Дисертації з теми "Total Internal Reflection Raman Spectroscopy"

Laser Raman spectroscopy has been used very effectively for some time to probe heterogeneous catalytic reactions in situ. TIR Raman is a variant of non-resonant Raman spectroscopy which uses a totally internally reflected light, i.e. an evanescent electric field acts as the excitation source. TIR Raman reduces or avoids some key limitations of bulk Raman spectroscopy, including reduction of laser induced sample damage. This thesis has therefore been investigating the possible application of TIR Raman spectroscopy to studying heterogeneous catalysts, in particular films of size controlled metal nanoparticle catalysts. Potential catalytic materials (such as platinum, supported either on optical elements or in mesoporous silica) were synthesised, characterised by techniques such as TEM, UV-VIS, deposited on substrates both at monolayer and thicker coverages, and subjected to both conventional and TIR Raman spectroscopy. This included a significant amount of synthetic development, in particular of copper nanoparticles. Results indicated that TIR Raman enables acquisition of spectra with improved sensitivity, compared to bulk Raman, below the damage threshold of the materials, even at low levels of surface coverage. Specifically, Raman bands indicating the presence and removal (by plasma cleaning) of organic capping agents on nanoparticles have been detected for a number of systems. This was not achieved using confocal Raman spectroscopy. This was extended to develop a system for studying gas/solid catalytic reactions in situ using a specially constructed gas cell to enable application of the technique under reaction conditions. Finally, the nanoparticles synthesised primarily for the TIR Raman study have also been demonstrated as catalysts in improving the understanding of several catalysed reactions, in particular direct amide bond formation from amines and alcohols, cascade oxidations, and selective furfural hydrogenation.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.
Title from electronic title page (viewed Mar. 27, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry.

The study of the binding between estrogen receptors (ER) and their ligands in vitro has long been of interest mainly because of its application in anti-estrogen drug discovery for breast cancer treatment as well as in the screening of environmental contaminants for endocrine disruptors. Binding strength was conventionally quantified in terms of equilibrium dissociation constant (KD). Recently, emphasis is shifting towards kinetics rate constants, and off-rate (koff) in particular. This thesis reported a novel method to measure such binding kinetics based on fluorescence polarization complemented with total internal reflection fluorescence (FP-TIRF). It used tethered receptors in a flow cell format. For the first time, the kinetics rate constants of the binding of full-length human recombinant ERα with its standard ligands were measured. koff was found to range from 1.3 10-3 to 2.3 10-3 s-1. kon ranged from 0.3 105 to 11 105 M-1 s-1. The method could also be used to screen potential ligands. Motivated by recent findings that ginsenosides might be functional ligands of nuclear receptors, eleven ginsenosides were scanned for binding with ER and peroxisome proliferator-activated receptor gamma (PPAR). None of the ginsenosides showed significant binding to ER, but Rb1 and 20(S)-Rg3 exhibited significant specific binding with PPAR.

