Research Projects – REU Program
Multiplexed microchip for neonatal sepsis
Faculty advisor: Dr. Cornelius F. Ivory (PI and team mentor)
For the past 10 years my research group has developed microchips for medical diagnostics, especially for cardiovascular diseases in collaboration with Prof. Wen-Ji Dong. A new collaboration with the University of New Mexico envisions a rapid, multiplex microchip platform for early detection of neonatal sepsis (NS). NS refers to a bacterial blood stream infection in newborns. Current tests for NS take several days and are prone to false negatives. The challenge here is in multiplexing the chip to detect two or more disease biomarkers from a heel-stick blood sample to improve accuracy. For neonatal sepsis, our triplex targets are the three most important disease biomarkers: calcitonin, MCP-1 and TNFR2. The guiding hypothesis underlying this project is that multiplex assays for a single disease will lead to more accurate diagnosis with fewer false positives and negatives. A secondary hypothesis germane to this project at this stage is that removing interferents from each of the target biomarkers, separating them from each other and concentrating them by several orders of magnitude will improve diagnostic accuracy.
The REU scholar will work as part of a team that includes a graduate student plus the co-PI to determine which separation/detection strategies can run simultaneously in a microchip format. Then, they will design, fabricate and test their chip, first using mixtures of model proteins, then blood serum and, finally, whole blood. We anticipate that the REU scholars will first try out their ideas at the bench, i.e., in slab gels, columns or capillaries, and then adapt their run protocols down to the nanoliter volumes typical of microchannels. Scholars will also be introduced to key engineering tools used in process scale-down. These include an array of software packages that aid design, e.g., SOLIDWORKS®, simulation, e.g., COMSOL®, and fabrication, e.g., 3D printing, as well as automation, e.g., LabView®. REU scholars will learn how to apply these tools to open-ended engineering design challenges.
Biomechanical modeling to estimate tissue loads related to neck pain
Faculty advisor: Dr. Anita Vasavada (Co-PI and team mentor)
Neck pain is a significant global health problem, especially in the workplace. Mechanical load and deformation in the spine have been linked to pain, and flexed postures increase the compressive and shear forces on spinal structures. The long-term goal of Vasavada’s lab is to develop mechanistic models to predict the potential of neck pain from posture and other biological and psychosocial factors. Specific to this study, our goal is to develop a relationship between posture and neck loads using biomechanical models. Dr. Vasavada developed the first and most complete musculoskeletal head and neck model that contained anatomically-obtained muscle architecture parameters. Using this tool, her lab found that postural changes associated with tablet computer use have significant biomechanical consequences. The REU scholar will conduct further studies to estimate compressive and shear loads on spinal discs.
The REU scholar will work with a graduate student to develop subject-specific biomechanical models based on subject size and posture from previously collected data. They will use engineering and mathematical methods including inverse kinematics, statics, multivariate regression and covariate estimation, and optimization algorithms to calculate compressive and shear on the spinal discs. They will use these models and tools to test hypotheses about the level of complexity needed in biomechanical models to predict how given postures and tablet computer use affect neck pain. Developing relationships between posture and tissue forces will contribute to the advancement of guidelines for the prevention of neck pain in the workplace.
Biofilms in the wound environment: Monitoring and detection
Faculty advisor: Dr. Haluk Beyenal (team mentor)
The role of biofilms in festering wounds has not yet been explored in great detail and the tools for detecting and monitoring biofilms grown on wounds are limited. Dr. Beyenal’s research group has developed many research tools for understanding biofilm processes at the microscale, including microelectrodes for exploring their chemistry and image analysis tools for monitoring their physiology. REUs will focus on imaging biofilms on model wounds and will monitor biofilm development over time using tools developed in Dr. Beyenal’s lab. REUs will work closely with Dr. Beyenal and his graduate students to learn how to grow biofilms, how to manufacture microsensors, and how to quantify a biofilm structure from images.
For this research, the REUs will use a newly developed electrochemical scaffold by Dr. Beyenal’s group (new references 1-3). We hypothesize that wound healing can be achieved by using electrochemical scaffolds and their effectiveness can be estimated by monitoring local, pH, redox and dissolved oxygen concentration near the wound surfaces. The planned experiments will be run in the laboratory using explant samples. The REUs will learn about how to commercialize the electrochemical scaffold. Finally, the REUs will participate in a week-long biofilm summer school organized by Dr. Beyenal’s group (http://www.biofilms.wsu.edu). This workshop will allow the REU scholars to meet industrial and academic researchers from all over the world. At the end of their training, the scholars will have a broad understanding of the tools used in identifying wound-related medical infections.
