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There is an increasing and serious problem of antimicrobial resistance emerging where previously useful antibiotics are no longer effective. This causes a significant monetary and human cost in the healthcare system, and is a direct result of the misuse and over prescription of antibiotics. The development and implementation of rapid point of care diagnostics to conclusively identify problem pathogens such as 'Staphylococcus aureus' could slow the emergence of antibiotic resistance by correctly targeted antibiotic therapy. The rapid and sensitive protease detecting LGX probe is an example of such a diagnostic test. The synthesis of this rhodamine based fluorogenic probe and the investigation of other fluorescence motifs for protease detection has been the focus of this project. Scale up and optimisation of the synthetic route to the previously produced LGX probe was achieved, allowing further testing in a healthcare environment. Previous work has failed to address the complexities associated with the use of conventional fluorescence motifs for probe generation, as well as the ways in which their fluorescence may be modulated. Herein we address these problems, and those encountered with the scale up of the 'LGX' probe. The lessons learned from the LGX synthesis provided the basis for development of a number of novel analogues of the orphan fluorophore class Singapore Green. A library of nine simplified and highly active fluorescent Singapore Greens were generated. These were exhaustively investigated and it was determined how structural variations effect both their spectral and fluorescence properties including quantum yield and extinction coefficient. The spectral characteristics of Singapore Green fluorescent motifs were found to be highly favourable allowing them to provide the central core of future probes. This work has also provided the synthetic methodology required to produce 'LGX' in the quantities required for eventual utilisation in healthcare environments. Furthermore it has provided the several novel platforms for the development of future probes. This is of potentially considerable value as it may not only contribute to the reduction of the problem pathogen 'S. aureus' as well as slow the emergence of resistance, but also facilitate generation of other important protease detection systems.

This dissertation presents theoretical, numerical, and experimental research into a technique for extending the observation time of a single molecule in solution, while also enabling measurement of its diffusion coefficient. A confocal microscope is used to observe the fluorescently labelled molecule in aqueous solution, which is confined within a nanochannel. By focusing a laser beam into the nanochannel and applying electrokinetic flow along the tube, a molecule passes through the laser beam and emits a burst of photons. The molecule then passes back and forth through the focus while the voltage is repeatedly reversed at a fixed delay after each detected burst. First, a Monte Carlo simulation of the single-molecule recycling (SMR) process is made to develop algorithms for timing the flow reversals and to study the choice of experimental parameters for diffusivity measurements. To detect fluorescence bursts from the background, a weighted sliding sum algorithm is applied, and the results show it has clear advantages over a previously used photon binning algorithm. Maximum-likelihood methods are developed to measure single-molecule diffusivities and their confidence limits from the variation in the times between detections. The simulations show that SMR can distinguish single molecules with diffusivities differing by a factor of ~1.3 or less, which is smaller than that resolvable in ensemble experiments by fluorescence correlation spectroscopy. Simulations are also developed to study both 1 and 2-photon excitation of ultrasmall CdSe quantum dots, which exhibit fluorescence intermittency or blinking. The simulation incorporates standard photophysics in such a way as to account for the known non-ergodic power-law dependence of the blinking intervals. For SMR experiments, two configurations are implemented: capillary microchannel devices are fabricated and used with a piezo system to provide motion, and nanochannels from a previous research project are used with applied voltage to give electroosmotic motion. A real-time control system that implements the weighted sliding sum and motion switching algorithms studied in the simulations is developed. The results from the experiments demonstrate SMR for 40 nm fluorescently labelled beads for hundreds of times and yield diffusion coefficients in the magnitude of 10–13 m2s–1 in a nanochannel.

The development of small molecule affinity-based chemical probes as research tools for studying the role of carbonic anhydrases (CAs) in their wider biological context has become an active field of research owing to an increasing awareness of the therapeutic relevance of this enzyme family, particularly in diseases such as glaucoma (CA II) and solid tumors (CA IX, CA XII). High CA isozyme selectivity, low nonspecific labeling, and efficient labeling yield are the characteristics of an ideal chemical probe, however achieving an effective balance of all three properties is challenging. A panel of chemical probes for two-step labeling of CA II or CA IX has been designed to study the impact of probe structural features on the efficiency and specificity of CA-selective labeling when in a challenging environment, including protein mixtures, cell lysates, or live cells. The panel comprised Generation 1 probes (P1 and novel probes P2–P5), Generation 2 linear PAL probes (P6 and novel probes P7–P15), and Generation 3 branched PAL probes (novel probes P16–P20).

