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1
Liu, Yang, and 刘洋. "Free energy simulations of important biochemical processes." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/196036.
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Free energy simulations have been widely employed to compute the thermodynamic properties of many important biochemical processes. In the first part of this dissertation, two important biochemical processes, protonation/deprotonation of acid in solution and solvation of small organic molecules, are investigated using free energy simulations.
Accurate computation of the pKa value of a compound in solution is important and challenging. To efficiently simulate the free energy change associated with the protonation/deprotonation processes in solution, a new method of mixing Hamiltonian, implemented as an approach using a fractional protonin the hybrid quantum mechanics/molecular mechanics (QM/MM) scheme, is developed. This method is a combination of a large class of λ-coupled free-energy simulation methods and the linear combination of atomic potential approach. Theoretical and technical details of this method, along with the calculation results of the pKa value of methanol and methanethiol molecules in aqueous solution, are discussed. The simulation results show satisfactory agreement with experimental data.
Though the QM/MM method is one of the most useful methods in the modeling of biochemical processes, little attention has been paid to the accuracy of QM/MM methods as an integrated unit. Therefore, the solvation free energies of a set of small organic molecules are simulated as an assessment of ab initio QM/MM methods. It shows that the solvation free energy from QM/MM simulations can vary over a broad range depending on the level of QM theory / basis sets employed. Diffuse functions tend to over-stabilize the solute molecules in aqueous solution. The deviations pose a pressing challenge to the future development of new generation of MM force fields and QM/MM methods if consistency with QM methods becomes a natural requirement.
In the second part of the dissertation, the dynamic and energetic properties of two molten globule (MG) protein molecules, α-lactalbumin(α-LA) and monomeric chorismate mutase (mCM) are investigated using molecular dynamics simulations. The exploring of the molecular mechanism of protein folding is a never-settled battle while the properties of MG states and their roles in protein folding become an important question. The MGs show increased side chain flexibility while maintain comparable side-chain coupling compared to the native state, which partially explains the preserving of native-like overall conformation. The enhanced sampling method, temperature-accelerated molecular dynamics (TAMD), is used for the study of the hydrophobic interactions inside both biomolecules. The results suggest that these hydrophobic cores could overcome energy barriers and repack into new conformation states with even lower energies.
The repacking of the hydrophobic cores in MGs might be served as a criterion for recognizing the MGs in large class of biomolecules.published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy
2
Richmond, Graham J. "Collision-induced energy transfer in ground and excited state free radicals." Thesis, Heriot-Watt University, 2006. http://hdl.handle.net/10399/2156.
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This thesis describes studies of collisional energy transfer in two small, combustion relevant free radicals. Collision-induced electronic energy transfer (EET) between the B2r and A2 fj. states of the CH and CD radicals was investigated with the collision partners He, Ar, H2, N2, CO and C02 using a dispersed laser induced fluorescence technique. CH or CD radicals were prepared photolytically, and excited into a single rotational level in either of the B2::E-, v ==°or A211, v =1 levels. Wavelength dispersed, time-resolved emission was then recorded from the initial and product states. Microscopic rate constants for vibronically resolved transfer between the two electronic states were determined for each collision partner, as well as those for vibrational energy transfer in the A state and total removal to other, unobserved states. EET was demonstrated to be ubiquitous, occurring with all of the collision partners used in the study, with varying efficiencies. These relative efficiencies were found to correlate well with long range attractive forces exerted by the collider. No special enhanced efficiency was observed with those colliders chemically reactive towards CHID. Transfer from B2::E- to A2 1::,. was not greatly affected by the significant differences to the vibronic energy level structure -between CH and CD, demonstrating the inapplicability of empirical energy gap scaling laws to this process. The collisional evolution of alignment and orientation moments in the OH radical was investigated using polarisation spectroscopy (PS). One-Colour PS measurements were made using the first five P branch lines in the A2::E+ - x2n (0,0) band of OH, in the presence of fixed pressures of He, Ar or N2. The time delay between pump and probe laser pulses was varied to inspect the remaining alignment or orientation in the sample as a function of time after the pump pulse. The results of these experiments were analysed using a detailed theoretical treatment developed previously in the laboratory, producing rate constants and cross-sections for the collisional decay of alignment and orientation over the range of quantum states and collision partners studied. While some uncertainty exists in the numerical values of the results, due to the observation of an apparently pressure-independent depolarisation process, clear phenomenological distinctions were found between the collision partners He, Ar and N2, over the range of studied quantum states.
3
Karis, Klas. "The Free Energy of Vesicular Transmembrane Lipid Transfer Studied With Molecular Dynamics Simulations." Thesis, KTH, Teoretisk fysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-152473.
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Crichton, Hilary Jane. "Spectroscopic probes of collisional energy transfer in ground and excited state free radicals." Thesis, Heriot-Watt University, 2004. http://hdl.handle.net/10399/342.
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Adrian, Marlene [Verfasser]. "Energy transfer in free-standing van der Waals heterostructures after optical excitation / Marlene Adrian." Kassel : Universitätsbibliothek Kassel, 2019. http://d-nb.info/1187165239/34.
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Ermantraut, Andreas [Verfasser], Ingo [Akademischer Betreuer] Krossing, and Thorsten [Akademischer Betreuer] Koslowski. "The experimental determination of the Gibbs free energy of transfer of single Ions without sxtra‐thermodynamic assumptions." Freiburg : Universität, 2018. http://d-nb.info/1165503239/34.
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Okazaki, S., N. Yoshii, and K. Fujimoto. "Molecular dynamics study of free energy of transfer of alcohol and amine from water phase to the micelle by thermodynamic integration method." AIP Publishing, 2012. http://hdl.handle.net/2237/20838.
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Okazaki, S., N. Yoshii, and K. Fujimoto. "Molecular dynamics study of solubilization of immiscible solutes by a micelle: Free energy of transfer of alkanes from water to the micelle core by thermodynamic integration method." AIP Publishing, 2010. http://hdl.handle.net/2237/20837.
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Öhman, Amanda. "Towards a fossil free steel sector : Conditions for technology transfer of hydrogenbased iron and steel in Europe." Thesis, KTH, Skolan för industriell teknik och management (ITM), 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-264106.