The accurate and safe diagnosis of breast cancer is a significant societal issue, with annual disease incidence of 48,000 women and around 370 men in the UK. Early diagnosis of the disease allows more conservative treatments and better patient outcomes. Microcalcifications in breast tissue are an important indicator for breast cancers, and often the only sign of their presence. Several studies have suggested that the type of calcification formed may act as a marker for malignancy and its presence may be of biological significance. In this work, breast calcifications are studied with FTIR, synchrotron FTIR, ATR FTIR, and Raman mapping to explore their disease specific composition. From a comparison between vibrational spectroscopy and routine staining procedures it becomes clear that calcium builds up prior to calcification formation. Raman and FTIR indicate the same size for calcifications and are in agreement with routine staining techniques. From the synchrotron FTIR measurements it can be proven that amide is present in the centre of the calcifications and the intensity of the bands depends on the pathology. Special attention is paid to the type of carbonate substitution in the calcifications relating to different pathology grades. In contrast to mammography, Raman spectroscopy has the capability to distinguish calcifications based on their chemical composition. The ultimate goal is to turn the acquired knowledge from the mapping studies into a clinical tool based on deep Raman spectroscopy. Deep Raman techniques have a considerable potential to reduce large numbers of normal biopsies, reduce the time delay between screening and diagnosis and therefore diminish patient anxiety. In order to achieve this, a deep Raman system is designed and after evaluation of its performance tested on buried calcification standards in porcine soft tissue and human mammary tissue. It is shown that, when the calcification is probed through tissue, the strong 960 cm-1 phosphate band can be used as a pseudo marker for carbonate substitution which is related to the pathology of the surrounding tissue. Furthermore, the first study in which human breast calcifications are measured in bulk tissue with a thickness of several millimetres to centimetres is presented. To date, measurements have been performed at 41 specimens with a thickness up to 25 mm. Measurements could be performed through skin and blue dye. The proposed deep Raman technique is promising for probing of calcifications through tissue but will need refinement before being adopted in hospitals.

This thesis focuses on investigations of transient dark states of fluorescentmolecules using spectroscopic techniques. The main purpose is to show andconvince the reader that transient dark states are not always a nuisance, butalso represent an additional source of information. Several studies with fluorescencecorrelation spectroscopy were performed, all related to non-fluorescentstates such as triplet state or isomerized states.Photobleaching is one of the main problems in virtually all of the fluorescencetechniques. In this thesis, mechanisms that retard photobleaching arecharacterized. Several compounds, antioxidants and triplet state quenchers,which decrease photobleaching, are studied, and guidelines for achieving optimalfluorescence brightness using these compounds are presented.Triplet state quenching by several compounds was studied. Detailed investigationsof the fluorescence quencher potassium iodide demonstratedthat for some of fluorophores, except of quenching, there is fluorescence enhancementmechanism present. In agreement with the first publication inthis thesis, antioxidative properties were found to play an important role inthe fluorescence enhancement. Quenching of the triplet state is proposedas a tool for monitoring diffusion mediated reactions over a wide range offrequencies.Specially designed fluorophores combining high triplet yields with reasonablefluorescence brightness and photostability were characterized forpossible applications in novel super-resolution imaging techniques based onfluorescence photoswitching. Except of benefits for imaging techniques, photoinducedswitching to non-fluorescent states could be used for monitoringmolecular diffusion, which was also demonstrated in this thesis.Studies of the triplet state kinetics of fluorophores close to dielectric interfaceswere performed using fluorescence spectroscopy. The analysis of thetriplet state kinetic can provide information about the local microenvironmentand electrostatic interactions near dielectric interfaces.
QC 20100414

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2007.
Committee Chair: Mizaikoff, Boris; Committee Member: Bottomley, Lawrence; Committee Member: Hunt, William; Committee Member: Janata, Jiri; Committee Member: Josowicz, Miroslawa.

Alzheimer’s disease (AD) is currently considered the most prevalent neurodegenerative disease and places a large financial burden on society as healthcare resources are limited and the disease does not have a cure. Alzheimer’s disease is characterized by the presence of amyloid beta (Aβ) plaques and neurofibrillary tangles; however current literature suggests Aβ oligomers are the main aggregating species leading to AD symptoms. Therefore, the underlying cause of Alzheimer’s, accumulation of amyloid beta, is currently being studied in hopes of developing treatment options. Our research aims at determining the mechanism and kinetics of Aβ oligomer dissociation into non-toxic monomers in the presence of denaturants or small molecule dissociators. These highly active small molecule dissociators, selected from the Apex Screen 5040 library, were previously identified by ELISA studies by the laboratory of Dr. Harry LeVine. We have used fluorescence correlation spectroscopy (FCS) to characterize the size distribution and mole fraction of synthetically prepared fluorescein labeled Aβ (1-42) oligomers. Our FCS results show that in the presence of denaturants or small molecule dissociators, oligomer dissociation may proceed by at least two different mechanisms; high order cooperative dissociation and linear dissociation. A cooperative mechanism is more desirable for therapeutics as oligomer directly dissociates into monomer rather than through various oligomer intermediates. Our FCS studies show the most efficient dissociators proceed through the cooperative dissociation mechanism. We also observed a large retardation of the oligomer dissociation in the presence of gallic acid. We also started preliminary work to develop a total internal reflection fluorescence (TIRF) spectroscopy method to image Aβ (1-42) oligomers. This technique if successful will help to verify the two distinct mechanisms seen by FCS or determine if there is one mechanism that occurs at different rates as TIRF allows for faster analysis.

