Bioassay Development for Molecular Diagnostics on an Optofluidic Platform
- Author(s): Stambaugh, Alexandra
- Advisor(s): Schmidt, Holger
- et al.
Optofluidics, the science that integrates photonics and microfluidics, has produced a number of promising devices for biosensing and bioanalysis. In the past couple of years, several optofluidic approaches have demonstrated the capability to optically detect single biological microparticles. However, despite the sensitivity, many other requirements must be met to fully realize a molecular diagnostic device on a lab-on-a-chip platform. These requirements include target specificity, large dynamic range of detection, a low limit of detection, and multiplex differentiated diagnosis of many relevant biomarkers in a single sample. To this end, we have previously developed an optofluidic biosensor based on liquid-core antiresonant reflecting optical waveguides (LC-ARROWs). Orthogonally intersecting liquid and solid core ARROWs deliver the appropriate architecture for a highly sensitive, reconfigurable, and portable to the point-of-care device for ultra-sensitive detection of fluorescently labeled biomarkers in flow. We have recently introduced multiplexing capabilities into the ARROW-based optofluidic platform by integrating multi-mode interference (MMI) excitation waveguides, allowing for spectral and spatial multiplexing on a single compact chip. As a proof of concept, influenza virions were non-specifically labeled and simultaneously and differentially detected on chip. The goal of this thesis was to expand the detection capabilities of the MMI waveguide-detection platform via developing different specific bioassays to analyze different molecular biomarkers on chip and in clinically relevant environments and concentrations. First, a bead-based nucleic acid capture assay is described, which specifically captures nucleic acids onto an immobilization magnetic microsphere and functionalizes the complex with fluorescent dyes. The first simultaneous, multiplex differentiated detection of non-amplified total RNA samples from Viral Hemorrhagic Fever (VHF) infected cell cultures is reported. The four VHF samples detected are from the Zaire Ebolavirus (EBOV), Lake Victoria Marburgvirus (MARV), Ravn Marburgvirus (RAVN), and Crimean Congo Hemorrhagic Fever (CCHF). This assay is then expanded to specifically trap viral protein antigens, creating a specific bead-based protein antigen capture assay. This expansion enabled the target agnostic differentiated detection of both non-amplified Zika virus (ZIKV) genomic material and Non-structural protein (NS1) antigens. Target agnostic detection is a profound consequence for an optofluidic molecular diagnostic platform as few devices have the capability to detect the different viral biomarkers that may occur at different times after infection. However, this thesis continues to push the limits of sensitivity in the ARROW optofluidic detection platform, culminating in an ultra-sensitive single antigen detection assay enabled by a newly developed bright fluorescent probe. The probes were designed and synthesized in house from a fluorescently functionalized 1kB strand of double-stranded DNA. Their brightness and specificity to viral antigens enabled specific detection of single SARS-CoV-2 and Influenza A antigens from clinical nasopharyngeal swabs at a clinically relevant concentration of 30 ng/mL.