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In order to meet the targets of the Paris Agreement, there is a need to significantly reduce emissions from energy-intensive industries, iron and steel included. One promising technology with the potential to reduce the emissions related to iron and steelmaking to basically none is direct reduction with fossil free hydrogen, which requires large amounts of fossil free electricity. This master thesis explores the conditions for this technology in a European context with an energy perspective as the main focus. Three primary steel producing countries in Europe are chosen as focus countries; Germany, France and Italy. The findings of the study conclude that neither of the focus countries is an optimal sociotechnical fit for hydrogen-based direct reduction for iron and steel production at present. France is the country with the best conditions from a solely energy perspective but lacks some important factors for an enabling environment for technology transfer. Germany on the other hand have the most promising characteristics for an enabling environment but still face large challenges when it comes to power sector decarbonisation. In order to overcome the barriers and create an enabling environment it is key that energy and industry transitions are aligned, that a policy framework that supports these transitions is in place and that key actors representing all aspects of the transition cooperate; from industry to research, academia, policymakers and others. The findings also show that the current locations of the primary steel plants are in many cases not where the most favourable conditions for renewable power generation are and given the renewable capacity and transmission limitations of today, merely switching to a hydrogenbased process is not likely viable. A future configuration could be decentralised value chains where the different processes are located where there are optimal conditions e.g. that either hydrogen or sponge iron is produced where there are favourable power conditions and then transported to steel plants for the remaining processes in the value chain.För att nå målen uppsatta i Parisavtalet behöver energiintensiva industrier kraftigt minska sina utsläpp, däribland järn- och stålindustrin. Direktreduktion med fossilfri vätgas är en teknologi med potential att minska utsläppen från järn och ståltillverkning till praktiskt taget noll men kräver stora mängder fossilfri el. Detta examensarbete undersöker de energimässiga förutsättningarna för denna teknik i en europeisk kontext. Tre länder som producerar primärstål är utvalda som fokusländer i studien; Tyskland, Frankrike och Italien. Resultaten av studien visar att inget av de utvalda länderna i dagsläget har optimala sociotekniska förutsättningar för tekniken. Frankrike är det land med de bästa energimässiga förutsättningarna men saknar några viktiga faktorer för att vara en möjliggörande socioteknisk miljö. Tyskland å andra sidan har de mest lovande förutsättningarna för en lämplig socioteknisk miljö men står inför utmaningar när det kommer till energisystemet och tillgången på fossilfri el. För att skapa förutsättningar för denna teknik är det viktigt med koordinerade omställningar i energisektorn och industrin, policys som möjliggör dessa omställningar samt ett väl fungerande samarbete mellan industrin, akademin, beslutsfattare och andra viktiga aktörer. Studien visar också att de platser där nuvarande stålverk för primärstål finns inte har de bästa förutsättningar för förnybar elproduktion och att en vätgasbaserad process inte är optimal, baserat på den förnybara kapaciteten och de transmissionsbegränsningar som finns idag i elsystemet. Det finns istället möjlighet till decentraliserade värdekedjor, där varje process placeras där de mest lämpliga förhållandena finns. Detta kan exempelvis innebära att vätgas eller järnsvamp produceras där tillgången till fossilfri el är god, för att sedan transporteras till stålverken för de resterande processtegen.
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Björk, Erik. "Energy Efficiency Improvements in Household Refrigeration Cooling Systems." Doctoral thesis, KTH, Tillämpad termodynamik och kylteknik, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-93061.
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This thesis is based on eight articles all related to the characteristics of the cooling system and plate evaporator of a household refrigerator. Through these articles, knowledge is provided that can be used to increase the operational efficiency in household refrigeration. Papers A, B and C focus on heat transfer and pressure drop in a commonly used free convection evaporator – the plate evaporator. Applicable correlations are suggested on how to estimate the air side heat transfer, the refrigerant side pressure drop and the refrigerant side heat transfer. Papers D, E and F hold a unique experimental study of the refrigerant charge distribution in the cooling system at transient and steady state conditions. From this cyclic losses are identified and estimated and ways to overcome them are suggested. In paper G the topic “charging and throttling” is investigated in an unparalleled experimental study based on more than 600 data points at different quantities of charge and expansions device capacities. It results in recommendations on how to optimize the capillary tube length and the quantity of refrigerant charge. Finally, Paper H holds a thermographic study of the overall cooling system operating at transient conditions. Overall, a potential to lower the energy use by as much as 25 % was identified in the refrigerator studied. About 10 % was found on the evaporator’s air side. 1-2 % was identified as losses related to the edge effect of the evaporator plate. About 8 % was estimated to be cyclic losses. About 5 % was found in cycle length optimization. It is believed that most of these findings are of general interest for the whole field of household refrigeration even though the results come from one type of refrigerator. Suggestions of simple means to reduce the losses without increasing the unit price are provided within the thesisQC 20120411
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Ryberg, Per. "Concerted or Stepwise? : β-Elimination, Nucleophilic Substitution, Copper Catalysed Aziridination and Ruthenium Catalysed Transfer Hydrogenation Studied by Kinetic Isotope Effects and Linear Free-Energy Relationships." Doctoral thesis, Uppsala universitet, Avdelningen för organisk kemi, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-2008.
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This thesis describes the use of kinetic isotope effects, linear free energy relationships and stereochamical studies to distinguish between different mechanistic alternatives and to obtain information about transition state structure. In the first part fluorine and deuterium kinetic isotope effects were determined for the base promoted HF elimination from 4-fluoro-4-(4’-nitrophenyl)butane-2-on. During this work a new method for the determination of fluorine kinetic isotope effects was developed. The results from the study demonstrates that the reaction proceeds via an E1cBip mechanism. In the second part the transition state structure for the SN2 reaction between ethyl chloride and cyanide ion in DMSO was studied. Kinetic isotope effects for six different positions in the reacting system, both in cyanide and ethyl chloride, were determined. The experimental isotope effects were then compared with the theoretically predicted isotope effects. The third part describes the use of Hammett type free-energy relationships and stereochemical evidence to study the mechanism of the copper catalysed alkene aziridination. The results from the study support a model that involves the simultaneous presence of two different copper nitrene intermediates. One which reacts non-stereospecifically via a radical intermediate and one which reacts stereospecifically via a concerted mechanism. In the fourth part a mechanistic study of the Ru(aminoalcohol) catalysed transfer hydrogenation of acetophenone in isopropanol is described. Kinetic isotope effects were determined for both proton and hydride transfer. The observation of significant primary deuterium kinetic isotope effects for both proton and hydride transfer support a mechanism where the proton and hydride are transferred simultaneously in a concerted mechanism.
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Han, Yunqing. "THEORETICAL STUDY OF THERMAL ANALYSIS KINETICS." UKnowledge, 2014. http://uknowledge.uky.edu/me_etds/35.
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In the past decades, a great variety of model fitting and model free (isoconversional) methods have been developed for extracting kinetic parameters for solid state reactions from thermally stimulated experimental data (TGA, DSC, DTA etc.). However, these methods have met with significant controversies about their methodologies. Firstly, model-fitting methods have been strongly criticized because almost any reaction mechanism can be used to fit the experimental data satisfactorily with drastic variations of the kinetic parameters, and no good criterion exists to tell which mechanism is the best choice. Secondly, previous model free methods originated from the isoconversional principle, which is often called the basic assumption; previous studies comparing the accuracy of model free methods have not paid attention to the influence of the principle on model free methods and, therefore, their conclusions are problematic.
This work gives, firstly, a critical study of previous methods for evaluating kinetic parameters of solid state reactions and a critical analysis of the isoconversional principle of model free methods. Then an analysis is given of the invariant kinetic parameters method and recommends an incremental version of it. Based on the incremental method and model free method, a comprehensive method is proposed that predicts the degree of the dependences of activation energy on heating programs, and obtains reliable kinetic parameters. In addition, this work also compares the accuracy of previous methods and gives recommendations to apply them to kinetic studies.
