Recent outbreaks of infectious diseases underscore the urgent need for ultrasensitive, high-throughput, and versatile sensors to diagnose and contain them at their early stage, thereby mitigating their immense threat to public health and preventing disruption to social and economic growth worldwide. Optofluidic biosensors can offer highly sensitive single molecule detection and precise particle manipulation by combining optics with microfluidics in a single platform. A specially designed anti-resonant reflecting optical waveguide (ARROW) based optofluidic platform offers compatibility for enabling optical interaction with biomarkers and small molecules in their native low-refractive index fluidic environment. Further integration with highly selective bioassays, electronics, and signal processing techniques can improve the performance of these platforms. The goal of this thesis is to develop integrated optofluidic platforms for low complexity, remote-controllable, high-throughput, and ultrasensitive biosensing at clinically relevant biomarker concentrations. First, we explore the potential of integrating programmable fast electronics such as field programmable gate array (FPGA) for enhancing detection throughput of the MMI waveguide-based ARROW optofluidic platform. With this framework, salient experimental parameters such as target concentration and detection rate are extracted in real-time, demonstrating a highly accurate (99%) detection scheme with fluorescent nanobeads covering the entire clinically relevant range (femto to attomolar) of particle concentrations. Subsequent validation with real-time fluorescence detection of single bacterial plasmid DNA at attomolar concentrations indicates the platform's potential as a point-of-care diagnostic tool. Next, a cloud-based, Internet of Things (IoT)-enabled polydimethylsiloxane (PDMS) optofluidic platform is demonstrated for the remote operation of automated bio sample preparation and detection. This platform offers a user-friendly and intuitive workflow for on-chip liquid handling and serves as a valuable collaboration and training tool for remote access from anywhere across the globe. The rest of my thesis will discuss solid-state nanopores, essentially nanoscopic holes in thin insulated membranes, as label-free single-molecule analysis tools integrated with the optofluidic platform. When combined with a modified solid-phase extraction (SPE) bioassay, the platform offers specificity and amplification-free rapid biomarker quantification at ultra-low concentrations. The optofluidic platform allows optical trapping of target-enriched microbeads near the nanopore detector, followed by a thermal release assisting the electrophoretic target capture process. This optical trapping enhanced nanopore capture rate enhancement (TACRE) process demonstrates ~2000x enhancement factor compared to the diffusion-limited nanopore capture process. Also, this high-throughput method enabled the successful detection of SARS-CoV-2 RNAs from human nasopharyngeal swabs covering entire clinically relevant concentrations. Next, this method was modified to detect Zika and SARS-CoV-2 RNAs from non-human primate biofluids in a longitudinal infection study. This direct detection method demonstrates qRT-PCR-like performance without requiring any intermediate complex bioreactions. The versatility of the TACRE assay in analyzing six different types of biofluids and the practicality of this platform as a sensitive molecular diagnostic tool are also manifested. Finally, the integrated nanopore-optofluidic platform was utilized to characterize organoid-derived exosomes, an important biomarker for monitoring intercellular communication. This thesis concludes with a report on another application of the high-throughput and sensitive TACRE platform in monitoring the ENO-1 gene marker, a key regulatory enzyme in glycolysis, from organoid-derived exosome cargo, showing promise for further application in the clinical evaluation of cell growth and health in cell culture.
