The thickness shear mode acoustic wave (TSM) sensor, operated in a flow-through format, has been widely used in bioanalytical research. My research is mainly focused on the study of surface-attached biomolecules and cells using the TSM sensor, including lesions in DNA, conformational change of calmodulin, as well as the properties and attachment of rat aortic smooth muscle cells. Aldehydic apurinic or apyrimidinic sites (AP sites) that lack a nucleobase moiety are one of the most common forms of toxic lesions in DNA. In this work, synthesized oligodeoxyribonucleotides containing one, two, or three abasic sites were hybridized to complementary sequences immobilized on the gold electrode of the TSM device by affinity binding. The influence of AP sites on local base stacking energy and geometry caused a dramatic destabilization of the DNA duplex structure, which was detected by the TSM sensor. The signals detected by TSM correlated well with the thermostability of DNA duplexes in solution. The results indicate that both the number of sites and their localization in the double-stranded structure influence the stability of a 19 b.p. duplex. TSM was also used to detect the binding of ions or peptides to surface-attached calmodulin. The interaction between calmodulin and ions induced an increase in resonant frequency and a decrease in motional resistance. In addition, these signal changes were reversible upon washing with buffer. The response was interpreted as a decrease in surface coupling induced by exposure of hydrophobic domains on the protein, and an increase in the length of calmodulin by approximately 3 Å. In addition, the interaction of the protein with peptide together with calcium ions was detected successfully, despite the relatively low molecular mass of the 2-kDa peptide. In addition, the attachment of smooth muscle cells to various surfaces has been monitored by TSM. These surfaces include laminin, fibronectin and bare gold. The results of these experiments in terms of changes of frequency (fs) and resistance (Rm) were analyzed. The responses of the surface-bound cells to the introduction of various ions, depolarisation events and damage subsequent to exposure to hydrogen peroxide were also observed. Morphological changes in the cells, as confirmed by atomic force microscopy and scanning electron microscopy, are correlated with results from the TSM sensor.

The thickness shear mode acoustic wave (TSM) sensor, operated in a flow-through format, has been widely used in bioanalytical research. My research is mainly focused on the study of surface-attached biomolecules and cells using the TSM sensor, including lesions in DNA, conformational change of calmodulin, as well as the properties and attachment of rat aortic smooth muscle cells. Aldehydic apurinic or apyrimidinic sites (AP sites) that lack a nucleobase moiety are one of the most common forms of toxic lesions in DNA. In this work, synthesized oligodeoxyribonucleotides containing one, two, or three abasic sites were hybridized to complementary sequences immobilized on the gold electrode of the TSM device by affinity binding. The influence of AP sites on local base stacking energy and geometry caused a dramatic destabilization of the DNA duplex structure, which was detected by the TSM sensor. The signals detected by TSM correlated well with the thermostability of DNA duplexes in solution. The results indicate that both the number of sites and their localization in the double-stranded structure influence the stability of a 19 b.p. duplex. TSM was also used to detect the binding of ions or peptides to surface-attached calmodulin. The interaction between calmodulin and ions induced an increase in resonant frequency and a decrease in motional resistance. In addition, these signal changes were reversible upon washing with buffer. The response was interpreted as a decrease in surface coupling induced by exposure of hydrophobic domains on the protein, and an increase in the length of calmodulin by approximately 3 Å. In addition, the interaction of the protein with peptide together with calcium ions was detected successfully, despite the relatively low molecular mass of the 2-kDa peptide. In addition, the attachment of smooth muscle cells to various surfaces has been monitored by TSM. These surfaces include laminin, fibronectin and bare gold. The results of these experiments in terms of changes of frequency (fs) and resistance (Rm) were analyzed. The responses of the surface-bound cells to the introduction of various ions, depolarisation events and damage subsequent to exposure to hydrogen peroxide were also observed. Morphological changes in the cells, as confirmed by atomic force microscopy and scanning electron microscopy, are correlated with results from the TSM sensor.