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Zelleke, Theodros Gaschau Tena [Verfasser], Dominik [Gutachter] Marx, and Lars [Gutachter] Schäfer. "Computer simulations of methylamine dehydrogenase : free energy landscape and proton transfer pathways in oxidative deamination of methylamine / Theodros Gaschau Tena Zelleke ; Gutachter: Dominik Marx, Lars Schäfer ; Fakultät für Chemie und Biochemie." Bochum : Ruhr-Universität Bochum, 2018. http://d-nb.info/1161942068/34.
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Zelleke, Theodros [Verfasser], Dominik [Gutachter] Marx, and Lars [Gutachter] Schäfer. "Computer simulations of methylamine dehydrogenase : free energy landscape and proton transfer pathways in oxidative deamination of methylamine / Theodros Gaschau Tena Zelleke ; Gutachter: Dominik Marx, Lars Schäfer ; Fakultät für Chemie und Biochemie." Bochum : Ruhr-Universität Bochum, 2018. http://d-nb.info/1161942068/34.
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Holmgren, Tomas. "Emissions of organic compounds from technosphere articles : Measurements and modeling of mass transfer from consumer goods and building materials to air and water." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-66363.
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This thesis describes the development of a generic model for predicting the emissions of organic compounds from materials used in the manufacture of various goods and products. Many products contain organic substances that are not bound to the matrix formed by their constituent materials and are thus able to dissociate from the material and become transferred into the surrounding environment. A wide range of materials and products are used in modern societies, and many compounds deriving from these materials are regarded as emerging pollutants in both indoor and outdoor environments. The model uses three components to describe the transfer of compounds from materials to the surrounding environment: partitioning of the compound between the material and its surroundings based on linear free energy relationships, diffusion within the material based on the Piringer equation, and convective mass transfer in air or water based on an empirical flat surface model. The model’s predictive capacity was tested against three experimental case studies: emissions of plasticizers from vinyl flooring and triphenyl phosphate from LCD screens into the air, and leaching of organophosphates from concrete into water. The rates of emission from vinyl flooring were clearly affected by the number of layers comprising the material. Triphenyl phosphate was found in the front surface of all tested flat screens and its rates of emission were related to the nature of the screen and its operating temperature. The model accurately predicted emissions into the air and leaching from concrete into water once modified to include modules that describe dissolution from surfaces and diffusion in water-filled pores. The model was then used to investigate emissions on the national scale. It was found that the rates of emission from vinyl flooring are not changing over time, and that the total mass of emitted material is dependent on annual sales volumes and the expected life span of the vinyl flooring. Moreover, the additive used has a large effect on the emitted mass. Emissions from flat screen displays depend strongly on their operating temperatures: displays with high working temperatures that are active for extended periods of time produce more emissions. The model was also used to study the release of organophosphates from the concrete used to make a bridge, which depended on the flow of water under the bridge, the temperature, the porosity of the concrete, and the additive’s water solubility. Data on annual sales volumes and the total surface area of sold goods are essential when studying emissions on a national scale. National retailers’ organizations are valuable sources of such information. When adequate data are not available, it is necessary to perform uncertainty analyses to determine the impact of uncertainty in the modeling of different stages of the emissions process in different scenarios.
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Herrera, Andrés Medina. "Estrutura eletrônica da amino- e dimetilamino-benzonitrila em meio usando métodos híbridos de QM/MM." Universidade Federal de Goiás, 2015. http://repositorio.bc.ufg.br/tede/handle/tede/5123.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
In this research we studied the structural and electronic properties of the ground state of molecules amino-benzonitrile (ABN) and dimethylamino-benzonitrile (DMABN), isolated and in different solvents.We performed computer simulations of those molecules in different solvents as cyclohexane, dichloromethane, acetonitrile and water. The structure electronic method MP2 (second order perturbation Møller-Plesset) was used to perform quantum calculations. To study the molecules in solvent we used the hybrid sequential QM/MM method combined with the free energy gradient method. The dual fluorescence to this type of molecules is a process that has been much studied but it is not well clarified that is the cause of the process. We performed the optimization of the molecules in an isolated state and in different solvents to determine the ground state structure. In the case of the DMABN molecule the optimization was performed both at room temperature and at low temperature, near the melting point of the solvent. We studied minimum energy point and some transition states of this molecules associated with the pyramidalization or the rotation of the amino group. The results showed that the molecules are pyramidal when they are isolated, and that in polar solvent they became less pyramidal. The rotation of amino group is unfavored in both molecules, increasing this effect in polar solvents.
Neste trabalho foram estudadas as propriedades estruturais e eletrônicas do estado fundamental das moléculas amino-benzonitrila (ABN) e dimetilamino-benzonitrila (DMABN), isoladas e em diferentes meios solventes. Foram realizadas simulações computacionais das moléculas em diferentes meios como, ciclohexano, diclorometano, acetonitrila e água. O método de estrutura eletrônica MP2 (Møller-Plesset em segunda ordem de perturbação) foi usado para fazer os cálculos quânticos. Para o estudo das moléculas em meio foi utilizado o método hibrido QM/MM sequencial combinado com o método de gradiente de energia livre. A dupla fluorescência para este tipo de moléculas é um processo que tem sido bastante estudado, mas ainda não está bem esclarecido qual é o causador do processo. Foram realizadas as otimizações das moléculas em estado isolado e nos diferentes meios, para determinar a estrutura do estado fundamental. No caso da molécula de DMABN a otimização foi feita tanto em temperatura ambiente como em baixas temperaturas, próximas do ponto de fusão dos solventes. Foram estudados pontos de mínimo e alguns estados de transição dessas moléculas associados à piramidalização ou à rotação do grupo amino. Os resultados mostram que essas moléculas são piramidais quando isoladas, e que em meio polar elas se tornam menos piramidais. A rotação do grupo amino é desfavorável em ambas as moléculas, aumentando esse efeito em meios polares.
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Blagoi, Gabriela. "Fluorescence resonance energy transfer (FRET) based sensors for bioanalysis." ScholarWorks@UNO, 2004. http://louisdl.louislibraries.org/u?/NOD,146.
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Thesis (Ph. D.)--University of New Orleans, 2004.Title from electronic submission form. "A dissertation ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry."--Dissertation t.p. Vita. Includes bibliographical references.
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Leveneur, Sébastien. "Catalytic synthesis and decomposition of peroxycarboxylic acids." Phd thesis, INSA de Rouen, 2009. http://tel.archives-ouvertes.fr/tel-00560883.
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L'objectif de cette thèse fut de développer un process pour la production d'acide peroxycarbolique à partir du peroxyde d'hydrogène et d'un acide carboxylique dans un réacteur continu. Dans un premier temps, la stabilité des espèces peroxydées fut étudiée en utilisant une méthode d'analyse en direct (spectromètre de masse). Un effort particulier a été apporté pour trouver un catalyseur hétérogène ne provoquant pas la décomposition des espèces peroxydées et ayant une activité catalytique similaire à l'acide sulfurique. Un réacteur en continu en lit fixe a été construit en utilisant des résines échangeuses de cation.
