Thesis (M. S.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006. / Bredas, Jean-Luc, Committee Member ; Sherrill, C. David, Committee Chair ; Hernandez, Rigoberto, Committee Member.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 180-198).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 185-203).

Title: Designing and testing of new metal nanosubstrates for biomolecular sensors based on surface-enhanced Raman scattering (SERS) spectroscopy Author: Vlastimil Peksa Department: Institute of Physics of Charles University Supervisor: doc. RNDr. Marek Procházka, Ph.D., Institute of Physics of Charles University Abstract: This experimental methodical work was aimed at the optimization of selected gold and silver substrates and their use in construction of SERS-based biosensors, including following practical application. Several types of substrates, fabricated via a combination of bottom-up techniques on solid surfaces, were tested. The properties of these substrates were examined with probe molecules, namely methylene blue, porphyrins and tryptophan, on a confocal Raman microspectrometer. Obtained findings about the influence of analyte application, objective focusing and internal intensity standard were exploited for optimization of measurement procedures with regard to sensitivity, accuracy and reproducibility. A method for quantitative detection of food dye azorubine (E 122) in commercially available drinks was developed, based on these findings. Its results have shown its potential as a pre-scan method for field application and preliminary testing. Keywords: Metal nanosubstrates, biomolecules,...

Biomolecule structural fluctuations determine function, regulating numerous biological processes My research has shed light on several interesting cases in which structural fluctuations have been identified to assess functional differences. Chapter 2 discusses the effects of structural rearrangement of the β2-β3 loop on the DNA binding affinity of the type 6 human papillomavirus E2 protein. Chapter 3 investigates the effects of phosphorylation on the C-terminal domain of Cdc37, a protein important in the Hsp90 chaperone cycle. Chapter 4 studies the effects on cyclycization on the conformational fluctuations of a γ-AApeptide used for high-throughput libraries. Chapter 5 is a structural study on a mini-fibril of spider dragline silk, in which a native-like ensemble was generated using temperature replica exchange. Chapter 6 investigates the structural features of repetitive motifs found in spider dragline silk when subject to both dope-like and fiber-like conditions. Chapter 7 elucidates conformational differences between the RXRα and the RXRβ ligand-binding domains and seeks to understand the atomic basis for different ligand binding affinities. This body of work has contributed to the understanding of conformational fluctuations and changes that occur in protein-DNA binding systems, drug-binding, regulation of chaperones via post-translations modifications and spider dragline silk.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Parte do custo de produÃÃo de biomolÃculas com elevada pureza e o rendimento deste processo depende das etapas de separaÃÃo e purificaÃÃo utilizadas. Nestas etapas, geralmente à necessÃrio utilizar tÃcnicas de cromatografia e um dos fatores que influenciam no seu elevado desempenho à o desenvolvimento de fases estacionÃrias ou adsorventes adequados. O presente trabalho investiga a adsorÃÃo de trÃs biomolÃculas-modelo (albumina de soro bovino - BSA, lisozima - LYS e celulase â CEL) em sÃlicas mesoporosas obtidas a partir de copolÃmeros tribloco como agentes direcionadores estruturais. Relatam-se diferentes procedimentos de sÃntese que visam modificar a quÃmica da superfÃcie e promover alteraÃÃes texturais como, por exemplo, o alargamento e/ou encurtamento dos poros, bem como a sua ordenaÃÃo mesoscÃpica. Na primeira parte da tese, foram sintetizadas (i) SBA-15 por duas rotas distintas, sol-gel e hidrotÃrmica, e (ii) SBA-16 por via hidrotÃrmica. Na segunda parte, amostras de SBA-15 com diferentes tamanhos de poros e comprimento de canais foram sintetizados utilizando 1,3,5-trimetilbenzeno (TMB), heptano e fluoreto de amÃnio (NH4F). O TMB foi utilizado para aumentar o diÃmetro dos poros e o heptano combinado com NH4F, para modificar o tamanho dos canais da SBA-15. Jà na terceira parte do trabalho, a acidez da SBA-15 foi modificada pela adiÃÃo de zircÃnio com trÃs razÃes molares Si/Zr distintas (5, 10 e 15). A adsorÃÃo das trÃs biomolÃculas-alvo foi estudada por experimentos em tanques agitados. Os resultados indicam que, para sÃlicas puras, maiores capacidades de adsorÃÃo sÃo obtidas quando o pH està prÃximo ao ponto isoelÃtrico da biomolÃcula. Resultados muito promissores foram encontrados para a adsorÃÃo de proteÃnas sobre as sÃlicas com maior