BME PhD Prospectus Defense - Elif Cevik

2:00 pm on Thursday, February 6, 2014
44 Cummington Mall, Room 203
Prof. M. Selim Unlu, BME/ECE (Advisor, Chair)
Prof. John H. Connor, MED
Prof. Ahmad S. Khalil, BME
Prof. Nese Lortlar Unlu, BME
Prof. Allison Dennis, BME

Title: A Universal Platform for Robust and Highly Sensitive Label-free Viral Diagnostics

Rapid and accurate diagnosis of viral infections is critical for limiting the spread of the infectious diseases, especially during sudden outbreaks of influenza and viral hemorrhagic fevers (VHFs). A number of different laboratory diagnostics tests can be used for clinical pathogen detection, including enzyme-linked immunosorbent assays (ELISAs) and reverse transcriptase-polymerase chain reaction (RT-PCR). While these tests are reliable, they require specialized equipment and personnel due to complex assay protocols and stringent sample preparation. Recent advances in interferometrically enhanced microscopy utilizing the IRIS (Interferometric Reflectance Imaging Sensor) have enabled the development of rapid and multiplexed assays for viral diagnostics using label-free protein microarrays. This technology achieves a high level of sensitivity through size based discrimination of individual captured viruses on an interferometric substrate without the need for secondary labels, thus reducing assay cost and complexity. Because IRIS allows for the quantification of individual binding events, it is compatible with low concentrations of analytes below the detection limit typical of ensemble-based methods. While preliminary data from IRIS assays indicates a lower limit of detection than ELISA assays, the endemic challenges associated with antibody immobilization, degradation, and storage obstacles that are native to all protein microarray platforms have limited the integration of this technology into robust microfluidic systems. Conversely, DNA microarrays offer high affinity binding, reproducible probe immobilization, and low non-specific adsorption on a robust platform capable of long-term storage. Additionally, because immobilized ssDNA probes are stable at temperatures up to 100C, conditions which rapidly denature proteins, they are compatible with the elevated temperatures required for the fabrication of robust microfluidic devices.

In this dissertation prospectus, I aim to develop a DNA-based universal platform for label-free, highly sensitive and multiplexed viral diagnostics utilizing IRIS. This platform uses a DNA-directed virus detection approach via antibody-DNA conjugates on a universal DNA chip. DNA chips can be stored for a long time and can be functionalized with proper antibody-DNA conjugates at the time of the assay, eliminating antibody degradation problem and providing a programmable sensor surface. My preliminary results indicate that the use of antibody-DNA conjugates enables noticeable increases in reproducibility and bound virus density compared to direct antibody surface immobilization. My dissertation will also focus on the sensor surface characterization using Spectral Self-interference Fluorescence Microscopy (SSFM), which determines the axial position of molecules on the sensor surface. This will help us understand the differences between the DNA assisted and direct antibody immobilization and engineer the sensor surface to achieve higher sensitivity. Such a chip will enable and accelerate the future development of simple, robust, and highly sensitive platforms for rapid detection of viral infections.