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Vaccaro, Carlos. "Use of luminescence energy transfer probes to detect genetic variants." Thesis, University of North Texas, 2004. https://digital.library.unt.edu/ark:/67531/metadc4566/.
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The purpose of this research was to study the hybridization of molecular beacons under different conditions and designs. Data collected suggest that the inconsistency found in the emission intensity of several of these probes may be caused by 3 important factors: length of the probe, nucleotide sequence and, the formation of an alternative complex structure such as a dimer. Of all three factors, dimer formation is the most troublesome, since it reduces the emission of the reporter molecules. A new probe design was used to reduce dimer formation. The emission signal of the improved probe was several folds stronger than those probes with the early design.
In this research, dimer formation is detected, furthermore a new probe with a different design was tested. If dimer formation can be reduced molecular beacons can be integrated into more complex hybridization systems providing an important tool in research and diagnosis of genetic disorders.
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Beck, Michael, Niko Hildebrandt, and Hans-Gerd Löhmannsröben. "Quantum dots as acceptors in FRET-assays containing serum." Universität Potsdam, 2006. http://opus.kobv.de/ubp/volltexte/2006/950/.
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Quantum dots (QDs) are common as luminescing markers for imaging in biological applications because their optical properties seem to be inert against their surrounding solvent. This, together with broad and strong absorption bands and
intense, sharp tuneable luminescence bands, makes them interesting candidates for methods utilizing Förster Resonance Energy Transfer (FRET), e. g. for sensitive homogeneous fluoroimmunoassays (FIA). In this work we demonstrate
energy transfer from Eu3+-trisbipyridin (Eu-TBP) donors to CdSe-ZnS-QD acceptors in solutions with and without serum. The QDs are commercially available CdSe-ZnS core-shell particles emitting at 655 nm (QD655). The FRET system was achieved by the binding of the streptavidin conjugated donors with the biotin conjugated acceptors. After excitation of Eu-TBP and as result of the energy transfer, the luminescence of the QD655 acceptors also showed lengthened decay times like the donors. The energy transfer efficiency, as calculated from the decay times of the bound and the unbound components, amounted to 37%. The Förster-radius, estimated from the absorption and emission bands, was ca. 77 Å. The effective binding ratio, which not only depends on the ratio of binding pairs but also on unspecific binding, was obtained from the donor emission dependent on the concentration. As serum promotes unspecific binding, the overall FRET efficiency of the assay was reduced. We conclude that QDs are good substitutes for acceptors in FRET if combined with slow decay donors like Europium. The investigation of the influence of the serum provides guidance towards improving binding properties of QD assays.
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Windsor, Kramer Michelle Anne. "Development of a FRET biosensor to detect the pathogen mycoplasma capricolum." Diss., Columbia, Mo. : University of Missouri-Columbia, 2005. http://hdl.handle.net/10355/4289.
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Thesis (M.S.)--University of Missouri-Columbia, 2005.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (January 11, 2006) Includes bibliographical references.
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Li, Xuelian. "USING CONJUGATED POLYMERS AS BIOLOGICAL SENSOR BASED ON FLUORESCENCE RESONANCE ENERGY TRANSFER." OpenSIUC, 2011. https://opensiuc.lib.siu.edu/dissertations/321.
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J E. coli On-Off &ldquo, &rdquo, °, &ndash The specific objectives of the work presented in this dissertation are to design novel molecular sensors based on fluorescence resonance energy transfer (FRET) between fluorophore (donor) and polydiacetylene (PDA, acceptor) for selective detection of biomolecules in solution. The work described in this dissertation is divided into three sections. In the first section, we report here two novel systems where the rate of energy transfer is based on changes in the spectral overlap between the emission of the donor and the absorption of the acceptor (J) as well as changes in the quantum yield of the acceptor. In the second section, we discuss modified these high sensitive molecular sensors based on FRET by using different receptors for selective detection of biomolecules such as proteins or bacteria in solution. The third section develops reversibility studies on FRET based sensors in solution or solid state. In the Chapters two and three, conjugated polydiacetylene (PDA) possessing stimuli-responsive properties have been intensively investigated for developing efficient sensors. Sensors based on FRET between conjugated polymers and fluorophores can be more sensitive than colorimetric based sensors. We use the fluorophore dansyl as the donor and polydiacetylene (PDA) as the acceptor to demonstrate the modulation of FRET efficiency through conformationally induced changes in the PDA absorption spectrum following thermal treatment that converts the PDA backbone of the liposome from the blue form to the red form. We have used steady-state electronic absorption, emission and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). Energy transfer was found to be significantly more efficient from dansyl to the red-form PDA. This is due to large increase in the J values between dansyl emission and absorption red-form of PDA. We also have found that the monomer ratio of acceptor to donor (Rad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. A decrease in Rad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. Increase in Rad led to increase probability of FRET from donor to acceptor as larger number of acceptors are present around a given donor. The competition between donor for energy transfer increases with decrease in Rad that contributed to lower FRET efficiency between donors and acceptors. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to the insertion of dansyl in the bilayer of the liposomes which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. Much larger emission amplification for FRET was observed as compared to direct-excitation of PDA. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. These findings support the basis of a new sensing platform that utilizes J-modulated FRET as an actuating mechanism. A FRET based protein sensor by using sulforhodamine 101 as donor and PDA as acceptors was developed. This novel FRET based system primarily utilizes changes in J values (the spectral overlap between the emission of the donor and absorption of the acceptor) for the modulation of FRET efficiency between donors and acceptors. These FRET based sensors can be modified by tagged receptors (for proteins, viruses, and bactria) onto PDA liposomes which can interact with ligands present on proteins or bacteria. The biotin-streptavidin interactions were used as a sensing model system to test our FRET sensor response. In chapter 4, four different biotin-tagged lipids were used as receptors to investigate the effect of interactions between ligand-receptors on the FRET efficiency. The biotin was covalently linked to the liposome surface when using biotin-tagged diacetylene; whereas the biotin-tagged lipids with hydrophobic chains but without diacetylene functionalities provided non-covalently inserted lipids in liposomes. These studies were used to elucidate the effect of molecular interactions on FRET sensor response. The conjugated polymerized liposomes consisted of sulforhodamine-tagged-diacetylene and receptors linked lipids in different molar ratios. The characterization of the liposomes and sensing mechanism was investigated using UV-Vis and steady-state emission spectroscopy. The liposome solution yielded a weak donor emission (sulforhodamine 101) from after photo-polymerization of diacetylene monomers. This is due to energy transfer from the donor to PDA backbone chains (acceptors). The addition of streptavidin which interacted with biotin receptors resulted in increase in the sulforhodamine 101 emissions. The stress, due to interactions between biotin and streptavidin, induced the chromatic shift in the absorption spectrum of PDA which led to a decrease in the spectral overlap (J) between the emission spectra of