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The focus of this thesis is to explore and use the benefits of time-varying excitation in fluorescence spectroscopy for studies of biomolecular dynamics. Two new techniques taking advantage of modulated excitation are presented. Also described are the first efforts in a project where single molecule FRET and multi-parameter fluorescence detection are used for characterization of the conformational dynamics of the retinoid X receptor (RXR). RXR is one of the most important proteins in the group of nuclear receptors. It is believed to be involved in many diseases and is hence most interesting as a potential drug target. Our study is at present at a very early stage and some sample issues are still to be resolved. However, single molecule measurements should give insights not attainable by previously applied ensemble methods and help explaining how RXR can regulate so many different processes. Long-lived transient states of fluorescent molecules can, because of their long lifetimes, be used to detect subtle changes in the microenvironment of the molecule. A method for determining the kinetic rates for transitions to and from such states by registration of changes in the average fluorescence intensity related to different modulation of the excitation source is introduced. It combines the sensitivity of fluorescence with the environmental sensitivity of the long-lived transient states and allows the use of slow detectors such as CCD cameras, making parallelization and imaging possible developments. The approach was experimentally verified by measurements of the triplet kinetics of rhodamine 6G (Rh6G) in aqueous solution and compared with fluorescence correlation spectroscopy (FCS). It should also be applicable to any other photoinduced transient states affecting the fluorescence intensity. A strategy to combine FCS with modulated excitation, in a way that allows extraction of correlation data for all correlation times, is presented. This enables the use of modulation to optimize the measurement conditions with respect to the photophysical properties of the dyes used. Measurements were made on Rh6G to verify the method. To illustrate its usefulness, it was applied to measurements of protonation kinetics of fluorescein at different pH. FCS with modulated excitation will most probably prove very useful in many future studies involving multiple kinetic processes occurring in overlapping time ranges. / QC 20101115

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Lab-on-a-chip devices are changing the way that medical testing is performed by allowing rapid testing with small samples. Optofluidic biosensors are a type of lab-on-a-chip device that use light excitation on a fluid sample. One such application of an optofluidic biosensor is a device that can detect antibiotic resistant bacteria by combining DNA from a sample with fluorescent beads, flowing that sample through a hollow channel, and shining laser light on the channel. If the bacteria tested for is present, the fluorescent beads will give off photons that can be detected as a positive signal. The main method for illumination for these devices has been coupling light through a fiber optic cable to a waveguide on the side of the chip. Though effective, this method is impractical in a real world setting such as a hospital due to the difficulty of aligning to the side of the device. One solution to this problem is the use of illumination from the top of the device. Top-down illumination allows for more alignment flexibility, but also introduces the risk of additional noise or false signal as extra light reflects of the device. This dissertation discusses the viability and development of top-down illumination for optofluidic biosensors. This includes the development of an anti-reflective layer compatible with optofluidic biosensors, comparison of top-down illumination to side illumination, and simulations of various methods of performing top-down illumination. Based on the research and findings discussed in this dissertation, it has been found that top-down illumination is a viable illumination method for optofluidic biosensors. Additionally, the use of a pattern of laser lines combined with a light blocking anti-reflective layer is the recommended method for top-down illumination.

An inexpensive detector for proteins is described. The detection technique was based on two-photon excitation intrinsic protein fluorescence using a visible 532 nm diode-pumped nano laser as the excitation source. Proteins that exhibit intrinsic fluorescence must contain at least one tryptophan, tyrosine, or phenylalanine residue in their amino acid sequences. The detector was characterized and was found to have a detection limit of 4 micro-molar for tryptophan, 22 micro-molar for tyrosine and 500 micro-molar for phenylalanine. Bovine serum albumin, a serum protein with 3 tryptophan residues in its amino acid sequence was also used to characterize the detector. It was found that the detection limit for this protein was 0.9 micro-molar. The detector volume was determined based on a photon counting histogram - a technique in fluorescence fluctuation spectroscopy. From the results of this analysis, the excitation volume was found to be 2.9 fL. With such an excitation volume, the detection limits were either within or below the atto-mole range.

