Luminescent quantum dots have attracted great interest in recent years among biological researchers since they provide solutions to problems associated with use of organic fluorophores in cellular studies. Quantum dots show high photostability, high emission quantum yield, narrow and symmetric emission peaks and size-dependent wavelength tunability. The objective of my PhD studies was to develop CdSe/ZnS quantum dot-based probes and utilize them in cellular assays. The first phase of the work was to develop luminescent quantum dot fluorescence resonance energy transfer (FRET) based probes for protease activity. The probes were based on FRET interactions between quantum dots that serve as donors and rhodamine molecular acceptors that were immobilized to the surface of the quantum dots through peptide linkers, which contained selective enzymatic cleavage sites. Upon enzymatic cleavage of the peptide linkers, the rhodamine molecules no longer provided an efficient energy transfer channel to the quantum dots, which brightened the previously quenched quantum dots. The probes were applied to detect enzyme activity, screen enzyme inhibitors, and discriminate between normal and cancerous cells primarily because of the difference in the proteolytic activity in extracellular matrices. The second phase of my work was to take advantage of FRET and quantum dots to develop pH sensor. First quantum dots were modified with metallothionein (MT) to be water-soluble and biocompatible. The MT-coated quantum dots were labeled with Rhodamine through the formation of amide bonds with å-amine group of lysine in MT peptide to form the probes. FRET efficiency between quantum dots (donor) and rhodamine (acceptor) was pH dependent. The final phase of my studies focused on the first preparation of reversible quantum dot-based cellular probes for labile iron. The MT coated quantum dots was modified with EDTA to form probes. When captured by the EDTA molecules, iron ions quenched the emission of quantum dots. Removal of iron from the quantum dot surface by free EDTA or other iron chelators with higher binding affinity resulted in a rise in the luminescence of quantum dots. The analytical properties of the probes including sensitivity, selectivity, and reversibility were characterized. Intracellular assays in iron-enriched astrocytes will be carried out.

Proteases are essential for regulating a wide range of physiological and pathological processes. The imbalance of protease activation and inhibition will result in a number of major diseases including cancers, atherosclerosis, and neurodegenerative diseases. Although fluorescence resonance energy transfer (FRET)-based protease probes, a small molecular dye and other methods are powerful, they still have drawbacks or limitations for providing significant information about the dynamics and pattern of endogenous protease activation and inhibition in a single living cell or in vivo. Currently protease sensors capable of quantitatively measuring specific protease activity in real time and monitoring activation and inhibition of enzymatic activity in various cellular compartments are highly desired. In this dissertation, we report a novel strategy to create protease sensors by grafting an enzymatic cleavage linker into a sensitive location for changing chromophore properties of enhanced green fluorescent protein (EGFP) following protease cleavage, which can be used to determine protease activity and track protease activation and inhibition with a ratiometric measurement mode in living cells. Our designed protease sensors exhibit large relative ratiometric optical signal change in both absorbance and fluorescence, and fast response to proteases. Meanwhile, these protease sensors exhibiting high enzymatic selectivity and kinetic responses are comparable or better than current small peptide probes and FRET-based protease probes. Additionally, our protease sensors can be utilized for real-time monitoring of cellular enzymogen activation and effects of inhibitors in living cells. This novel strategy opens a new avenue for developing specific protease sensors to investigate enzymatic activity in real time, to probe disease mechanisms corresponding to proteases in vitro and in vivo, and to screen protease inhibitors with therapeutic effects. Strong fluorescence was still retained in the cleaved EGFP-based protease sensors, which stimulated us to identify the EGFP fragment with fluorescence properties for further understanding chromophore formation mechanisms and investigating protein-protein interactions through fluorescence complementation of split EGFP fragments. Through fusing EF-hand motifs from calbindin D9k to split EGFP fragments, a novel molecular probe was developed to simultaneously track the calcium change or calcium signaling pathways and calcium-dependent protein-protein interaction in living cells in real time.

Quantum Dot (QD) nanobeacons distinguish themselves from molecular beacons with the promise of non-linear activation, tunability, and multi-functionality. These unique features make them highly attractive for cancer detection imaging with opportunities for increased signal-to-background ratio and tunable sensitivity. In this thesis, a nanobeacon was designed to target matrix metalloproteinase-7 (MMP-7), known to be over-expressed by a wide array of tumours. The nanobeacon is normally dark until specifically activated by MMP-7. The overall design strategy links single QDs to multiple energy acceptors by GPLGLARK peptides that can be cleaved specifically by MMP-7. However, design details such as the choice of energy acceptor and conjugation method was found to drastically alter the function of the nanobeacon. Studies of nanobeacons synthesized with Black Hole Quencher-1 or Rhodamine Red by either covalent conjugation or electrostatic self-assembly revealed that peptide conformation and bonding flexibility are both important considerations in nanobeacon design due to QD sterics.

Quantum Dot (QD) nanobeacons distinguish themselves from molecular beacons with the promise of non-linear activation, tunability, and multi-functionality. These unique features make them highly attractive for cancer detection imaging with opportunities for increased signal-to-background ratio and tunable sensitivity. In this thesis, a nanobeacon was designed to target matrix metalloproteinase-7 (MMP-7), known to be over-expressed by a wide array of tumours. The nanobeacon is normally dark until specifically activated by MMP-7. The overall design strategy links single QDs to multiple energy acceptors by GPLGLARK peptides that can be cleaved specifically by MMP-7. However, design details such as the choice of energy acceptor and conjugation method was found to drastically alter the function of the nanobeacon. Studies of nanobeacons synthesized with Black Hole Quencher-1 or Rhodamine Red by either covalent conjugation or electrostatic self-assembly revealed that peptide conformation and bonding flexibility are both important considerations in nanobeacon design due to QD sterics.