donor and the absorption spectra of acceptor, leading to a decrease in the FRET efficiency from sulforhodamine 101 to PDA. These sensors, thus, show an "On-Off" type optical mechanism based on FRET between fluorophores and PDA where the donor emission was highly quenched in the "Off" state but was turned "On" due to receptor-ligand interactions. Large electronic absorbance and emission intensity differences between covalently and non-covalently bound biotin liposome systems were observed which indicated that the molecular interactions between biotin and PDA backbone play a crucial role in the FRET sensor response. In Chapter 5, we also developed FRET sensor for the detection of E. coli in aqueous media. Two glucose-based receptors were used in this study: (1) glucose-tagged lipid which can be inserted non-covalently in the bilayer of liposome, and (2) glucose-tagged diacetylene monomer in which the receptors were covalently bound to the backbone of the PDA liposome. The steady-state UV/Vis absorbance and fluorescence emission spectroscopy, and the fluorescence microscopy analysis of the receptors-containing liposomes were investigated for the detection of E. coli. The blue shift in the absorption spectrum of the conjugated PDA backbone induced through the interactions between receptors and bacteria resulted in decrease in the spectral overlap between the emission of SR-101 (donor) and the absorption of PDA (acceptor). This, ultimately, led to change in FRET efficiency between SR-101 and PDA after glucose - E. coli binding and caused increase in the emission intensity of SR-101. Polydiacetylenes have been exploited because of their sensitivity to external stimuli, such as temperature, pH, ions, and ligands. Unfortunately, the majorities of the sensors developed are not reversible but used as a one-time use. Here we report our preliminary results of a benzoic acid monomer of polydiacetylene (PDA-mBzA) to investigate reversible FRET characteristics between fluorophore and PDA. The LS films containing dansyl-tagged-diacetylene monomers and m-aminobenzoic acid derivatized- diacetylene monomers in different molar ratios were self-assembled and polymerized. The UV/Vis and steady-state fluorescence emission analysis of these LS films were investigated. These systems have shown partial reversible FRET over many "on-off" cycles. We believe that this incomplete FRET reversibility is due to liposomes preparation conditions used for liposomes which decreased PDA-mBzA amount in liposomes. We also reported reversible FRET studies on the liposome solutions, made from the monomer of m-aminobenzoic acid derivatized-10,12-pentacosadiynoic acid (PDA-mBzA) monomers and 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA) or dansyl-tagged diacetylene. After photo-polymerization, the solution appeared blue in color at room temperature. Heating and cooling cycles (between 25 ºC and 95 ºC temperature range), illustrated a visible color change from blue to red and a complete return to blue over many thermal cycles. Our preliminary reversible absorption and emission measurements showed that there exist opportunities for reversibility in FRET response. We are now performing more experiments to increase the FRET reversibility in these experiments. Although our system does not display full reversibility, the preliminary absorption and emission measurements strongly suggest that there exist opportunities for fully reversible selective and sensitive FRET-based sensors after further optimization of the system.
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Balloi, Eleonora. "Investigating conformational changes of proteins using Förster Resonance Energy Transfer." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/investigating-conformational-changes-of-proteins-using-frster-resonance-energy-transfer(fd5f53e7-9464-40bf-9c87-6fbd0a3d8804).html.
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Förster Resonance Energy Transfer (FRET)-based techniques are gaining an increasing importance in cell biology and cell-matrix adhesion studies because they allow both the detection of conformational changes of target proteins and their localisation in cells. Frequency Domain-Fluorescence Lifetime Microscopy (FD-FLIM) is currently considered one of the most reliable methods to measure FRET in live cells. However, due to its dependence on many technical prerequisites, its use is not yet widespread. The purpose of this work was to first establish FD-FLIM measurements of FRET on a new FD-FLIM microscope module. Then we aimed to apply FD-FLIM-FRET measurements to the study of conformational changes of the cell matrix-adhesion proteins vinculin and integrin and of the growth factor receptor Tie-2. In the first part of the work, published FRET probes including distance-sensors and two sets of vinculin-based probes were extensively tested with FD-FLIM, sensitised emission and ratiometric FRET. FD-FLIM was shown to be the most accurate method in approximating molecular distances between fluorophores. Moreover this study unveiled specific caveats associated with both existing vinculin FRET probes. FD-FLIM was then used to study conformational changes of the extracellular matrix receptor alphavβ3 integrin and of the angiopoietin receptor Tie-2 using specific FRET probes designed by us. While data showed that the alphav-integrin-FRET probe localised to adhesion sites, more experiments will be required to evaluate its full functionality. The Tie-2-FRET probe was fully functional and, upon ligand binding, allowed the detection of a bending movement of the extracellular domain towards the cell membrane. Finally, a combination of FRET, immunofluorescence and tension release experiments were used to show that intracellular tension is not required to maintain integrins in their activated conformation. However, intracellular tension is required to recruit other key proteins such as vinculin, talin and tensin to adhesions sites. Overall this work demonstrates the importance of FD-FLIM-FRET as a tool to investigate conformational changes of adhesion proteins and transmembrane receptors within the cell environment.
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Merckx, R., Thomas Swift, R. Rees, Guyse J. F. R. Van, E. Schoolaert, Clerck K. De, H. Thienpont, and V. V. Jerca. "Förster resonance energy transfer in fluorophore labeled poly(2-ethyl-2-oxazoline)s†." Royal Society of Chemistry, 2020. http://hdl.handle.net/10454/18374.
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YesDye-functionalized polymers have been extensively studied to understand polymer chain dynamics, intra or inter-molecular association and conformational changes as well as in practical applications such as signal amplification in diagnostic tests and light-harvesting antennas. In this work, the Förster resonance energy transfer (FRET) of dye-functionalized poly(2-ethyl-2-oxazoline) (PEtOx) was studied to evaluate the effect of dye positioning and polymer chain length on the FRET efficiency. Therefore, both α (initiating terminus)- or ω (terminal chain end)-fluorophore single labeled and dual α,ω-fluorescent dye labeled PEtOx were prepared via cationic ring opening polymerization (CROP) using 1-(bromomethyl)pyrene as the initiator and/or 1-pyrenebutyric acid or coumarin 343 as the terminating agent, yielding well-defined PEtOx with high labeling efficiency (over 91%). Fluorescence studies revealed that intramolecular FRET is most efficient for heterotelechelic PEtOx containing both pyrene and coumarin 343 fluorophores as chain ends, as expected. A strong dependence of the energy transfer on the chain length was found for these dual labeled polymers. The polymers were tested in both dilute organic (chloroform) and aqueous media revealing a higher FRET efficiency in water due to the enhanced emissive properties of pyrene. The application of dual labeled polymers as fluorescent probes for temperature sensing was demonstrated based on the lower critical solution temperature behavior of the PEtOx. Furthermore, these polymers could be successfully processed into fibers and thin films. Importantly, the fluorescence properties were retained in the solid state without decreasing the FRET efficiency, thus opening future possibilities for application of these materials in solar cells and/or sensors.
The full-text of this article will be released for public view at the end of the publisher embargo on 8 Sep 2021.
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Dennis, Allison Marie. "Quantum dot-fluorescent protein pairs as fluorescence resonance energy transfer pairs." Diss., Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/37079.