In this thesis, cyclic biamperometry (CB) as a new method in electrochemistry, has been introduced and investigated. The hallmark of this method is the absence of a reference electrode which potentially allows simplification and miniaturization of the measurement apparatus. Similarities and differences of this method and cyclic voltammetry (CV) have been studied and it was shown that under conditions of using standard electrodes, CB has a better sensitivity and a lower detection limit than CV. A new equivalent circuit model for the cell has been proposed and parameters affecting the sensitivity of CB, such as keeping the concentration of one redox species in excess and having a larger W2 electrode, have been described. The redox cycling effect in biamperometric systems has been investigated and it is shown that improvements of at least two orders of magnitude in sensitivity can be achieved by using interdigitated electrodes (IDEs). In addition, an example for applications of this method, including biamperometric dead-stop titration of 1-naphthol with ferricyanide, has been presented and possible fields in which CB can be incorporated (e.g. monitoring the activity of alkaline phosphatase) have been illustrated. Finally, a few suggestions for future studies and further improvements have been outlined.

In this thesis, cyclic biamperometry (CB) as a new method in electrochemistry, has been introduced and investigated. The hallmark of this method is the absence of a reference electrode which potentially allows simplification and miniaturization of the measurement apparatus. Similarities and differences of this method and cyclic voltammetry (CV) have been studied and it was shown that under conditions of using standard electrodes, CB has a better sensitivity and a lower detection limit than CV. A new equivalent circuit model for the cell has been proposed and parameters affecting the sensitivity of CB, such as keeping the concentration of one redox species in excess and having a larger W2 electrode, have been described. The redox cycling effect in biamperometric systems has been investigated and it is shown that improvements of at least two orders of magnitude in sensitivity can be achieved by using interdigitated electrodes (IDEs). In addition, an example for applications of this method, including biamperometric dead-stop titration of 1-naphthol with ferricyanide, has been presented and possible fields in which CB can be incorporated (e.g. monitoring the activity of alkaline phosphatase) have been illustrated. Finally, a few suggestions for future studies and further improvements have been outlined.

The data presented in this thesis highlights how sea water composition and biological activity can affect the morphology, composition, organic volume fraction, and hygroscopicity of individual sea spray aerosols (SSA). A variety of techniques were used to measure seawater and aerosol composition with the emphasis placed on spatial chemical composition obtained through Scanning Transmission X-ray Microscopy-Near Edge X-ray Absorption Fine Structure (STXM-NEXAFS). Through NEXAFS data, organic volume fractions were derived from Beer's Law, and spatially resolved chemical composition for individual particles were determined through application of singular value decomposition and principal component analysis. These methods were applied to two specific studies: a 30 day mesocosm study using a wave flume termed Investigation into Marine PArticle Chemistry and Transfer Science (IMPACTS) and a smaller scale collection of SSA generated from a miniature Marine Aerosol Reference Tank (mini-MART) with bacteria enriched sea water. For IMPACTS, two consecutive phytoplankton blooms were observed; however, organic enrichment in sea spray aerosols only occurred during one of the blooms. STXM-NEXAFS measurements revealed four distinct particle types: sea salt-organic particles with a distinct NaCl core and an organic carbon coating, homogenously mixed organic-inorganic particles, calcium-rich needle-like particles, and agglomerations of optically thick organic material with inorganic salts. Organic enrichment was correlated with aliphatic-rich organic species as detected by an intense Cls—•a(C-H)* exciton excitation. This enrichment was unique to particles collected in the aerodynamic size range 0.18-0.32 µm and corresponded with a depression in the hygroscopicity of small particles. This depression can significantly suppress the number of cloud condensation nuclei thus influencing cloud properties. Results of the mini-MART collection revealed that whole bacterial inclusions are ejected into SSA via jet drops. Bacterial inclusions are rich in protein and can be identified through Principal Component Analysis (PCA) on image stacks acquired at the carbon K edge. Vesicles were not identified in SSA but could be resolved in standard liquid cell samples in which they exhibited a strong phospholipid spectrum that could also be resolved spatially usually PCA coupled with k-means clustering. Bacterial inclusions in SSA may affect SSA physical properties by serving as ice nuclei.1,2,3

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.

