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Tsai, Hsin-Yun

University of British Columbia

Förster resonance energy transfer (FRET) is widely used for developing fluorescent probes for bioanalysis and imaging. Typically, multiplexed sensing with FRET involves the deployment of multiple probes. In contrast, concentric FRET (cFRET) is a design motif that enables multiplexed sensing with a single probe. Prior to this thesis, cFRET probes had only been developed around metal chalcogenide quantum dots (QDs), which may limit the scope of biological applications. This thesis focuses on expanding the materials palette of cFRET, with the goal of exploring and evaluating new capabilities and enhancing its suitability for more diverse applications. To avoid metal chalcogenide QDs, an alternative cFRET probe was developed using poly(amidoamine) dendrimers labelled with a luminescent terbium complex (Tb). This material was functionalized with three types of dye-labelled oligonucleotides to establish a network of FRET pathways. The new cFRET probes were characterized, and a time-gated multiplexed assay for DNA targets was demonstrated. The time-gating offered also enabled the rejection of background autofluorescence from serum. A derivative FRET probe was compatible with a smartphone-based time-gated imaging device, suggesting potential applications in point-of-care diagnostics. Silicon QDs, also called silicon nanocrystals (SiNCs), are another potential alternative to metal chalcogenide QDs. The size and PL properties of SiNCs were characterized via spectroelectrophoresis and time-resolved and time-gated spectroscopies. SiNCs were then paired as FRET donors with a series of dark quenchers or a fluorescent dye for systematically studying energy transfer. The polydispersity of the SiNCs and resulting heterogeneity of PL properties made these materials non-ideal for cFRET. Lastly, metal chalcogenide QD-based cFRET probes were structurally integrated with a hydrogel toward sensing cell-secreted protease activity. The hydrogel mimics the extracellular matrix to provide a more physiological-like microenvironment for three-dimensional cell culture. The PL of the QD-based cFRET probes was characterized and compared between bulk solution and hydrogel environments. As an initial proof of concept, QD-based FRET probes were integrated with hydrogel and used to measure the activity of a model protease that diffused into the hydrogel. Overall, this thesis advances the concept of cFRET for bioanalysis and informs future research in this area.

Text

2024-06-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/85174

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/