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Fluorescence resonance energy transfer (FRET)-based biosensors have been designed to fluorometrically detect everything from proteolytic activity to receptor-ligand interactions and structural changes in proteins. While a wide variety of fluorophores have demonstrated effectiveness in FRET probes, several potential sensor components are particularly notable. Semiconductor quantum dots (QDs) are attractive FRET donors because they are rather bright, exhibit high quantum yields, and their nanoparticulate structure enables the attachment of multiple acceptor molecules. Fluorescent proteins (FPs) are also of particular interest for fluorescent biosensors because design elements necessary for signal transduction, probe assembly, and device delivery and localization for intracellular applications can all be genetically incorporated into the FP polypeptide.
The studies described in this thesis elucidate the important parameters for concerted QD-FP FRET probe design. Experimental results clarify issues of FRET pair selection, probe assembly, and donor-acceptor distance for the multivalent systems. Various analysis approaches are compared and guidelines asserted based on the results. To demonstrate the effectiveness of the QD-FP FRET probe platform, a ratiometric pH sensor is presented. The sensor, which uses the intrinsic pH-sensitivity of the FP mOrange to modulate the FP/QD emission ratio, exhibits a 20-fold change in its ratiometric measurement over a physiologically interesting pH range, making it a prime candidate for intracellular imaging applications.
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Martin, Sarah Friede. "Fluorescence resonance energy transfer studies of protein interactions." Thesis, St Andrews, 2008. http://hdl.handle.net/10023/537.
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Chen, Chi. "Lanthanide Energy Transfer Donors on Nanoparticles Surfaces : From Fundamental Mechanisms to Multiplexed Biosensing." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS196/document.
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Le multiplexage optique basé sur des nanoparticules offre de nombreux avantages pour la biodétection et l'imagerie à multiparamètres. Toutefois, les modifications apportées à un paramètre entraînent également la modification d’autres paramètres. Par conséquent, la couleur, la durée de vie ou l’intensité ne peuvent pas être utilisées, respectivement, comme paramètre indépendant. Cette thèse peut être divisée en deux aspects. Le premier concerne le développement d'un multiplexage à une seule nanoparticule avec un temps résolu, basé sur le transfert d'énergie par résonance de type Förster (FRET) des complexes de lanthanides aux points quantiques (QD) et ensuite aux colorants fluorescents. Une investigation systématique de toutes les différentes combinaisons avec une large gamme de donneurs et d'accepteurs sur le QD est présentée, et les résultats expérimentaux sont comparés à la modélisation théorique. Le résultat ne contribue pas seulement à une compréhension complète de ces voies de transfert d'énergie compliquée entre multi donneurs / accepteurs sur des nanoparticules, mais offre également la possibilité d'utiliser les modèles pour développer de nouvelles stratégies permettant de préparer le QD avec une couleur, une durée de vie et une intensité réglables de manière indépendante. Le deuxième aspect porte sur le mécanisme de transfert d'énergie du Tb à la nanoparticule d'or (AuNP). Le transfert d'énergie par nanosurface (NSET) s'est révélé être un mécanisme opérationnel pour l'extinction des PL par les AuNP, une information importante pour le développement, la caractérisation et l'application de nanobiocapteurs basés sur l'extinction des PL par les AuNPOptical multiplexing based on nanoparticles provides many advantages for multiparameter biosensing and imaging. However, the changes in one parameter also lead to changing of other parameters, and thus, color, lifetime, or intensity could not be used as an independent parameter, respectively. This thesis can be divided into two aspects. The first one focuses on developing time-resolved single-nanoparticle multiplexing based on Förster resonance energy transfer (FRET) from lanthanide complexes to quantum dot (QD) to fluorescent dyes. Systematical investigation of all different combinations with a broad range of numbers of donors and acceptors on QD are presented, and the experimental results are compared with theoretical modelling. The result do not only contribute to a full understanding of such complicated multi donor-acceptor energy transfer pathways on nanoparticles but also open the opportunity to use the models for developing new strategies to achieve the QD with independent tunable color, lifetime and intensity. The second aspect focuses on the energy transfer mechanism from Tb to gold nanoparticle (AuNP). Nanosurface energy transfer (NSET) proved to be an operational mechanism in PL quenching by AuNPs, which is important information for the development, characterization, and application of nanobiosensors based on PL quenching by AuNPs
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Guo, Jiajia. "Time-resolved Multiplexed Förster Resonance Energy Transfer for Nucleic Acid Biosensing." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS162/document.
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Les biomarqueurs à base d’acides nucléiques, qui sont impliqués dans le contrôle de l'expression génétique, sont spécifiques de nombreux types de cancers. Les applications basées sur le transfert d'énergie par résonance de Förster (FRET) sont parmi les plus prometteuses pour la biodétection d’acides nucléiques. Comme la détection simultanée de plusieurs acides nucléiques est très demandée et que le multiplexage spectral est limité par des interférences (optical crosstalk), le multiplexage temporel est utilisé ici pour ajouter de nouvelles possibilités de multiples détections simultanées. La thèse porte sur le développement de systèmes comprenant différentes distances entre molécules donneuses et acceptrices de FRET (Terbium vers fluorophores) pour créer des signaux d'intensité spécifiques correspondant à différentes séquences d'acides nucléiques. Les distances Tb-to-dye peuvent être arrangées en positionnant spécifiquement le donneur Tb sur des molécules d’ADN de différentes longueurs. Les technologies d'amplification d’acides nucléiques, telles que la réaction d'hybridation en chaîne (HCR) et l’amplification circulaire de l’ADN (RCA), ont été utilisées pour obtenir simplicité, rapidité, sélectivité et sensibilité dans la détection d’acides nucléiques. Le multiplexage temporel du signal de FRET a également été combiné avec le multiplexage spectral (couleur) pour le démultiplier. De plus, la possibilité d'un multiplexage temporel à base de nanoparticules a été démontréeNucleic acid biomarkers, which involve in gene expression control, are found specific for many kinds of cancers. Förster Resonance Energy Transfer (FRET) based applications are one of the most promising for nucleic acid biosensing. As parallel detection of multiple nucleic acids is highly demanded and spectral multiplexing is limited by optical crosstalk, temporal multiplexing is used for opening another dimension of the multiplexing. The thesis focuses on developing different Tb-to-dye FRET distances to create specific intensity signals corresponding to different nucleic acid sequences. The Tb-dye distances can be tuned by specific location of the Tb donor using different lengths of DNA. Amplification technologies, such as hybridization chain reaction (HCR) and rolling circle amplification (RCA), are used to achieve simplicity, rapidity, selectivity, and sensitivity of nucleic acid detection. Temporal multiplexing FRET was also combined with spectral (color) multiplexing for higher order multiplexed detection. Moreover, a single Tb-QD FRET modeling demonstrated the possibility of nanoparticle-based temporal multiplexing
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Wu, Yu-Tang. "Förster Resonance Energy Transfer Immunoassays Using Engineered Proteins for Breast Cancer Biomarker Detection." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS340/document.
