Fluorescence has proven to be a robust and powerful method of analysis in numerous fields: forensics, pharmaceutics, geology, food science, and environmental sciences have all developed a large collection of fluorescence instruments and methods to overcome application-specific challenges. Biological applications saw the same development of a fluorescence toolkit with methods capable of multiplexing, in vivo sensing, or surpassing the diffraction limit. Using these new technologies, a new biomarker – microRNA (miRNA) – emerged as playing an important role in post- transcriptional gene regulation. Since then, miRNA have closely studied as early indicators of cellular diseases such as cancer. However, the several miRNA will need to be monitored if any useful information about how gene expression and cancer progression are related. This research is two-fold: 1) characterize the signal generation quenching by a biosensor developed in the Burrows group, and 2) reduce spectral cross-talk to increase the number of available colors for analysis.The development of sensitive, selective, and robust miRNA biosensors is a principle objective of the Burrows group. Our current understanding is that over- or under- expression of certain miRNA are indicative of the current state of a cell’s gene expression. However, most in situ miRNA detection methods are complicated by false signals arising from sensor degradation, off-analyte binding, and poor spatiotemporalresolution. The Burrows group initially developed a ‘Reporter+Probe Complex’ (R+P) miRNA biosensor that reduced degradation and off analyte binding. Chapter 2. of this dissertation focuses on the differences in analytical figures of merit when the using this biosensor in two opposing detection mechanisms: Signal generation vs. signal quenching. It was found that signal generation is slightly more sensitive, but the (R+P) biosensor (a signal quencher) had less off-analyte binding than the current ‘standard’ signal generation method, molecular beacons (MB). Furthermore, the comparison of theoretical thermodynamic (∆G) and equilibrium (K) values to experimental limits of detection (LOD) showed that the LOD could be further lowered by lowering the initial reporter and probe concentrations. However, there is a lower limit, as too dilute will eventually reduce the binding and total signal generated.Chapters 3. and 4. will discuss the development of an in silico prediction model and in situ experimental system that work together to reduce spectral cross-talk from samples with several co-localized fluorophores (dyes). CLEER (CoLocalized Excitation Emission Resolution) uses a custom MATLAB script to compare excitation-emission matrices (EEM) of dyes and generate a set of Dye-Specific Excitation Emission Coordinates (DyeSEECs). These coordinates are dye-specific excitation and emission wavelengths that are tuned in situ with a tunable ultra-fast laser and novel continuous variable filters (CVF). This research will show how some DyeSEECs are very effective at reducing cross-talk, while other DyeSEECs fail at this goal. The conclusion of this work will demonstrate how the fluorescent peak shape and emission filter cut-off profile play an important role in peak position accuracy, and how that eventually affects the spectral cross-talk.
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