Highly sensitive bioassays for novel cardiac biomarker detection
Faculty advisor: Dr. Wen-Ji Dong (team mentor)
Understanding the molecular mechanism of cardiac regulation in healthy and diseased states is a major research thrust for Dr. Dong’s group. Heart failure (HF) is responsible for billions of dollars in healthcare costs with 50% of newly diagnosed patients predicted to die within 5 years. It is widely agreed that the pathology must be detected during subclinical progression for medical intervention to be effective. Mounting evidence suggests that the ultra-low ratio of circulating Troponin I (p-cTnI) to total Troponin I (cTnI) could serve as a cardiac biomarker to assist physicians in the predictive assessment of HF progression. Based on the results of current clinical studies, we hypothesize that once the heart enters into a pathological condition, the level of protein kinase A (PKA) phosphorylated cTnI will start to decrease. This reduction will continue as the condition worsen until it reaches a minimal concentration at which the heart will completely fail. Since no commercial assay able to measure p-cTnI at a clinical level is currently available, to test this hypothesis, Dr. Dong’s lab is devoted to developing a highly integrated sensitive device to measure the ratio of p-cTnI to total cTnI in serum at subclinical concentrations. A step to this goal is to integrate on-board purification and concentration of targets with iTIRF, an iPhone device, for rapid and ultra-sensitive quantification of this ratio in clinical serum samples.
Assisted by graduate students, the REU scholars in Dong’s lab will use the developed technique to investigate and quantify the interactions between cTnI isoforms and their antibodies. Ultimately, they will measure the ratios from blood samples from different patients at different stages of heart failure. Once the hypothesis is verified, more research will be performed to identify intervention pathways to increase the detection level of cardiac p-cTnI. REU scholars will learn various biochemical, biophysical, spectroscopic techniques as well as engineering fabrication skills. Furthermore, the REU scholars will attend journal club and participate in weekly lab meetings to present their experimental results.
Manufacturing bio-scaffolds for articular cartilage generation & osteoarthritis treatment
Faculty advisor: Dr. Arda Gozen (team mentor)
Dr. Gozen’s group focuses on additive manufacturing with functional soft materials. In collaboration with Drs. Abu-Lail and Van Wie, Dr. Gozen is working on a NSF funded project that aims at engineering articular cartilage (AC) for effective treatment of osteoarthritis (OA). Injury and degeneration of AC leads to OA in one of two Americans. In Gozen’s lab, REU scholars will be involved in the fabrication of bio-scaffolds to be placed in a novel centrifugal bioreactor to grow AC tissues in the presence of mechanical and chemical stimuli representative of the in vivo AC environment.
Bio-scaffolds will be manufactured via the direct-ink-writing (DIW) method that has been widely used for fabrication of artificial tissue scaffolds. Here, various nutraceutical hydrogel inks blended with bio-matter (articular chondrocyte, mesenchymal stem cell and growth factors) or rigid additives will be deposited layer-by-layer to form three-dimensional, composite structures mimetic of the AC anatomy. The REUs will work with a team including a graduate student and the mentor to test the hypothesis that the 3D-printed scaffolds can precisely mimic the compressive modulus gradients of the AC through control of the hydrogel ink composition and scaffold design.
The initial research tasks of the scholars will involve the design, construction and implementation of a multi-nozzle print-head for a direct-ink-writing platform which will be used to print multiple hydrogel inks. They will then take part in experimental studies where various composite test AC sub-cm scale three-dimensional hydrogel structures are fabricated and their mechanical properties are tested. Throughout these activities, the scholar will be trained on hydrogel ink synthesis, rheology and direct-ink-writing, fluid dispensing and valve systems, LabView-based automation control, and compressive mechanical testing of soft-materials.
Nanobiocatalysts in non-invasive diabetes detection
Faculty advisor: Dr. Su Ha (team mentor)
Design, synthesis and characterization of nanobiocatalysts (NBs) for detecting accurate levels of glucose in blood streams for diabetic patients is an important research thrust in Dr. Ha’s group. Implantable glucose bio-sensors show great promise for enabling non-invasive, portable wireless and continuous monitoring of diabetic patients. To effectively utilize enzymes for practical sensing applications, they need to be immobilized over suitable nanomaterials to form NBs. We hypothesize that nanomaterials with different surface properties significantly affect the nature and degree of protein-metal surface interactions, which determine both final enzyme activity and stability of NBs.