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Les protéines modifiées ont suscité un grand intérêt en raison de leur taille extrêmement petite par rapport à l'anticorps entier. Ces petites protéines de liaison ont démontré de nombreux avantages tels qu'une bio distribution rapide, une bonne pénétration dans le tissu tumoral et une élimination rapide du sérum et des tissus non-infectés. Ainsi, ces protéines devraient être d'excellentes alternatives aux anticorps pour les applications cliniques. Cette thèse présente le développement de biocapteurs basés sur des anticorps synthétiques et le transfert d'énergie par résonance de type Förster (FRET) résolu en temps par la détection de biomarqueurs. Les tests immunologiques à base de FRET sont établis en utilisant des complexes de terbium (Tb) comme donneurs de FRET et des boîtes quantiques semi-conducteurs (QDs) comme accepteurs de FRET. Les propriétés photophysiques exceptionnelles de ce couple de FRET Tb-QD permettent une détection quantitative ultrasensible. Des anticorps monocaténaires (single-domain antibody, sdAb) et des petites protéines d’affinité synthétiques (albumin-binding domain-derived affinity protein, ADAPT) sont utilisés pour étudier différentes stratégies de conjugaison d'anticorps, et quantifier des biomarqueurs cliniques (EGFR, HER2). Ce travail peut être considéré comme une condition préalable à l’utilisation des QDs en diagnostic cliniqueEngineered affinity proteins have raised great interest due to their extremely small size compared to full length antibodies. Such small binding proteins have demonstrated many advantages such as quick biodistribution, good penetration into tumor tissue, and fast elimination from serum and nondiseased tissues. Thus, they are expected to be excellent alternatives to antibodies for clinical applications. This thesis focuses on the development of biosensors based on engineered antibodies and time-resolved Förster resonance energy transfer (FRET) through biological recognition of biomarkers. FRET-based immunoassays are established using terbium complexes (Tb) as FRET donors and semiconductor quantum dots (QDs) as FRET acceptors. The exceptional photophysical properties of the Tb-QD FRET pair allow for ultrasensitive quantitative biosensing. Single-domain antibodies (sdAb) and small engineered scaffold antibodies (ADAPT) are used to investigate different antibody-conjugation strategies for quantifying human epidermal growth factor receptors (EGFR, HER2) as clinical biomarkers. This work can be considered as a prerequisite to implementing QDs into applied clinical diagnostics
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Linden, Stina. "Terbium-based time-gated Förster resonance energy transfer imaging for evaluating protein-protein interactions on cell membranes." Thesis, Paris 11, 2015. http://www.theses.fr/2015PA112094.
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Cette thèse étudie l'utilisation de la microscopie FRET en décalage temporelle pour la détection de co-localisation des deux protéines membranaires E- et N-cadhérine. Ces protéines sont importantes pour les contacts cellule-cellule et jouent un rôle important dans la transition épithélio-mésenchymateuse (EMT), un processus clé dans la métastase du cancer. Dans EMT cellules perdent leurs marqueurs épithéliaux (par exemple E-cadhérine) et acquièrent des marqueurs mésenchymateuses (par exemple N-cadhérine), ce qui augmente leur motilité et le caractère invasif pour échapper à la tumeur primaire dans la circulation sanguine en tant que ce qu'on appelle des cellules tumorales circulantes (CTC). Ce manuscrit porte sur la détection des CTC qui ont subi une EMT partielle, montrant un phénotype hybride (épithélio-mésenchymateuse) et co-expriment E et N-cadhérine, par des études de co-localisation en utilisant le FRET sur une lignée modèle de cellules. FRET (Transfer d’énergie par résonance de type Förster) est un transfert d'énergie non-radiatif entre deux molécules qui sont en résonance et à proximité (environ 1-20 nm). Une co-localisation d’E- et N-cadhérine en clusters serait donc détectable par FRET. La coloration des cadhérines qui a été fait en utilisant des anticorps spécifiques marqués avec une donneur qui a un longue durée de vie de fluorescence, le complexe de terbium Lumi4-Tb (TbL4) de Lumiphore, Inc., et diverses accepteurs. La longue durée de vie du donneur et la longue durée de vie d’accepteur sensibilisé (FRET) pourraient être imagés dans une configuration de microscopie en décalage temporelle. L’imagerie en décalage temporelle présente plusieurs avantages par rapport à l'imagerie stationnaire en termes de suppression efficace du bruit de fond dans des échantillons biologiques. L'installation décrite dans ce manuscrit est basée sur l'utilisation d'une caméra CCD intensifiée, une source d'excitation laser pulsé en UV et un décalage temporel défini de quelques microsecondes entre l'excitation et l'acquisition d'image. L’imagerie de FRET en décalage temporelle a été utilisée pour étudier des différents échantillons biologiques (intracellulaire et située à la membrane). Bien que les deux marqueurs protéiques pourraient imager correctement sur les mêmes cellules, FRET entre E- et N-cadhérine ou E- et E-cadhérine ne pouvaient pas être détectés. Des expériences de contrôle avec des anticorps contre le même anticorps primaire ont révélé des signaux de FRET forts à cause de la reconnaissance des anticorps donneurs et accepteurs aux mêmes anticorps primaires. Ces résultats de FRET entre deux anticorps différents séparés par quelques nanomètres démontrent la faisabilité de mesurer des interactions protéine-protéine et la co-localisation sur des membranes à l'aide de l’imagerie de FRET en décalage temporelle en utilisant TbL4. Imagerie en décalage temporelle de quelques microsecondes est particulièrement intéressante pour l'enquête des interactions protéine-protéine dans des échantillons biologiques hautement autofluorescentes, tels que les tissus cancéreuxThis thesis investigates the use of time-gated FRET microscopy for detection of colocalization of two membrane proteins, E- and N-cadherin. These proteins are important for cell-cell contacts and have an important role in the epithelial to mesenchymal transition (EMT), a key process in cancer metastasis. In EMT cells lose their epithelial markers (such as E-cadherin) and gain mesenchymal markers (such as N-cadherin), increasing their motility and invasiveness, enabling escape from the primary tumor into the bloodstream as so called circulating tumor cells (CTCs). This manuscript focuses on the detection of CTCs that have undergone partial EMT, displaying a hybrid phenotype (epithelial-mesenchymal) and co-express E- and N-cadherin, by FRET co-localization studies on a model cell line. FRET (Förster resonance energy transfer) is a non-radiative energy transfer between two molecules that are in resonance and in close proximity (ca. 1-20 nm). A co-localization of E- and N-cadherin in clusters would therefore be detectable by FRET. The staining of the cadherins was done by using specific antibodies labelled with a long lifetime donor, the terbium complex Lumi4-Tb (TbL4) from Lumiphore, Inc., and various acceptors. The long lifetime donor and long lifetime sensitized acceptor emission (FRET) could be imaged in a time-gated microscopy setup. Time -gated imaging has several advantages compared to steady state imaging in terms of efficient background suppression in biological samples. The setup described in this manuscript is based on the use of an intensified CCD camera, a pulsed UV-laser excitation source, and a defined (µs) delay between excitation and image acquisition. In addition to the E- and N-cadherin FRET experiments the time-gated FRET imaging microscopy was used to investigate different biological samples (intracellular and membrane located). Although both protein markers could be successfully imaged on the same cells, FRET between E- and N-cadherin or E- and E-cadherin could not be detected. Control experiments with antibodies against the same primary antibody revealed strong time-gated FRET signals due to binding of both donor and acceptor antibodies to the same primary antibodies. The successful time-gated imaging of two different antibodies separated by a few nanometers demonstrates the feasibility of probing protein-protein interaction and co-localization at membranes using TbL4 based time-gated FRET imaging. Microsecond time-gated imaging is especially interesting for the investigation of protein-protein interactions in highly autofluorescent biological samples such as cancer tissues
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Thomson, Cameron Ian. "Probing the Nature of Cellulosic Fibre Interfaces with Fluorescence Resonance Energy Transfer." Diss., Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/16277.