Thesis advisor: Mary F. Roberts
Thesis advisor: Steven D. Bruner
The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is specifically activated by low concentrations of a non-substrate lipid, phosphatidylcholine (PC), presented as an interface. However, if the PC concentration in the interface is too high relative to substrate, the enzyme exhibits surface dilution inhibition. Understanding this bacterial enzyme, which shares many kinetic features with the larger and more complex mammalian PI-PLC enzymes, requires elucidating the mechanism for PC activation and inhibition. Various techniques were applied to study the interaction of the protein with vesicles composed of both the activator lipid PC and the substrate lipid (or a nonhydrolyzable analogue). Fluorescence correlation spectroscopy (FCS), used to monitor bulk partitioning of the enzyme on vesicles, revealed that both the PC and the substrate analogue are required for the tightest binding of the PI-PLC to vesicles. Furthermore, the tightest binding occurred at low mole fractions of substrate-like phospholipids. Field cycling 31P NMR (fc-P-NMR) spin-lattice relaxation studies provided information on how bound protein affects the lipid dynamics in mixed substrate analogue/PC vesicles. The combination of the two techniques could explain the enzyme kinetic profile for the PC activation and surface dilution inhibition: small amounts of PC in an interface enhanced PI-PLC binding to substrate-rich vesicles while high fractions of PC tended to sequester the enzyme from the bulk of its substrate leading to reduced specific activity. FCS binding profiles of mutant proteins were particularly useful in determining if a specific mutation affected a single or both phospholipid binding modes. In addition, an allosteric PC binding site was identified by fc-P-NMR and site directed spin labeling. A proposed model for PC activation suggested surface-induced dimerization of the protein. Experiments in support of the model used cysteine mutations to create covalent dimers of this PI-PLC. Two of these disulfide linked dimers, formed from W242C or S250C, exhibited higher specific activities and tighter binding to PC surfaces. In addition, single molecule total internal reflection fluorescence microscopy was used to monitor the off-rate of PI-PLC from surface tethered vesicles, providing us with a direct measure of off-rates of the protein from different composition vesicles
Thesis (PhD) — Boston College, 2009
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