Proteins are of primary importance to the structure and function of all living cells. Study of proteins relies on the ability to separate a complex mixture so that individual proteins can be more easily processed by other techniques. Since protein samples often exist at low concentration in a small volume, the trend in chemical analysis is toward micro total analysis systems (µTAS) or lab-on-a-chip devices. Among µTAS separation methods, the relatively new electric field gradient focusing (EFGF) technique has shown potential. It focuses and separates analytes based on their electrophoretic migration in an opposing hydrodynamic flow. The detection principles that are compatible with µTAS separation may not always scale down. This thesis represents the development of laser-induced two-photon fluorescence detection on a microchip separation device. This detection is based on excitation of native fluorescence of aromatic amino acids by simultaneous absorption of two photons. First, a compact two-photon prototype detector was investigated. Its sensitivity was improved after discovery of the source of the background and subsequent reduction of background levels. Simple CE separation on a square capillary was coupled to this detector to demonstrate its ability for micro-scale detection. However, this detector did not provide a way to view the location illuminated by a laser and was difficult to use for on-microchip detection. A two-photon microscope was constructed on the frame of a commercial Olympus microscope to solve this problem. The eyepiece of this microscope enabled viewing of the detection volume, and the removal of a glass compensator from the trinocular head allowed for UV detection. This detection system was carefully aligned and optimized before coupling to microchip CE. Two microchip substrates including poly (methyl methacrylate) (PMMA) and glass, and two chip layouts were explored for their compatibilities with the microscope detector. It was found that the PMMA chip with conventional chip layout was not suitable for two-photon detection; therefore, a novel chip layout on PMMA was designed. Through testing the new design, it was concluded that precise focusing of the laser was essential to successful detection on microchips. Although the precise focusing of the laser inside microchip channels was not achieved completely in the limited research period, it is believed that this new design should be an appropriate solution to coupling PMMA chips with the two-photon microscope. Finally, glass chips were employed to successfully demonstrate the detection of amino acids.

Protein structure affects the bioactivity and functionality of whey protein ingredients in food systems. Bioactivity of whey proteins and their derivatives are highly dependent upon primary, secondary and tertiary structure. The degree of denaturation of whey proteins is an important factor for determining how whey protein ingredients will perform in a food system. Several analytical methods have been developed to quantify protein denaturation of whey proteins. The goal of this project was to use a variety of analytical methods to quantify whey protein denaturation and to evaluate the correlation of denaturation to the functionality of whey protein powders. The objective of the first series of experiments was to compare three different analytical methods to measure denaturation of whey proteins in liquid whey obtained by various methods of separation and with varying degrees of heat treatment. A split plot experimental design was used. Raw bovine milk was skimmed and liquid whey was separated from the skim milk at natural pH. Three separation methods: 1) centrifugation, 2) membrane filtration and 3) enzyme coagulation, made up the first split plot. Each sub-plot of liquid whey was then divided into three split plots to receive heat treatment. Heat treatments were no heat, 76°C for fifteen seconds and 85°C for three minutes. Each of the resulting nine treatment combinations was analyzed by 1) polyacrylamide gel electrophoresis, 2) bicinchoninic acid-soluble protein assay and 3) fluorescence spectroscopy to determine the amount of denatured protein in the liquid whey. Fluorescence spectroscopy was found to be the most sensitive and reliable method for detecting differences in structure due to denaturation, while native polyacrylamide gel electrophoresis was found to be the least sensitive method. The sample which received the centrifugal treatment of isolation with no heat was found to be the most undenatured in structure while the sample which received the enzyme treatment of isolation with high heat was found to be the most denatured in structure. The objective of the second series of experiments was to evaluate the effect of denaturation on whey protein solubility in dried whey protein powders. Solubility is one of the most important functional properties to consider when selecting a whey protein ingredient, especially for beverage systems. Processing parameters are often manipulated in efforts to improve solubility. The protein structures of whey are considered to have an effect on solubility. Specifically, the degree of denaturation of whey proteins is thought to play a role in solubility. In this experimental design, raw bovine milk was skimmed and pasteurized then enzyme-coagulated at natural pH to separate the whey. Liquid whey was then split into three aliquots and each received one of the following treatments: 1) mild heat/ freeze dry, 2) mild heat/spray dry and 3) high heat/spray dry. Heat treatment was applied to liquid whey prior to concentration. Heat treated whey was then concentrated and dried. Powders were reconstituted and analyzed for denaturation using 1) bicinchoninic acid assay for soluble protein and 2) fluorescence spectroscopy and for solubility using an insolubility index. pH 4.6 solubility and fluorescence spectroscopy for quantifying denaturation correlated well to one another. Both found that the low heat treated samples were less denatured in structure than the sample which received the high heat treatment, regardless of drying method. However, the drying method of the protein powders was correlated to solubility rather than heat treatment. A correlation of denaturation measured in whey protein powders and solubility was apparent for the low heat, freeze dried sample and the high heat, spray dried sample. Several conclusions were made in this research. 1) Centrifugal force causes less denaturation than membrane filtration and enzyme coagulation, thus unheated liquid whey obtained by centrifugal force can be used as a control in research on denaturation. 1) Fluorescence spectroscopy is a better method for quantifying denaturation in liquid and powdered whey compared to native PAGE and pH 4.6 solubility measured by BCA. 3) Functional solubility is dependent on denaturation and can be correlated to analytical methods of measuring denaturation.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Single molecule studies on CdSe quantum dots functionalized with oligo-phenylene vinylene ligands (CdSe-OPV) provide evidence of strong electronic communication that facilitate charge and energy transport between the OPV ligands and the CdSe quantum dot core. This electronic interaction greatly modify, the photoluminescence properties of both bulk and single CdSe-OPV nanostructure thin film samples. Size-correlated wide-field fluorescence imaging show that blinking suppression in single CdSe-OPV is linked to the degree of OPV coverage (inferred from AFM height scans) on the quantum dot surface. The effect of the complex electronic environment presented by photoexcited OPV ligands on the excited state property of CdSe-OPV is measured with single photon counting and photon-pair correlation spectroscopy techniques. Time-tagged-time-resolved (TTTR) single photon counting measurements from individual CdSe-OPV nanostructures, show excited state lifetimes an order of magnitude shorter relative to conventional ZnS/CdSe quantum dots. Second-order intensity correlation measurements g(2)(τ) from individual CdSe-OPV nanostructures point to a weak multi-excitonic character with a strong wavelength dependent modulation depth. By tuning in and out of the absorption of the OPV ligands we observe changes in modulation depth from g(2)(0) ≈ 0.2 to 0.05 under 405 and 514 nm excitation respectively. Defocused images and polarization anisotropy measurements also reveal a well-defined linear dipole emission pattern in single CdSe-OPV nanostructures. These results provide new insights into to the mechanism behind the electronic interactions in composite quantum dot/conjugated organic composite systems at the single molecule level. The observed intensity flickering, blinking suppression and associated lifetime/count rate and antibunching behaviour is well explained by a Stark interaction model. Charge transfer from photo-excitation of the OPV ligands to the surface of the CdSe quantum dot core, mixes electron/holes states and lifts the degeneracy in the band edge bright exciton state, which induces a well define linear dipole behaviour in single CdSe-OPV nanostructures. The shift in the electron energies also affects Auger assisted hole trapping rates, suppress access to dark states and reduce the excited state lifetime.