Abstract: A large number of exogenous and endogenous agents can result in DNA damage leading to mutagenesis and cell death. A thorough knowledge and understanding about the effects of these agents on different sequences of DNA is important for elucidating the fundamental chemical mechanisms of DNA damage. Therefore, developing a rapid and inexpensive technique for the detection of damage in multiple samples is of great importance. Numerous DNA detection techniques, involving polymerase chain reaction and gel electrophoresis, have been developed. However, most of these techniques are cumbersome and/or expensive. In this thesis, we develop a simple, sensitive, inexpensive, mix-and-read assay to detect DNA damage with higher throughput and better ease-of-use. The goal of this work is to design simple hybridization assays for DNA damage detection of multiple sequences in single and double stranded DNA on a 96-well microplate platform. We first replaced the conventional cuvette method with the well plate method in order to study UVC-induced ssDNA damage on four sequences simultaneously. In this study, we measured the change in fluorescence intensity of a series of sequence-specific smart probes (SPs) for quantifying the extent of damage. The results show that this method has similar reproducibility as the cuvette method, but designing SPs complementary to each sequence makes the method tedious and expensive. In the second approach we developed a microplate assay coupled with EvaGreen (EG), an intercalating dye to quantify both ssDNA and dsDNA damage in an inexpensive way. This gives maximum sensitivity for detecting damage since the dye gives zero/minimum fluorescence with ssDNA and a maximum fluorescence with dsDNA. This dye gave good sensitivity and selectivity for quantification of dsDNA damaged by both UVC and a Ru cis-platin analog. The calibration curve for the EG probe shows good linearity (R2 = 0.99) with a limit of detection of 2.3 nM for dsDNA. Confirmation that EG can detect damage was done by melting curve and matrix-assisted laser desorption/ ionization time-of-fight mass spectroscopy (MALDI-TOF-MS). We also used our method to study the effect of the number and position of mismatches on the stability of dsDNA with another intercalating dye, Hoechst 33258 (H258). Results show that the sensitivity of the dsDNA detection is determined more by the position of mismatches than by the number of mismatches. We also compared the UVC-induced DNA damage kinetics of ssDNA and dsDNA. Our results are in agreement with in vitro and in vivo studies, which show ssDNA to be more vulnerable to damage than dsDNA. The H258 dye was much cheaper than the EG dye, but its use is limited because of its high selectivity for A-T rich regions of dsDNA. Finally, we explored the ability of our method to detect DNA damage in multiple samples of K-Ras and N-Ras proto-oncogenes. Results show that the K-Ras sequences are more mutagenic than the N-Ras sequences. This result is in excellent agreement with past biological studies performed on K-Ras and N-Ras genes. Thus, our method proves as a simple, inexpensive, mix-and-read assay for the reproducible quantification of DNA damaged induced by different etiological agents.

Abstract: This thesis describes a study on tapered air-core Bragg waveguides for use as integrated micro-spectrometers in lab-on-a-chip systems. Position-dependent cutoff of the leaky waveguide modes results in spatial dispersion of polychromatic input light. These devices may be viewed as side-coupled Fabry-Perot cavities of varying core thickness. Chip-scale spectrometers have a number of applications, including point-of-care diagnostics, biochemistry, and field-deployable threat detectors. Bragg cladding mirrors with an omnidirectional band (for TE-polarized light) between ~490 nm and ~570 nm were deposited using both e-beam evaporation and reactive sputtering. Using these claddings, tapered hollow Bragg waveguides were assembled using two different methods: buckling self-assembly and a wafer-bonding process. The tapers exhibited resolutions ranging from ~2.2 nm to ~0.8 nm, for mode orders m = 1 to m = 8. Implementing the tapers in a prototype sensing system, the fluorescence spectra of individual fluorescent beads were successfully measured over the ~70 nm wide operating range of the device.