Click chemistry is a molecular synthesis strategy based on reliable, highly selective reactions with thermodynamic driving forces typically in excess of 20 kcal mol-1. The 1,3-dipolar cycloaddition of azides and alkynes developed by Rolf Huisgen saw dramatic rate acceleration using Cu(I) as a catalyst in 2002 reports by Barry Sharpless and Morten Meldal enabling its click chemistry eligibility. Since these seminal reports, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has become the quintessential click reaction finding diverse utility. The popularity of the CuAAC has naturally led to interest in new catalyst systems with improved efficiency, robustness, and reusability with particular focus on nanomaterial catalysts, a common trend across the field of catalysis. The high surface area of nanomaterials lends to their efficacy as colloidal and heterogeneous nanocatalysts, but the latter boasts the added benefit of easy separation and recyclability. With any heterogeneous catalyst, a common question arises as to whether the active catalyst species is truly heterogeneous or rather homogeneous through metal ion leaching. Differentiating these processes is critical, as the latter would result in reduced efficiency, higher cost, and inevitable environmental and heath side effects. This thesis explores the CuAAC from an interdisciplary approach. First as a synthetic tool, applying CuAAC-formed triazoles as functional, modular building blocks in the synthesis of optical cation sensors by combining azide and alkyne modified components to create a series of sensors selective for different metal cations. Next, single-molecule spectroscopy techniques are employed to observe the CuNP-catalyzed CuAAC in real time. Combining bench-top techniques with single-molecule microscopy to monitor single-catalytically generated products proves to be an effective method to establish catalysis occurs directly at the surface of copper nanoparticles, ruling out catalysis by ions leached into solution. This methodology is extended to mapping the catalytic activity of a commercial heterogeneous catalyst by applying super-localization analysis of single-catalytic events. The approach detailed herein is a general one that can be applied to any catalytic system through the development of appropriate probes. This thesis demonstrates single-molecule microscopy as an accessible, effective, and unparalleled tool for exploring the catalytic activity of nanomaterials by monitoring single-catalytic events as they occur.

Single-stranded DNA binding proteins (SSB) bind to single-stranded DNA (ssDNA) that is generated by molecular machines such as helicases and polymerases. SSBs play crucial roles in DNA translation, replication and repair and their importance is demonstrated by their inclusion across all domains of life. The homotetrameric E. coli SSB and the heterotrimeric human RPA demonstrate how SSBs can vary structurally, but all fulfil their roles by employing oligonucleotide/oligosaccharide binding (OB) folds. Nucleofilaments of SSB proteins bound to ssDNA sequester the ssDNA strands, and in doing so protect exposed bases, keep the ssDNA in conformations favoured by other proteins that metabolise DNA and also recruit other proteins to bind to ssDNA. This thesis focuses on the SSB from the archaeon S. solfataricus (SsoSSB), and has found SsoSSB to be a monomer that binds cooperatively to ssDNA with a binding site size of 4-5 nucleotides. Tagging ssDNA and SsoSSB with fluorescent labels allowed the real time observation of single molecule interactions during the initial nucleation event and subsequent binding of an adjacent SsoSSB monomer. This was achieved by interpreting fluorescent traces that have recorded combinations of FRET, protein induced fluorescent enhancement (PIFE) and quenching events. This novel analysis gave precise measurements of the dynamics of the first and second monomers binding to ssDNA, which allowed affinity and cooperativity constants to be quantified for this important molecular process. SsoSSB was also found to have a similar affinity for RNA, demonstrating a promiscuity not found in other SSBs and suggesting further roles for SsoSSB in the cell - possibly exploiting its capacity to protect nucleic acids from degradation. The extreme temperatures that S. solfataricus experiences and the strength of the interaction with ssDNA and RNA make exploring the application of SsoSSB for industrial uses an interesting prospect; and its rare monomeric structure provides an opportunity to investigate the action of OB folds in a more isolated environment than in higher order structures.