The REU scholar will team with a graduate student and Dr. Ha to test this hypothesis by determining how various nanomaterials with different physicochemical properties and immobilization strategies can influence both the activity and stability of glucose oxidase (GOx)-based NBs. Based on this interaction, activity and stability relationship, they will design and fabricate high performance GOx-based electrochemical sensor, first using model glucose solutions, then blood serum and, finally, whole blood. We anticipate that the REU scholars will first test out the hypothesis using a standard three electrode system and then adapt their ideas into a more sophisticated microchannel-based sensor platform. A key skillset for REU scholars will be nanomaterial fabrication, enzyme immobilization, enzyme assays and electrochemical characterization.
Protein engineering for molecular mechanisms of leiomodin-induced nemaline myopathies
Faculty advisor: Dr. Alla Kostyukova (team mentor)
The Kostyukova lab studies mechanisms that regulate actin cytoskeleton rearrangement in muscle and non-muscle cells. In muscles, alterations in thin (actin) filament lengths and architecture are linked to the development of myopathies. Recently, several pathogenic mutations in leiomodin (Lmod), an actin-binding protein, were identified in patients with extremely severe nemaline myopathy (NM). NM is a disorder that weakens skeletal muscles throughout the body but most severe in the muscles of the face, neck, and limbs. With the exception of one-point mutation, the identified NM-linked mutations are all stop codons or frame-shift mutations. All of the mutations affect the length of thin filaments. A mechanistic understanding of how Lmod functions at the molecular level will provide essential insights into how actin filament lengths are precisely regulated.
The REU scholar will team with a graduate student to study the effect of mutations on actin assembly. Specifically, we hypothesize that one-point mutations affect thin filaments’ lengths by changing Lmod’s interactions with its binding partners. To test our hypothesis, REUs will design and engineer Lmods mimicking NM-linked mutations. REU scholars will participate in the creation of constructs for protein expression; protein purification; protein primary analysis (structure, functionality and purity); and testing engineered Lmods in binding and polymerization assays. This process will allow the REU scholars to understand correlations between gene sequences, protein structure, structure-function relationships and molecular mechanisms of some myopathies.
Bioanalytical sensors for differential diagnosis for prostate cancer
Faculty advisor: Dr. Bernard Van Wie (team mentor)
A non-invasive bioassay for differential diagnosis of aggressive versus non-aggressive prostate cancer (PC) is challenging because of the vanishingly small numbers of prostate circulating tumor cells (PCTCs) in the blood. Proper diagnosis affects whether androgen deprivation is used (for non-aggressive PC) or whether PCTCs have shifted to an aggressive, androgen-independent phenotype where antibody therapy is needed. Dr. Van Wie’s group is pursuing micro-sensing technologies to detect PCTCs expressing prostrate surface membrane antigen (PSMA) versus those expressing hepatocyte growth factor receptor kinase, c-Met. Over the REU time frame, different students will test each of three hypotheses: 1) that dual ionophore ion-selective electrodes (ISEs) can be created to substantially shift the ISE voltage when PCTCs bind to ligands associated with the membrane half containing one of the ionophore types; 2) that an antigen specific lysis (ASL) technique can be developed in which PCTCs are coated with different protective polymers depending on whether ligands for PSMA or c-Met are used and that lysis of the remaining cell types will leave behind the polymer coated PCTCs which can then be counted; and 3) that novel sensor packaging will lead to practical devices and protocols.
The hypotheses will be tested by REU scholars as they design and run experiments to determine: 1) optimal ligand concentrations for detecting PSMA or c-Met expressing PCTCs; 2) optimal concentrations of antibody or ligand molecule, monomer, and colored reagents for distinguishing between PSMA and c-Met expressing PCTCs; and 3) methods to miniaturize hand-held ISE packages and inexpensive imaging microscopy for monitoring PCTC numbers.
|Mentor Photo||Mentor Name||Research Project|
|Cornelius F. Ivory||Multiplexed microchip for neonatal sepsis|
|Anita Vasavada||Biomechanical modeling to estimate tissue loads related to neck pain
|Haluk Beyenal||Biofilms in the wound environment: Monitoring and detection
|Wen-ji Dong||Highly sensitive bioassays for novel cardiac biomarker detection
|Arda Gozen||Manufacturing bio-scaffolds for articular cartilage generation & osteoarthritis treatment
|Su Ha||Nanobiocatalysts in non-invasive diabetes detection
|Alla Kostyukova||Protein engineering for molecular mechanisms of leiomodin-induced nemaline myopathies
|Bernard Van Wie||Bioanalytical sensors for differential diagnosis for prostate cancer