiotics such as kanamycin and neomycin have been known to cause severe hearing loss in some patients. The Rubel Lab in the UW Department of Otolaryngology has recently reported a molecule, termed PROTO1, which has been shown to protect cochlear hair cells from aminoglycoside induced degradation. However, due to the poor vascularization of the cochlea, it is difficult to deliver ototherapeutic drugs systemically, and thus other methods of delivery must be explored. This study reports on the development of a PROTO1-loaded hydrogel based on the amphiphilic triblock copolymer poly(ethylene oxide)-poly[(r)-3-hydroxybutyrate]-poly(ethylene oxide) (PEO-PHB-PEO). In this study, two cross linking mechanisms were explored. The first uses the oligosaccharide α-cyclodextrin to thread α-cyclodextrin onto the terminal PEO chains of the micelles resulting in a supramolecular structure termed Pluronic/aCD pseudorotaxanes. Hydrogels formulated in this manner showed controlled linear release of PROTO1 over 25 days but are difficult to manipulate and requires sonication and heating to achieve gelation. An ideal hydrogel would be able to undergo in situ gelation, in which the hydrogel components remain liquid prior to being introduced to the active site, upon which they immediately cross-link to form a hydrogel. In this work, we synthesize materials for an alternative approach that is based on crosslinked cyclodextrin-modified polymers. The synthesis and characterization of the polymer components are reported.

Paper-based diagnostic assays have become popular for their use in the detection of both pregnancy and infectious disease. The majority of lateral flow tests are run using a paper-like membrane called nitrocellulose on which liquid can flow using capillary action until it reaches the spot or line containing capture reagents. They provide an inexpensive, user-friendly alternative to expensive lab-based analysis. However, commercial paper-based assays typically only run single step processes that can lead to inaccurate results. Our goal is to create more sophisticated assays that can perform multi-step processes for higher performance using paper networks. In order to achieve this goal, we need to create paper fluidic valves to regulate fluid flow in these devices. For a simple shut-off valve, we came up with the idea of placing a sugar cube in a gap between two strips of paper so it would act as a temporary bridge and allow a certain amount of fluid to flow through until the sugar dissolved. This would enable specific volumes of fluid to flow through different channels -- something normally regulated by trained personnel operating pipettes in the lab -- with an untrained user merely having to add excess fluid to a single well. Dissolvable sugar bridges can serve as a method for manipulating fluid/reagent volumes within paper-based devices. We've been able to demonstrate and characterize their operation, including tunability using parameters such as geometry and composition. We've also demonstrated the utility of dissolvable bridges in the important context of automated delivery of different volumes of the same reagent (e.g. water) from a common source to multiple locations in an assay for simple device loading and activation. Sugar bridges have the potential to help bring advanced testing using paper networks to limited-resource settings.

While red blood cell transfusions require basic blood type matching (A+, B-, O+, etc...), effective platelet transfusions require complex histocompatibility matching, testing to make sure specific alleles of genes are the same between donor and recipient so as to prevent rejection; this is similar to the testing required for organ transplants. Currently, platelets are transfused from random donors until a patient shows signs of rejection, at which point a blood center must attempt to find a histocompatible donor out of its registry to donate on short notice. Using a canine model, the Slichter lab has created a treatment, through a combination of filtration leukoreduction to remove antigen presenting white cells (APCs) and irradiation to inactivate residual APCs, which may prevent transfused recipients from rejecting non-histocompatible donor platelets. In my research, I will focus on canine immune responses to platelet transfusions. The lab transfuses canines with mismatched, treated platelets and measures the recovery and survival of radiolabeled donor platelets to document rejection; antibody levels are also measured, to correlate decreased donor platelet recoveries and survivals as being immune mediated. I perform an Enzyme-Linked Immunoabsorbent spot (ELI-spot) assay to test the production of gamma interferon (IFN-g). IFN-g is produced primarily by activated T-h1 and CD8 lymphocytes as well as other cells such as macrophages and mast cells. As such, the ELI-spot serves as a direct indication of the very beginning of an immune response. My research aims to expand on the current antibody data and directly correlate the recipient’s response to transfused platelets with the results of the ELI-spot assay. This research allows a direct link between cell interactions and immune responses, which can then be compared to the donor’s platelet recovery and survival data to see if a correlation exists. This will allow us to better judge the effectiveness of various platelet treatments by identifying significant correlations between in vitro data and post-transfusion platelet responses. Not only can this lead to breakthroughs in platelet delivery, but the results may also be useful in detecting early organ transplant rejection.