diÃmetro de poros (acima de 10 nm), chegando a centenas de miligramas por grama de adsorvente. No caso dos materiais com zircÃnio, os melhores resultados foram encontrados para os materiais com a menor quantidade do referido heteroÃtomo (Si/Zr = 15), que apresentam textura similar ao suporte original. Isto sugere que pode haver uma acidez moderada Ãtima para a adsorÃÃo das biomolÃculas estudadas ou que o âexcessoâ de Ãtomos de zircÃnio leva a impedimentos estÃricos que causam uma reduÃÃo na capacidade de adsorÃÃo da biomolÃcula. / Part of the cost of production of biomolecules with high purity and yield depends on the separation and purification steps used. In these steps, it is usually necessary to use chromatography techniques and one of the factors that influence on its high performance is the development of stationary phases or suitable adsorbents. The present study investigates the adsorption of biomolecules three model (bovine serum albumin - BSA, lysozyme - LYS cellulase and - CEL) in mesoporous silica obtained from tri-block copolymers as agents drivers structure. We report different synthesis procedures aimed at modifying the surface chemistry and promoting textural changes, such as enlargement and/or pores, and the mesoscopic ordering. In the first part of this thesis were synthesized (i) SBA-15 by two different routes, sol-gel and hydrothermal and (ii) SBA-16 by hydrothermal. In the second part, SBA-15 samples with different pore sizes and channel length were s ynthesized using 1,3,5-trimethylbenzene (TMB), heptane and ammonium fluoride (NH4F). TMB was used to increase the pore diameter and heptane combined with NH4F to modify the size of the channels of SBA-15. In the third part of the work, the acidity of SBA-15 was modified by the addition of zirconium with three molar ratios Si/Zr distinct (5, 10 and 15). The adsorption of the three target biomolecules was studied by experiments in stirred tanks. The results indicate that for pure silicas, higher adsorption capacities are obtained when the pH is close to the isoelectric point of biomolecule. Very promising results for were found the adsorption of proteins on silicas with larger pore diameters (above 10 nm), up to hundreds of milligrams per gram of adsorbent. In the case of materials with zirconium, the best results were fou nd for the materials with the lowest amount of said heteroatom (Si/Zr = 15), which have similar texture to the original support. This suggests that there may be a moderate acidity which-enhances the adsorption of the studied biomolecules or excess zirconium atoms lead to steric hindrances causing a decrease in the adsorption capacity of the biomolecule

No description available.

The main goals in electroanalytical sensing are towards improved sensitivity and selectivity, or specificity, of an analyte. There are several approaches to achieving these goals with the main approach being modification of an electrode surface with synthetic or natural catalysts (enzymes), polymers and also utilisation of nanostructured materials. At present, there is a strong movement towards hybrid sensing which couple different properties of two or more surface modification approaches. In this thesis, a range of these surface modifications were explored for analysis and detection of two main analytes: the amino acid, tryptophan (Trp); and, the neurotransmitter, dopamine (DA). Specifically, this thesis aimed to utilise these methods to enhance the sensitivity and selectivity for Trp over an interferent, the indoleamine, melatonin (Mel); and, DA over the vitamin, ascorbic acid (AA). For Trp detection, immobilisation of an enzyme, Tryptophanase (Trpase) resulted in poor selectivity for the analyte. However, enhanced sensitivity and selectivity was achieved through pH manipulation of the electrolyte medium at a Nafion®-modified electrode surface for both Trp and Mel. At pH 3.0, the Mel and Trp anodic peak potentials were sufficiently resolved allowing for an LOD of 1.60 and 1.62 nM,respectively, and permitting the accurate analysis of Trp in a dietary supplement containing Mel. Multi-walled carbon nanotubes (MWCNTs) suspended in Nafion® exhibited further increases in the signal responses of these analytes at pH 3.0 and 7.4 with minimal change in the resolution of the anodic peaks. A lower sensitivity was, therefore, observed at the Nafion® and MWCNT modified electrode compared to the Nafion®-modified electrode at pH 3.0 with LODs of 0.59 and 0.80 nM exhibited for Trp and Mel, respectively. Enhanced selectivity for Trp in the presence of Mel can be achieved with MWCNTs in the presence of metallotetrasulphonated phthalocyanines (MTSPcs) particularly at pH 3.0, owing to cation exchange effects. However, the lack of sensitivity towards Trp, and even Mel, at this CoTSPc and MWCNT modified electrode remains a drawback. For DA, detection