L'exocytose régulée, est un processus qui permet la communication entre les cellules à travers la sécrétion des hormones et des neurotransmetteurs. Dans les neurones et les cellules neuroendocrines, l'exocytose est strictement contrôlée par des signaux extracellulaires tels que le potentiel trans-membranaire et la fixation des ligands sur des récepteurs. Des progrès substantiels ont été effectués afin de comprendre le mécanisme moléculaire de l'exocytose. Les composants majeurs de la machinerie de sécrétion ont été dévoilés. Maintenant, la question qui émerge concerne le rôle de la plateforme de protéines qui semble avoir une action coordonnée entre chaque protéine. Dans le cas de la famille des annexines, qui est bien connue pour son action dans l'exocytose, leurs modes d'interactions séquentielles ou concertées avec d'autres protéines ainsi que leurs effets régulateurs sur l'exocytose ne sont pas encore bien établis. Des résultats précédents indiquent que l'Annexine A6 (AnxA6) affecte l'homéostasie du calcium et la sécrétion de la dopamine à partir des cellules PC12, utilisées comme un modèle cellulaire de neurosécrétion (Podszywalow Bartnicka et al., 2010). Afin de déterminer l'effet inhibiteur de l'AnxA6 sur l'exocytose de la dopamine, nous cherchons des partenaires moléculaires de l'AnxA6 dans les cellules PC12. Nous faisons l'hypothèse que l'AnxA6 interagit avec la PLD1, une enzyme active dans l'étape de la fusion des vésicules avec la membrane plasmique. En utilisant la microscopie confocale et la microscopie à onde évanescente, nous avons trouvé que l'isoforme 1 de l'AnxA6 et la PLD1 sont tous les deux recrutés sur la surface des vésicules au cours de la stimulation des cellules PC12. AnxA6 inhibait l'activité de la PLD comme indiqué par notre méthode d'analyse enzymatique au moyen de la spectroscopie infrarouge. En conclusion, nous proposons que l'AnxA6 n'est pas seulement impliquée dans la réorganisation des membranes par ses capacités à se lier avec des phospholipides négativement chargés et avec le cholestérol, mais elle influence également l'activité de la PLD1, changeant la composition lipidique des membranes
The regulated exocytosis is a key process allowing cell-cell communication through the release of hormone and neurotransmitters. In neurons and neuroendocrine cells, it is strictly controlled by extracellular signal such as transmembrane potential and ligand bindings to receptors. Substantial progress has been made to understand the molecular mechanism of exocytosis. Major components of secretory machinery have been brought to light. Now the emergent question concerns the role of scaffolding proteins that are thought to coordinate the action of each other. In the case of annexin family well known to be involved in exocytosis, their modes of –sequential or concerted- interactions with other proteins, and their regulatory effects on exocytosis are not very well established. Previous findings indicated that Annexin A6 (AnxA6) affected calcium homeostasis and dopamine secretion from PC12 cells, used as cellular model of neurosecretion (Podszywalow-Bartnicka et al., 2010). To determine the inhibitory effect of AnxA6 on exocytosis of dopamine, we were looking for molecular partners of AnxA6 in PC12 cells. We hypothesized that AnxA6 interacts with phospholipase D1 (PLD1), an enzyme involved in the fusion step. By using confocal microscopy and total internal reflection fluorescence microscopy, we found that isoform 1 of AnxA6 and Phospholipase D1 are both recruited on the surface of vesicles upon stimulation of PC12 cells. AnxA6 inhibited phospholipase D activity as revealed by our enzymatic assay based on infrared spectroscopy. To conclude, we propose that AnxA6 is not only implicated in membrane organization by its capacity to bind to negative charged phospholipids and to cholesterol, but AnxA6 is also affecting PLD1 activity, changing membrane lipids composition