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.

abstract: Microfluidics has shown great potential in rapid isolation, sorting, and concentration of bioparticles upon its discovery. Over the past decades, significant improvements have been made in device fabrication techniques and microfluidic methodologies. As a result, considerable microfluidic-based isolation and concentration techniques have been developed, particularly for rapid pathogen detection. Among all microfluidic techniques, dielectrophoresis (DEP) is one of the most effective and efficient techniques to quickly isolate and separate polarizable particles under inhomogeneous electric field. To date, extensive studies have demonstrated that DEP devices are able to precisely manipulate cells ranging from over 10 μm (mammalian cells) down to about 1 μm (small bacteria). However, very limited DEP studies on manipulating submicron bioparticles, such as viruses, have been reported. In this dissertation, rapid capture and concentration of two different and representative types of virus particles (Sindbis virus and bacteriophage M13) with gradient insulator-based DEP (g-iDEP) has been demonstrated. Sindbis virus has a near-spherical shape with a diameter ~68 nm, while bacteriophage M13 has a filamentous shape with a length ~900 nm and a diameter ~6 nm. Under specific g-iDEP experimental conditions, the concentration of Sindbis virus can be increased two to six times within only a few seconds, using easily accessible voltages as low as 70 V. A similar phenomenon is also observed with bacteriophage M13. Meanwhile, their different DEP behavior predicts the potential of separating viruses with carefully designed microchannels and choices of experimental condition. DEP-based microfluidics also shows great potential in manipulating blood samples, specifically rapid separations of blood cells and proteins. To investigate the ability of g-iDEP device in blood sample manipulation, some proofs of principle work was accomplished including separating two cardiac disease-related proteins (myoglobin and heart-type fatty acid binding protein) and red blood cells (RBCs). Consistent separation was observed, showing retention of RBCs and passage of the two spiked protein biomarkers. The numerical concentration of RBCs was reduced (~70 percent after one minute) with the purified proteins available for detection or further processing. This study explores and extends the use of the device from differentiating similar particles to acting as a sample pretreatment step. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2017

No description available.

Enzymes are implicated in many diseases including neurodegenerative, cancer, immune deficiency, and inflammatory disorders. There is a constant need to develop novel drug compounds that target enzymes in order to modulate their function, thus treating the disease state. These compounds are typically small molecules with affinity to the enzyme active site or an allosteric site. In order to discover novel compounds for treating disease, the interaction between an enzyme and a small molecule must first be identified and then characterized. With the target enzyme known, it is beneficial to screen libraries of compounds against the target. Immobilizing the enzyme allows for pre-concentration of ligands on the surface and therefore increased signal enhancement, as well as permitting multiple wash steps and enzyme reuse. Immobilized enzyme columns are optimal for coupling to a variety of detection devices by way of liquid chromatography, including absorbance or mass spectrometric detection. Immobilized enzyme reactors (IMERs) were generated and optimized for two target molecules, acetylcholinesterase (AChE) and adenosine deaminase (ADA), for rapid function-based screening of enzyme inhibitors in mixtures. The IMER mode is useful for increasing throughput and facilitating the identification of hit mixtures, but it is slow and tedious to manually deconvolute hit compounds from mixtures and the IMER method is not amenable to natural product extracts, which are good sources of structurally diverse compounds that are more likely to result in a hit compound. Bio-selective solid-phase extraction (BioSPE) is an orthogonal method of isolating and identifying enzyme inhibitors in a single step, and was used to easily deconvolute complex mixtures, rapidly identifying to key compounds EHNA and MAC-0038732 out of mixtures using ADA columns. A data dependent acquisition MS method was developed and used to screen a set of fungal endophyte extracts, identifying two potentially novel inhibitors that were confirmed by IMER-MS/MS. / Thesis / Doctor of Philosophy (PhD) / The discovery of new drug compounds is crucial for the treatment of diseases. Enzymes are proteins that turn a substrate into a product; and in diseases they can often malfunction, overproducing the product. Small molecule compounds can sometimes inhibit enzyme function and can be further developed into therapeutic drugs. This thesis describes a method for detecting small molecule inhibitors that bind to an enzyme that is immobilized in a small column. Once the small molecule is bound to the immobilized enzyme, it can be detected by either showing that enzyme function is inhibited or by removing the compound from the enzyme and identifying the compound by mass spectrometry. These methods can quickly identify compounds at extremely low levels from complex mixtures, such as natural product extracts.