<p> This thesis represents a detailed assessment of the McMaster KN Accelerator site for the performance of a nuclear resonance absorption and fluorescence phenomenon in the 28Si nucleus. The main focus of this work is the 27Al(p, γ)^28 Si reaction, although other nuclear reactions are explored, such as: 27Al(p, p'γ)^27Al and 27Al(p, αγ)^24Mg. The gamma yield experiments from all these reactions suggest a repeatable and steady results, as well as very good agreement with the present literature. This is seen in chapter 2. Chapter 3 represents concrete nuclear resonance experiments with a direct ground state transition of the 12.33 MeV gamma energy from the 27Al(p, γ)^28Si reaction. These experiments are reproducible and repeatable with either HPGe or NaI(T1) (NaI elsewhere in text) detectors. Also, they are in close agreement with the literature.</p> <p> However, the main part of this work is described in chapter 4, where the first excited level of Si at 1.78 MeV is studied thoroughly. This is a pilot work that has never been attempted before. A thorough empirical approach is undertaken and described in section 4.1. This approach describes rationale for attempting nuclear resonance experiments with the first excited state of Si. The calculations suggest very close agreement between 12.33 MeV and 1.78 MeV experiments. Based on that, 7 different experimental sets, with several subsets ( within some of the sets) are performed. Very interesting results are obtained. However, so far, it cannot be concluded whether NRA/NRF experiments can be performed using the first excited state of Si. Most likely, hight current proton accelerators should be used and the experiments with 1.78 MeV lines should be repeated. These accelerators are described in chapter 5 and have the proton current output close to 1000 times higher than the McMaster KN accelerator. At the end, the dosimetry measurements suggest a negligible radiation dose from KN accelerator, as well as from these powerful accelerators.</p> / Thesis / Doctor of Philosophy (PhD)