Minimally invasive surgical (MIS) techniques, such as laparoscopic surgery (performed through small incisions using specialized tools), are an integral part of modern medicine and the future of surgery. Technologically advanced tools such as MIS robots are promising to replace or augment existing MIS manual tools. MIS robots improve on manual surgery, as they can incorporate increasingly sophisticated features such as force feedback for surgeons, automatic identification of diseased tissues, and partial automation of surgical tasks. These features can reduce patient pain, recovery time and thus healthcare costs by eliminating unintended damage to tissue and providing surgeons with tools to perform surgery more effectively. Each of these new features either requires or would greatly benefit from an accurate mathematical description of the dynamic mechanical properties for the tissues with which surgeons interact. Dynamic tissue property quantification requires that we apply a range of forces to the tissue and measure the resulting displacements. We have designed, built, and calibrated a device capable of collecting the necessary data to obtain an equation relating applied force to tissue displacement for any tissue of interest. We will process the data obtained from this device using an advanced signal processing and system identification algorithm (unscented Kalman filtering), which will evaluate the fit to the data recursively over time. This will allow us to maintain accurate results as the tissue properties change over time. This setup allows us to collect and analyze data from tissues including (but not limited to) in vivo abdominal organs in pigs. In addition to being an important study that has not been performed in the literature, the real-time collection and analysis of this data could have profound implications in partially automating surgery.

A reversible tissue adhesive is a surface that is capable of sticking to tissues repeatedly without losing adhesive ability, similar to how geckos' feet can adhere to smooth and rough dry surfaces. This project seeks to combine the reversible adhesion exhibited by geckos and the bacterium E. coli, a model for receptor-ligand tissue adhesives. This technology has the potential to benefit laparoscopic forceps designs, which are required to manipulate delicate tissues easily punctured or torn during a medical procedure. FimH is an adhesive protein expressed at the end of E. coli fimbriae that binds reversibly to mannose, a sugar expressed in human endothelial tissues. A FimH receptor-ligand adhesive has worked on small length scales (μm²), but adhesion fails at larger scales (cm²), possibly due to the propagation of a single crack at the adhesive interface. My project addresses this failure by 1) fabricating the geometry of the existing adhesive in PDMS allowing an interface size without damaging force instruments and 2) combining E. coli fimbriae with polymer molds of micron-sized pillar arrays that mimic the topology of gecko footpads and arrest crack propagation on dry surfaces. Spherical PDMS slices of increasing size (40μm – 300μm radii) were coated with mannose and brought down to fimbriated cell-culture plates. Results show that PDMS measures higher adhesion (226 nN) than the existing receptor-ligand adhesive (25 nN), indicating a successful increase in adhesive interface size. The PDMS surface can be used to measure and compare the adhesive capabilities of various micropillar array geometries coated with E. coli fimbriae. In conclusion, micropillar geometries that measure higher adhesion than plates coated with fimbraie have the potential to be developed into a tissue adhesive. Future studies may show reversible adhesion is achieved when fimbriated-micropillar substrates are brought in contact with cultured and dissected tissues.

A key issue in synthetic biology is the evolutionary robustness of synthetic circuits. Creation of robust circuits requires the development of design criteria to extend evolutionary timescales, and insight into the relative evolutionary fitness of different circuit designs. Experimentally, this research requires the use of multiple cell culture runs that are time-consuming and laborious. To accelerate this work, development of a high-throughput, automated, and parallel mini-chemostat and turbidostat device is proposed. While the development of nutrient-replenishing chemostat devices dates back to 1950, and the usage of such devices is well-documented, even modern chemostats are not well-suited to evolutionary experiments. Current designs face significant issues of equipment fouling, specialized complexity, and prohibitive cost. Presently there is a lack of cost-effective options for highly parallel milliliter-scale chemostat devices which can be applied to problems in synthetic biology. The proposed device is small and modular, utilizing multiple standard petri dishes for parallel culture experiments. The design features integrated media refreshment and temperature control mechanisms. Visible and fluorescent direct-view, real-time imaging systems are included. The device is constructed of readily-available parts to maintain low cost. Parts are modular such that used petri dishes can be easily removed, disposed of, and replaced. The design is scalable such that numerous devices can be stacked or tiled for parallel experiments. At present, design and fabrication of the device hardware have been completed, and the device has been verified functional for an operational period of at least 24 hours. Directed evolution experiments to measure of loss-of-functionality mutations in engineered E. coli will be conducted to assess live-culture performance of the device. An open-source software package and electronic microcontroller system are being developed to allow for automated real-time measurement and customization of growth and measurement parameters.

The Paul Yager Lab in the University of Washington’s Bioengineering Department has been researching the dynamics of fluids on two dimensional paper networks of nitrocellulose. Knowledge of this science will allow for the development of distributed diagnostics, akin to pregnancy tests, that can be used in remote settings without access to electricity or medical supplies. By testing the storage and flow of various proteins and carbohydrates on nitrocellulose, we are developing lateral flow enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of various diseases. My research involves the control of proteins once they are already on the nitrocellulose membrane. By printing proteins and carbohydrates in various conformations onto the nitrocellulose, we were able to test the effectiveness of our methods for delaying and mixing protein flow. We showed that you can delay protein flow by printing a wall of sugar around the protein that needs to dissolve before the protein can flow with the liquid. We tested the flow patterns of two consecutive patterns of proteins and found that we can predict the time and efficiency with which they arrive at the downstream mixing site. We also developed ways to effectively load up to 1µl of solution onto the nitrocellulose strips. This research will be useful in the eventual development of a working ELISA, when combined with other results from researchers in the lab.