at the MWCNT and Nafion® surface resulted in improved sensitivity over that of both the bare electrode (613.0 nM) and the Nafion® modified electrode (1045.1 nM) with a calculated LOD of 133.9 nM at this layer. Furthermore, improvements in the selectivity of DA were achieved at the Nafion® and MWCNT modified electrode as exclusion of AA (150 μM) was achieved. At the MWCNT and CoTSPc surface, AA was excluded up to 130 μM with sensitivity for DA extending as low as 14.3 nM, far greater than observed for Trp and Mel. These concentrations are well within physiological concentration ranges and represent the most significant solution yet in terms of AA exclusion and enhanced sensitivity for DA. An examination of the surface layering by impedance spectroscopy and atomic force microscopy indicates that the success of the hybrid sensor utilising CoTSPc and MWCNTs lay in improved dispersion of MWCNTs and improved electron transfer kinetics, facilitated by the net charge of the materials present. This thesis, thus, showed the utility of a judicious selection of synthetic and biological catalysts, polymers and carbon nanomaterials towards a hybrid approach to the electrochemical sensing of Trp, Mel, DA and AA with focus on sensitivity and selectivity of these analytes.

L’objectif de cette thèse est de synthétiser de nouvelles surfaces spécifiques nécessaires à l’immobilisation des biomolécules ; visant à développer à terme un biocapteur pour la détection de pathogènes en industrie agroalimentaire. Cette nouvelle procédure de fonctionnalisation de surface consiste d’une part à greffer des molécules organiques sur un substrat métallique à partir d’une réaction électrochimique et d’autre part de synthétiser un monomère photopolymérisable sur tout type de surface. Ces surfaces sont enfin utilisées pour immobiliser les biomolécules. Ce procédé ainsi développé permet d’éliminer les multiples étapes, l’utilisation excessive de réactifs observés dans les protocoles classiques de fonctionnalisation de surface pour la capture de microorganismes. Deux stratégies de fonctionnalisation ont été investiguées : la polymérisation sur une plaque de platine et le dépôt de monocouche sur une surface d’or. La fonctionnalisation de surfaces ainsi que l’immobilisation de biomolécules ont été caractérisées par la spectroscopie Raman, la microbalance à cristal de quartz, la microscopie à force atomique (AFM) pour le premier et en plus la microscopie à fluorescence pour le second. Les résultats de la fonctionnalisation de surfaces par dépôt de polymère ont montré, une déstabilisation du polymère en présence de l’eau. Afin d’optimiser la synthèse, nous avons travaillé en milieu inerte, sous alumine activée. De plus, on note une large couverture de la zone spectrale des biomolécules par les signaux du polymère ; Pour le dépôt de monocouche, l’on a obtenu une surface très réactive, homogène. La diffusion Raman est la principale technique de caractérisation utilisée. Elle présente l'avantage d'être une méthode de caractérisation physico-chimique non destructive et non invasive. Longtemps délaissée dans les sciences du vivant, cette méthode apparaît maintenant particulièrement prometteuse grâce à un développement récent de spectromètres intégrés performants. La diffusion Raman sur la monocouche déposée montre une intensité accrue des signaux par l’utilisation de la surface d’or et un spectre plus dégagé conduisant à l’identification aisée des biomolécules après fixation. Elle permet non seulement d’identifier les bandes de vibrations de chaque groupement mais aussi la conformation des structures. Les résultats d’immobilisation ont montré que l’accroche des biomolécules sur les surfaces fonctionnalisées était spécifique. La fonctionnalisation de surface d’or par dépôt de monocouche constitue finalement une technique très rapide à mettre en œuvre, peu coûteuse permettant d’ancrer efficacement les biomolécules et peut être utilisée pour diverses applications. La synthèse du monomère photopolymérisable a été abordée et est en cours d’investigation. / In food processing industry, detecting bacteria or viruses is crucial. Nowadays, it can be achieved with microbiological tests but, it requires several days. The objective of the project was to synthesize new specific surfaces capable of biomolecules immobilization in order to develop a biosensor for the detection of various pathogenics in food-processing industry. This new procedure of surface functionalisation consists on one hand in anchoring organic molecules on a metallic substrate by an electrochemical reaction and on the other hand to synthesize a photocrosslinkable monomer on every type of surface. These surfaces are finally used to immobilize biomolecules. Two strategies of surface functionalisation were investigated: the polymerization on a platinium surface and the