This work demonstrates and characterizes a novel cross-polarization method of filtering the leakage excitation light from the emission signal of a fluorescent dye. The cross-polarization scheme was incorporated in an integrated on-chip fluorescence detection system, using a metal-halide lamp as excitation source and an organic photodiode as a detector. The benefits of cross-polarization are the significantly-reduced intensity of the leakage excitation light (from 5 µA to 19 nA, or 24 dB), and no wavelength specificity. These benefits make this approach suitable for applications with various dyes, and enable multi-analyte detection. With this setup, detection limits as low as 10 nM of Rhodamine 6G and fluorescein dyes (S/N = 1.5) have been achieved. These results represent a substantial improvement in the detection limit from previous reports in literature using organic photodiodes as fluorescence intensity detectors. This strongly suggests the feasibility of an inexpensive, highly sensitive, compact and disposable lab-on-a-chip fluorescence sensor for point-of-care medical diagnostics, on-site analysis for environmental monitoring and homeland security applications.

High performance liquid chromatography (HPLC) coupled with fluorescent detection was demonstrated for the analysis of amoxicillin residues in raw milk. Factors affection the fluorescent derivative formation (e.g. trichloroacetic acid (TCA), formaldehyde concentrations, reaction time and temperature) and the HPLC separation (e.g. buffer concentrations and pH and concentrations of organic solvent) were investigated. The derivative with the highest fluorescent intensity was obtained when amoxicillin was reacted with 20% w/v TCA, 7% v/v formaldehyde at 100oC for 30 min. Separation of the derivative was achieved in phosphate buffer (50 mM, pH 5.6) containing 24% v/v acetonitrile. Percent relative standard deviations (%RSDs) for injection, intra-day and inter-day precision were within 3.1% for the retention time and 2.3% for peak area. The percent recovery was 100.6% with the %RSD of 1.8%. The limit detection (LOD) and limit of quantitation (LOQ) were 20 and 50 ppb, respectively. The developed method was rapid, simple and sensitive for the analysis of amoxicillin residues in raw milk in trace amounts.

Since the early 1990's researchers have applied the principles of capillary electrophoresis to microfabricated devices. Separations of amino acids in free solutions as well as DNA in polymer solutions have been performed with high efficiencies in these devices. The majority of these devices have been fabricated using bulk micromachining of glass substrates and employ costly optical systems to detect fluorescent analytes. The real power of miniaturization is lost however, when these devices rely on a room full of equipment. This dissertation explores the use of surface micromachining for the fabrication of integrated capillary electrophoresis systems with on-chip fluorescence detection that can be incorporated into complex DNA diagnostic chips. A plastic surface micromachined technology is developed for the fabrication of long surface micromachined channels. This technology uses photoresist sacrificial layers in conjunction with vacuum deposited parylene-C. Channels up to 20 pm in height and nearly 2 mum in length have been fabricated. The low temperature fabrication process allows these channels to be fabricated on top of heaters, electrodes, photodiodes, temperature sensors, etc. without affecting device performance. Channels can be constructed on top of virtually any topography due to the conformal deposition of parylene-C. The channels fabricated show an extremely low autofluorescence as well as nearly 100% transparency, making them ideal for electrophoresis applications using fluorescence detection. Using this process electrophoresis devices fabricated on polycarbonate substrates are presented. A simple 4 mask process is developed that incorporates electrophoresis channels, electrodes, and reservoir structures. By using photodefinable silicone rubber for the reservoir structures, devices can be fabricated in a entirely batch manner. Steps typical in electrophoresis devices such as bonding, assembling of electrodes, and drilling of holes are replaced by surface micromachining. The use of lateral etch holes in long channels overcomes the limitations of sacrificially released channel structures. Separations of dsDNA in these devices have achieved over 100,000 theoretical plates. The use of plastics targets these devices for low cost, disposable DNA diagnostic applications. Finally, a monolithic surface micromachined electrophoresis device with integrated on-chip fluorescence detection is presented. An on-chip fluorescence detection system is developed using on-chip photodiodes, a thin film interference filter and a blue LED excitation source. DNA concentration levels of detection were as low as 225 pg/mul for dsDNA labeled with YOYO-1 intercalating dye. Separations of dsDNA were performed in these devices with on-chip DNA band detection. (Abstract shortened by UMI.)

In this study, reversed phase HPLC with dual UV photodiode (PDA) and fluorescence (FL) detection were used to investigate copper complexes with fulvic, caffeic, vanillic, salicylic, and adipic acids. Application of the RE method provided valuable information on the retention behavior and spectral characteristics of FA and model compounds. Even though the method was only applicable to VA, the use of the PDA detector allowed the UV-V is scanning of the separated peaks. This allowed the comparison between the UV-Vis spectra of uncomplexed species. The overall results provide an experimental framework for validation of the proposed Cu-humate interaction models.