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Abstract

This dissertation presents a set of contributions to engineer a new technology for multiplexed fluorescence detection using a series of nanostructures designed to have unique photophysics. This approach can be easily integrated with existing bioassay platforms for next-generation molecular diagnostics, imaging, and biosensing applications, and improves upon their sensitivity, resolution, and multiplexing capabilities. Single-molecule approaches to biophysics and biomedical sensing have emerged as a crucial technique for uncovering the molecular heterogeneity in molecular samples. In principle, real-time multiplexed fluorescence detection at this level could provide vital insights into the stoichiometry, dynamics, and interactions of biomolecular species in complex mixtures. Yet conventional single-molecule fluorescence detection faces limitations due to low signal-to-noise ratios, spectral overlap, and issues with chemical compatibility, which typically limit the distinguishable colors to only three or four channels, severely restricting multiplexing potential for state-of-the-art assays. This dissertation will first provide a systematic review of modern single-molecule optical multiplexing strategies, emphasizing the strengths and fundamental limitations of each. Next, it documents the design, implementation, and characterization of FRETfluors, a novel class of fluorescence tags central to this dissertation work that employ Förster Resonance Energy Transfer (FRET) to enhance the number of uniquely identifiable spectral signals at the single-molecule level. These DNA-based fluorescent labels present an easy-to-manufacture yet spectroscopically varied solution for high-accuracy single-molecule detection. As a proof-of-concept, we show that detecting tag identity with the Anti-Brownian Electrokinetic (ABEL) trap enables the simultaneous identification of up to about 30 distinct molecular species at sub-picomolar concentrations in a mixture, a significant leap beyond state-of-the-art fluorescence multiplexing methods. Beyond engineering fundamental advances in single-molecule detection technology, this dissertation work lays the groundwork for future practical applications of FRETfluors to biomedical assays and imaging, including Enzyme-Linked Immunosorbent Assay (ELISA) and Points Accumulation for Imaging in Nanoscale Topography (PAINT). The incorporation of FRETfluors into ELISA significantly improves sensitivity and multiplexing capability, enabling low-volume, high-throughput biomarker detection. In DNA-PAINT super-resolution imaging, FRETfluors can facilitate multi-target labeling with fewer total wash rounds, enhancing the efficiency of live-cell molecular profiling. These advancements show the wide adaptability of FRETfluors, positioning them as a groundbreaking tool for next-generation single-molecule analysis, molecular diagnostics, and super-resolution imaging.

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