deposition of monolayer on a gold surface. Both processes were characterized by spectroscopy Raman, Quartz Crystal Microbalance, Atomic Force Microscopy and Fluorescence Microscopy. The results of the functionalisation of surfaces by deposition of polymer showed a destabilization of the polymer in presence of water. To optimize the synthesis, we worked in sluggish middle, under activated alumina. Furthermore, we noted a wide coverage of the spectral zone of biomolecules by the signals of the polymer; For the monolayer deposition, we obtained a very reactive and homogeneous surface. The Raman spectroscopy was the main technique used to the characterization. It presented the advantage to be a non-destructive and non invasive physico-chemical method. This method seemed now particularly promising due to a recent development of successful integrated spectrometers. Raman Spectroscopy showed an enhanced intensity of the signals by the use of the gold surface and a more clear spectrum well-to-do identification of biomolecules after binding. It allowed not only the identification of the bands of vibrations of every connection but also the conformation of the structures. The results of the immobilization showed that the grafting of biomolecules on functionalised surfaces was specific and efficient. The functionalisation of gold surface by monolayer deposition constituted at the end an efficient and low cost technique allowing to anchor biomolecules and can be used for multitude applications. The last step consisting of the synthesis of photocrosslinkable monomer was started and still investigated.

Biological molecules, or molecular machines, visit a continuum of conformational states as they go through work cycles required for their metabolic functions. Single-molecule cryo-EM of suitable in vitro systems affords the ability to collect a large ensemble of projections depicting the continuum of structures. This information, however, comes buried among typically hundreds of thousands of unorganized images formed under extremely noisy conditions and microscopy aberrations. Through the use of machine-learning algorithms, it is possible to determine a low-dimensional conformational spectrum from such data, with leading coordinates of the embedding corresponding to each of the system’s degrees of freedom. By determining occupancies—or free energies—of the observed states, a free-energy landscape is formed, providing a complete mapping of a system’s configurations in state space while articulating its energetics topographically in the form of sprawling hills and valleys. Within this mapping, a minimum-energy path can be derived representing the most probable sequence of transitions taken by the machine between any two states in the landscape. Along this path, an accompanying sequence of 3D structures may be extracted for biophysical analysis, allowing the basis for molecular function to be elucidated. The ability to determine energy landscapes and minimum-energy paths experimentally from ensemble data opens a new horizon in structural biology and, by extension, molecular medicine. The present work is based on a geometric machine-learning approach using manifold embedding to obtain this desired information, which has been shown possible on two experimental systems—the 80S ribosome and ryanodine receptor—through a previously-established framework termed ManifoldEM. First, this framework is incorporated into an advanced graphic user interface for public release, and augmented with a new method, POLARIS, for determining minimum-energy pathways. ManifoldEM is next applied on two new systems: vacuolar ATPase and the SARS-CoV-2 spike protein, and for both systems, several novel aspects of the machine’s function are observed. During this exposition, critical limitations and uncertainties of the framework are also presented, as have been found throughout its extended development and use. However, in the absence of ground-truth data, testing and validation of ManifoldEM is infeasible. As recourse, a protocol is next proposed for generating simulated cryo-EM data from an atomic model subjected to multiple conformational changes and experimental conditions, with several Hsp90 synthetic ensembles generated for analysis by ManifoldEM. Guided by results of these ground-truth studies, new insights are made into the origin of longstanding ManifoldEM problems, further motivating and informing the development of a new, comprehensive method for correcting them, termed ESPER. The ESPER method operates within the ManifoldEM framework and, as will be shown using both synthetic and experimentally-obtained data, ultimately results in substantial improvements to the previous work. Finally, numerous recommendations are laid out for guiding future work on the ManifoldEM suite, particularly aimed at its next public release.