Idiosyncratic drug reactions (IDRs) represent a significant medical problem and pose a great challenge to drug development. Circumstantial evidence suggests that, in most cases, reactive metabolites of the drug are responsible. The major focus of this thesis is the identification of reactive metabolites and the synthesis of analogs required to test several hypotheses related to involvement of metabolism and covalent binding in the mechanisms of IDRs. Minocycline is unique among tetracyclines in causing a significant incidence of a lupus-like syndrome and autoimmune hepatitis. In this study, we demonstrated that minocycline is oxidized to reactive intermediates by myeloperoxidase/H2O2/Cl-, HOCl, horseradish peroxidase/H2O2, or hepatic microsomes. When trapped with N-acetylcysteine (NAC), two adducts with protonated molecular ions at m/z 619 were isolated and analyzed by NMR. One represents attack of the aromatic D ring by NAC meta to the N, N-dimethylamino group, implying that the reactive intermediate was a quinone iminium ion. The other adduct, which was not observed when minocycline was oxidized by hepatic microsomes, indicates that the NAC is attached at the junction of the B and C rings, suggesting that the HOCl added across the double bond of the B ring leading to a reactive molecule, and then NAC displaced the chloride ion. Nevirapine, an anti-HIV drug, is associated with idiosyncratic skin rashes in humans. The goal of this project was to investigate whether the 12-hydroxylation pathway is responsible for the skin rash. To test a part of this hypothesis, 12-trideuteronevirapine, 12-OH-NVP sulfate, and several other analogs of nevirapine were synthesized. D-penicillamine is known to cause idiosyncratic autoimmune reactions in humans. The goal of this project was to test whether D-penicillamine covalently binds to macrophages and triggers downstream events leading to autoimmunity. To test a part of this hypothesis, D-penicillamine conjugated to biotin was synthesized. In summary, reactive metabolites of minocycline were found that likely explain why minocycline has an IDR profile unique among the tetracyclines. In addition, analogs of nevirapine and D-penicillamine required for mechanistic studies of nevirapine and D-penicillamine-induced IDRs were synthesized. These studies provide additional support for the involvement of reactive metabolites in the mechanisms of IDRs.

Research toward a multiplexed nucleic acid biosensor that uses quantum dots (QDs) as donors in a fluorescence resonance energy transfer (FRET) assay is described. Optical fibers were modified with mixed films composed of different colours of QDs and different oligonucleotide probes that served as scaffolds for the hybridization of the corresponding target nucleic acid sequences. Fluorescent dyes that were suitable as acceptors for each QD donor were associated with hybridization and provided an analytical signal through FRET-sensitized emission. Different detection channels were achieved through the combination of different donors and acceptors: green emitting QDs with Cyanine 3 or Rhodamine Red-X; and red emitting QDs with Alexa Fluor 647. A detection channel that used the direct excitation of Pacific Blue complemented the FRET pairs. One-plex, two-plex, three-plex and four-plex hybridization assays were demonstrated. A sandwich assay format was adopted to avoid target labeling. Detection limits were 1-10 nM (1-12 pmol) and analysis times were 1-4 h. Single nucleotide polymorphisms were discriminated in multiplexed assays, and the potential for reusability was also demonstrated. Non-selective interactions between QDs and oligonucleotides were characterized, and routes toward the optimization of the QD-FRET hybridization assays were identified. A basic model for multiple FRET pathways in a mixed film was also developed. In addition to the advantages of solid-phase assays, the combination of QDs and FRET was advantageous because it permitted multiplexed detection using a single excitation source and a single substrate, in the ensemble, and via ratiometric signals. Spatial registration or sorting methods, imaging or spatial scanning, and single molecule spectroscopy were not required. The research in this thesis is expected to enable new chip-based biosensors in the future, and is an original contribution to both bioanalytical spectroscopy and the bioanalytical applications of nanomaterials.

Tuberculosis (TB) is an important disease worldwide. Currently, one-third of the world’s population is infected with TB, and it is a leading cause of death among people living with HIV. Immediate but also accurate diagnosis is required for disease control, yet available diagnostics cannot do both simultaneously. Therefore, designing a technique that can diagnose the disease correctly in the shortest possible time is in great demand in order to stop its spread. Diffraction-based sensing is a novel technique for measuring of biomolecular interaction that has potential for disease diagnosis. In this study, diffraction-based sensing successfully demonstrated its usefulness for diagnostics of TB using recombinant TB antigen, or by detection of interferon-γ that is produced from white blood cells when the immune system activates. The feasibility of the technology was also evaluated in terms of providing real time observation, reducing diagnostic duration, and increasing sensitivity of detection.

