Optimization of a microarray-based biosensor for detection of viral pathogens
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Rapid and sensitive detection of viral infections is of significant importance for improving patient care and containing outbreaks that threaten public health. Although there has been an enormous effort to develop point-of-care biosensors for viral diagnostics applications, sensitive, robust and easily portable platforms have yet to be realized. This dissertation focuses on optimization of a multiplexed immunoassay platform for viral diagnostics applications using a label-free optical biosensor termed Single-Particle Interferometric Reflectance Imaging Sensor (SP-IRIS). SP-IRIS utilizes an antibody microarray that captures the target viruses and an optical instrument that allows visualization of individual captured virus particles. Since this platform relies on capture of whole viruses, it is crucial to identify high-affinity antibodies that are capable of recognizing intact virions. For this purpose, we screened various antibodies for their performance on the SP-IRIS platform. By screening 43 different antibodies for three different viruses, we demonstrated specific and sensitive detection of different viruses and different subtypes of the same virus. This work allowed us to assemble an antibody microarray capable of multiplexed detection that has been tested in our laboratory as well as at two separate high-containment facilities. Next, we adapted a different antibody immobilization technique, DNA-directed antibody immobilization (DDI), to the SP-IRIS platform as a means to improve the sensitivity and robustness of the assay. First, we characterized the elevation of the antibodies conjugated to a DNA sequence on a three-dimensional polymeric surface using a fluorescence axial localization technique, Spectral Self-Interference Fluorescence Microscopy, determining the optimal length of the DNA linkers for SP-IRIS substrates. We subsequently showed the specific detection of Vesicular Stomatitis Virus (VSV) expressing Ebola glycoprotein on SP-IRIS platform using the DDI approach. We showed that DNA-conjugated antibodies improve the capture efficiency resulting in over a ten-fold improvement in assay sensitivity compared to directly immobilized antibodies. To demonstrate the feasibility of the DDI technique for multiplexed virus detection utilizing SP-IRIS, we used VSVs expressing Ebola, Marburg or Lassa surface glycoproteins and successfully demonstrated specific and multiplexed detection using a DNA microarray surface. We also combined this approach with a passive microfluidic cartridge, demonstrating the feasibility of SP-IRIS as a rapid testing technique that is well suited for point-of-care applications.