In conventional tribological experiments that study friction and wear behavior of materials, the contact interface is hidden by the contacting bodies. The physical or chemical phenomena taking place at the interface must then be examined through intermittent tests. These kinds of postmortem studies have remained speculative and model based. The inadequacy of such analysis has long been recognized. For a fundamental understanding of interfacial phenomena, it is important to study interactions in real-time. The present work attempts to overcome this hidden-interface problem by developing a tribometer using optically transparent surfaces which can carry out real-time tribological experiments under a microscope equipped with a Raman spectrometer. Such a micro-Raman system enables combined optical imaging capability with chemical mapping and is consequently a promising tool for in situ studies in tribology. Chapter 2 describes the development of a novel in situ total internal reflection (TIR) Raman tribometery, a technique based on TIR Raman spectroscopy combined with tribological experiments. The in situ TIR-Raman tribometer test rig is based on a ball-on-flat geometry. The rotating ball is immersed in an oil bath and carries a film of lubricant while rotating as it rubs against a flat (transparent) surface. Raman spectra of the lubricant are recorded from the contact region through the transparent window as a function of load, time, and velocity. A unique advantage of the TIR technique is that the sample can be analyzed from a known depth beneath the interface using evanescent waves. The capabilities of this TIR Raman tribometer have been demonstrated by carrying out experiments with known solid lubricant material molybdenum disulfide (MoS2). MoS2 is known for its solid lubricant properties. Nanoparticles of MoS2 are used as an anti- wear additive in engine oil. The commissioning of the tribometer rig was carried out using MoS2 nanoparticles to lubricate a steel-steel contact. Before carrying out in situ Raman tribometry on MoS2 nanoparticles, a set of characteristic friction and wear data was obtained from MoS2 nanoparticle-lubricated steel- steel contact (chapter 3) and compared with the data obtained from a commercial tribometer. In addition, friction characteristics of dry MoS2 and MoS2- hexadecane lubricated steel-steel contact were studied as a function of temperature. The friction in the dry particle lubrication was found to increase with temperature while the friction in wet condition was found to decrease with increasing temperature. Micro-Raman and FTIR spectroscopy are used to explore the roles of environmental moisture and chemical degradation of oil on the formation of antifriction films on the steel substrate. Ex situ optical and Raman analysis revealed the formation of an anti-friction film on the steel substrate. To understand the underlying mechanism of nanoparticle lubrication, in situ Raman tribometry was performed and results are presented in Chapter 4. By combining in situ optical imaging and Raman spectroscopy, the sliding history in friction-induced material transfer of dry 2H-MoS2 particles in a sheared contact was studied. Video images in contact showed the fragmentation of lubricant particles and build-up of a transfer film. Contact imaging was used to measure the speed of fragmented particles in the contact region. Total internal reflection (TIR) Raman spectroscopy was used to follow the build-up of the MoS2 transfer film. The combination of in situ and ex-situ analysis of the mating bodies was used to understand the mechanism of transfer film formation in the early stages of sliding contact. Application of in situ TIR Raman tribometry for the study of liquid lubricants was demonstrated by using PAO to lubricate SF10 glass – silica contact and results are discussed in Chapter 5. It is well established that under high shear rates, synthetic base oils undergo shear thinning. Earlier studies of shear thinning relied mainly on viscosity measurements from which it is not possible to obtain information on molecular alignments or ordering. In the present study, TIR Raman spectra were used to estimate the thickness of the lubricant film under sliding conditions and polarized Raman data was used to study the alignment of molecules during shear thinning. The experimentally obtained film thickness data was superposed with theoretical calculations and a transition from Newtonian to non-Newtonian behavior was observed at high shear rates. Also, the effect of additive molecules on the shear thinning behavior of poly alpha olefin lubricant was studied. The in situ TIR Raman tribometer is a powerful tool to detect the physiochemical changes lubricants undergo at the hidden interface under sliding conditions. The capability of this technique in enabling tribological processes to be observed dynamically in real time with concomitant chemical changes at the interface has the potential to make it an indispensable tool in fundamental studies of tribological interactions. The application of total internal reflection resulted in significant signal enhancement which makes the technique developed in this study an important addition to the already available ones for future tribological studies. The information and insight generated in a range of tribological phenomena taking place at the hidden interface of contact will be useful in developing new lubricant materials.

Thesis (B.S.) Summa Cum Laude--Butler University, 2008.
Includes curriculum vitae. Appendix 2 includes the article: Kraning CM, Benz TL, Bloome KS, Campanello GC, Fahrenbach VS, Mistry SA, Hedge CA, Clevenger KD, Gligorich KM, Hopkins TA, Hoops GC, Mendes SB, Chang H-C, Su M-C (2007) Determination of surface coverage and orientation of reduced cytochrome c on a silica surface with polarized ATR spectroscopy. J. Phys. Chem. C 2007, 111:13062-13067. Includes bibliographical references (88-91).