Tuberculosis (TB) is an important disease worldwide. Currently, one-third of the world’s population is infected with TB, and it is a leading cause of death among people living with HIV. Immediate but also accurate diagnosis is required for disease control, yet available diagnostics cannot do both simultaneously. Therefore, designing a technique that can diagnose the disease correctly in the shortest possible time is in great demand in order to stop its spread. Diffraction-based sensing is a novel technique for measuring of biomolecular interaction that has potential for disease diagnosis. In this study, diffraction-based sensing successfully demonstrated its usefulness for diagnostics of TB using recombinant TB antigen, or by detection of interferon-γ that is produced from white blood cells when the immune system activates. The feasibility of the technology was also evaluated in terms of providing real time observation, reducing diagnostic duration, and increasing sensitivity of detection.

Since the introduction of electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI), there has been an unprecedented growth of biomolecule analysis using mass spectrometry (MS). One of the most popular applications for mass spectrometry is the field of proteomics, which has emerged as the next scientific challenge in the post-genome era. One critical step in proteomic analysis is sample preparation, a major bottleneck that is attributed to many time consuming and labor-intensive steps involved. Microfluidics can play an important role in proteome sample preparation due to its ability to handle small volumes of sample and reagent, and its capability to integrate multiple processes on a single chip with the potential for high-throughput analysis. However, to utilize microfluidic systems for proteome analysis, an efficient interface between microfluidic chip and mass spectrometry is required. This thesis presents several methods for coupling of microfluidic chips with ESI-MS and MALDIMS. III Three microfluidic-ESI interfaces were developed. The first interface involves fabricating a polymer based microchannel at the rectangular corners of the glass substrates using a single photolithography step. The second interface was build upon the previous interface in which a digital microfluidic platform was integrated with the microchannel in a “top-down” format. The integrated microfluidic system was used for inline quantification of amino acids in dried blood spots that have been processed by digital microfluidics. The third interface was formed by sandwiching a pulled glass capillary emitter between two digital microfluidic substrates. This method is a simpler and more direct coupling of digital microfluidics with ESI-MS as compared to the method used for second interface. Finally, a strategy using a removable plastic “skin” was developed to interface digital microfluidics with MALDI-MS for offline sample analysis. We demonstrated the utility of this format by implementing on-chip protein digestion on immobilized enzyme depots.

Detection of conformational and structural shifts in biomolecules is of great importance in bioanalytical chemistry and pharmaceutical sciences. Transverse shear mode acoustic wave devices have been used as real-time, label-free detectors of conformational shifts in biomolecules on surfaces. However, material changes in the biochemical monolayer and coupling between the substrate and the surrounding liquid make it difficult to isolate the desired signal, so an understanding of these phenomena is required. In this thesis, interfacial slip, viscoelasticity, and structural changes are used to model acoustic signals due to surface adsorption of the protein neutravidin, immobilisation of HIV-1 TAR RNA, and subsequent interaction of the RNA with tat peptide fragments. Binding of tat peptides induces conformational changes in the TAR. Similar modelling is performed to describe experiments involving the binding of calcium to surface-attached calmodulin, which is also known to result in a conformational shift. The aim of the modelling is to isolate the sensor response due to conformational shifts. The biomolecules are described as hydrated, viscoelastic monolayers and slip is allowed at all interfaces. All models are numerically fit to experimental values using a two-parameter minimisation algorithm. Slip is found on the electrode surface prior to neutravidin adsorption. Neutravidin and TAR are described as distinct viscoelastic monolayers. Binding of tat peptide fragment to the TAR monolayer is modelled using a complex slip parameter and a change in length, corresponding to a straightening of the molecule. Similarly, numerical modelling of calmodulin results reveals a length change in the molecule upon calcium binding. Molecular dynamics (MD) simulations of the TAR-tat fragment system are performed to corroborate the modelling results. Starting structures are computed by molecular docking, and MD simulations of TAR complexed with various length tat fragments are described. The simulations are in general agreement with the modelling results and literature values from similar molecular dynamics experiment. A new parameter is introduced to describe biomolecule-solvent affinity, and is compared to interfacial coupling values obtained from modelling. This research demonstrates that acoustic wave devices can be used to detect conformational shifts in surface-attached biomolecules, provided molecular details about the shifts are known.

Idiosyncratic drug reactions (IDRs) represent a significant medical problem and pose a great challenge to drug development. Circumstantial evidence suggests that, in most cases, reactive metabolites of the drug are responsible. The major focus of this thesis is the identification of reactive metabolites and the synthesis of analogs required to test several hypotheses related to involvement of metabolism and covalent binding in the mechanisms of IDRs. Minocycline is unique among tetracyclines in causing a significant incidence of a lupus-like syndrome and autoimmune hepatitis. In this study, we demonstrated that minocycline is oxidized to reactive intermediates by myeloperoxidase/H2O2/Cl-, HOCl, horseradish peroxidase/H2O2, or hepatic microsomes. When trapped with N-acetylcysteine (NAC), two adducts with protonated molecular ions at m/z 619 were isolated and analyzed by NMR. One represents attack of the aromatic D ring by NAC meta to the N, N-dimethylamino group, implying that the reactive intermediate was a quinone iminium ion. The other adduct, which was not observed when minocycline was oxidized by hepatic microsomes, indicates that the NAC is attached at the junction of the B and C rings, suggesting that the HOCl added across the double bond of the B ring leading to a reactive molecule, and then NAC displaced the chloride ion. Nevirapine, an anti-HIV drug, is associated with idiosyncratic skin rashes in humans. The goal of this project was to investigate whether the 12-hydroxylation pathway is responsible for the skin rash. To test a part of this hypothesis, 12-trideuteronevirapine, 12-OH-NVP sulfate, and several other analogs of nevirapine were synthesized. D-penicillamine is known to cause idiosyncratic autoimmune reactions in humans. The goal of this project was to test whether D-penicillamine covalently binds to macrophages and triggers downstream events leading to autoimmunity. To test a part of this hypothesis, D-penicillamine conjugated to biotin was synthesized. In summary, reactive metabolites of minocycline were found that likely explain why minocycline has an IDR profile unique among the tetracyclines. In addition, analogs of nevirapine and D-penicillamine required for mechanistic studies of nevirapine and D-penicillamine-induced IDRs were synthesized. These studies provide additional support for the involvement of reactive metabolites in the mechanisms of IDRs.

Research toward a multiplexed nucleic acid biosensor that uses quantum dots (QDs) as donors in a fluorescence resonance energy transfer (FRET) assay is described. Optical fibers were modified with mixed films composed of different colours of QDs and different oligonucleotide probes that served as scaffolds for the hybridization of the corresponding target nucleic acid sequences. Fluorescent dyes that were suitable as acceptors for each QD donor were associated with hybridization and provided an analytical signal through FRET-sensitized emission. Different detection channels were achieved through the combination of different donors and acceptors: green emitting QDs with Cyanine 3 or Rhodamine Red-X; and red emitting QDs with Alexa Fluor 647. A detection channel that used the direct excitation of Pacific Blue complemented the FRET pairs. One-plex, two-plex, three-plex and four-plex hybridization assays were demonstrated. A sandwich assay format was adopted to avoid target labeling. Detection limits were 1-10 nM (1-12 pmol) and analysis times were 1-4 h. Single nucleotide polymorphisms were discriminated in multiplexed assays, and the potential for reusability was also demonstrated. Non-selective interactions between QDs and oligonucleotides were characterized, and routes toward the optimization of the QD-FRET hybridization assays were identified. A basic model for multiple FRET pathways in a mixed film was also developed. In addition to the advantages of solid-phase assays, the combination of QDs and FRET was advantageous because it permitted multiplexed detection using a single excitation source and a single substrate, in the ensemble, and via ratiometric signals. Spatial registration or sorting methods, imaging or spatial scanning, and single molecule spectroscopy were not required. The research in this thesis is expected to enable new chip-based biosensors in the future, and is an original contribution to both bioanalytical spectroscopy and the bioanalytical applications of nanomaterials.

Since the introduction of electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI), there has been an unprecedented growth of biomolecule analysis using mass spectrometry (MS). One of the most popular applications for mass spectrometry is the field of proteomics, which has emerged as the next scientific challenge in the post-genome era. One critical step in proteomic analysis is sample preparation, a major bottleneck that is attributed to many time consuming and labor-intensive steps involved. Microfluidics can play an important role in proteome sample preparation due to its ability to handle small volumes of sample and reagent, and its capability to integrate multiple processes on a single chip with the potential for high-throughput analysis. However, to utilize microfluidic systems for proteome analysis, an efficient interface between microfluidic chip and mass spectrometry is required. This thesis presents several methods for coupling of microfluidic chips with ESI-MS and MALDIMS. III Three microfluidic-ESI interfaces were developed. The first interface involves fabricating a polymer based microchannel at the rectangular corners of the glass substrates using a single photolithography step. The second interface was build upon the previous interface in which a digital microfluidic platform was integrated with the microchannel in a “top-down” format. The integrated microfluidic system was used for inline quantification of amino acids in dried blood spots that have been processed by digital microfluidics. The third interface was formed by sandwiching a pulled glass capillary emitter between two digital microfluidic substrates. This method is a simpler and more direct coupling of digital microfluidics with ESI-MS as compared to the method used for second interface. Finally, a strategy using a removable plastic “skin” was developed to interface digital microfluidics with MALDI-MS for offline sample analysis. We demonstrated the utility of this format by implementing on-chip protein digestion on immobilized enzyme depots.

Seeds are complex entities, within which the intricate processes of germination and early growth occur. We describe here a novel technique of our group in 2012 which is capable of simultaneous solution-, gel-, and solid-state analysis. CMP-NMR was applied to intact seeds where all components are studied and differentiated in situ. Characterization, germination and early growth of seeds were studied by variety of 1D and 2D 1H and 13C CMP-NMR experiments. Various metabolites, lipids, carbohydrate biopolymers and structural carbohydrates were first identified and further studied in germination and early growth stages. This research demonstrates the utility of CMP- NMR as a powerful tool to better understand the composition of seeds and processes underlying early seed growth.