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The material properties of fibre networks and fibre reinforced composites are strongly influenced by fibre-fibre interactions. Stress transfer between load bearing elements in such materials is often dictated by the nature of the fibre-fibre interface. Inter-fibre bonding is solely responsible for internal cohesion in paper, because all stresses transferred between fibres operate through fibre-fibre bonds. . The future development of cellulosic fibre materials will require an improved understanding of the fibre-fibre interface. Fluorescence resonance energy transfer (FRET) was proposed as a new tool for the study of fibre interfaces.
A protocol for covalent linkage of fluorophores to natural and regenerated cellulosic fibres was developed and the absorptive and emissive properties of these dyes were characterized. The fluorescent response of these dyed fibres in paper sheets was studied using steady-state fluorescence spectroscopy. Fluorescence micrographs of fibre crossings on glass slides were analyzed using the FRETN correction algorithm. Energy transfer from coumarin dyed fibres to fluorescein dyed fibres at the interface was observed. The FRETN surfaces for spruce and viscose rayon fibre crossings were distinctly different. The FRET microscopy method was able to detect statistically significant differences in spruce fibre interface development when fibre fraction and wet pressing were varied. The coalescence of natural cellulosic fibre interfaces during drying was also observed with the technique.
Polysaccharide films were employed as model systems for the natural and regenerated cellulose fibre interfaces. It was found that pressing cellulose films did not result in significantly increased FRETN either due to resistance to deformation or the inability to participate in interdiffusion. Conversely, xylan films demonstrated a drastic increase in the FRETN signal with increased wet pressing. These results support the previously observed differences between regenerated cellulose fibres and natural wood fibres. The results of the FRETN analysis of the polysaccharide film model systems suggest that lower molecular weight amorphous carbohydrates are likely to be significant contributors to fibre interface development.
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Kalinin, Stanislav. "Electronic Energy Transfer within Asymmetric Pairs of Fluorophores: Partial Donor-Donor Energy Migration (PDDEM)." Doctoral thesis, Umeå : Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-338.
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Wren, David Andrew. "Measuring calcium in Pseudomonas aeruginosa using a genetically encoded fluorescence resonance energy transfer (FRET) sensor." Connect to online resource, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:1460883.
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Durach, Maxim. "Giant Plasmonic Energy and Momentum Transfer on the Nanoscale." Digital Archive @ GSU, 2009. http://digitalarchive.gsu.edu/phy_astr_diss/42.
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We have developed a general theory of the plasmonic enhancement of many-body phenomena resulting in a closed expression for the surface plasmon-dressed Coulomb interaction. It is shown that this interaction has a resonant nature. We have also demonstrated that renormalized interaction is a long-ranged interaction whose intensity is considerably increased compared to bare Coulomb interaction over the entire region near the plasmonic nanostructure. We illustrate this theory by re-deriving the mirror charge potential near a metal sphere as well as the quasistatic potential behind the so-called perfect lens at the surface plasmon (SP) frequency. The dressed interaction for an important example of a metal–dielectric nanoshell is also explicitly calculated and analyzed. The renormalization and plasmonic enhancement of the Coulomb interaction is a universal effect, which affects a wide range of many-body phenomena in the vicinity of metal nanostructures: chemical reactions, scattering between charge carriers, exciton formation, Auger recombination, carrier multiplication, etc. We have described the nanoplasmonic-enhanced Förster resonant energy transfer (FRET) between quantum dots near a metal nanoshell. It is shown that this process is very efficient near high-aspect-ratio nanoshells. We have also obtained a general expression for the force exerted by an electromagnetic field on an extended polarizable object. This expression is applicable to a wide range of situations important for nanotechnology. Most importantly, this result is of fundamental importance for processes involving interaction of nanoplasmonic fields with metal electrons. Using the obtained expression for the force, we have described a giant surface-plasmoninduced drag-effect rectification (SPIDER), which exists under conditions of the extreme nanoplasmonic confinement. Under realistic conditions in nanowires, this giant SPIDER generates rectified THz potential differences up to 10 V and extremely strong electric fields up to 10^5-10^6 V/cm. It can serve as a powerful nanoscale source of THz radiation. The giant SPIDER opens up a new field of ultraintense THz nanooptics with wide potential applications in nanotechnology and nanoscience, including microelectronics, nanoplasmonics, and biomedicine. Additionally, the SPIDER is an ultrafast effect whose bandwidth for nanometric wires is 20 THz, which allows for detection of femtosecond pulses on the nanoscale.
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Hildebrandt, Niko. "Lanthanides and quantum dots : time-resolved laser spectroscopy of biochemical Förster Resonance Energy Transfer (FRET) systems." Phd thesis, Universität Potsdam, 2006. http://opus.kobv.de/ubp/volltexte/2007/1268/.
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Förster Resonance Energy Transfer (FRET) plays an important role for biochemical applications such as DNA sequencing, intracellular protein-protein interactions, molecular binding studies, in vitro diagnostics and many others. For qualitative and quantitative analysis, FRET systems are usually assembled through molecular recognition of biomolecules conjugated with donor and acceptor luminophores. Lanthanide (Ln) complexes, as well as semiconductor quantum dot nanocrystals (QD), possess unique photophysical properties that make them especially suitable for applied FRET. In this work the possibility of using QD as very efficient FRET acceptors in combination with Ln complexes as donors in biochemical systems is demonstrated. The necessary theoretical and practical background of FRET, Ln complexes, QD and the applied biochemical models is outlined. In addition, scientific as well as commercial applications are presented.
FRET can be used to measure structural changes or dynamics at distances ranging from approximately 1 to 10 nm. The very strong and well characterized binding process between streptavidin (Strep) and biotin (Biot) is used as a biomolecular model system. A FRET system is established by Strep conjugation with the Ln complexes and QD biotinylation. Three Ln complexes (one with Tb3+ and two with Eu3+ as central ion) are used as FRET donors. Besides the QD two further acceptors, the luminescent crosslinked protein allophycocyanin (APC) and a commercial fluorescence dye (DY633), are investigated for direct comparison. FRET is demonstrated for all donor-acceptor pairs by acceptor emission sensitization and a more than 1000-fold increase of the luminescence decay time in the case of QD reaching the hundred microsecond regime. Detailed photophysical characterization of donors and acceptors permits analysis of the bioconjugates and calculation of the FRET parameters. Extremely large Förster radii of more than 100 Å are achieved for QD as acceptors, considerably larger
than for APC and DY633 (ca. 80 and 60 Å). Special attention is paid to interactions with different additives in aqueous solutions, namely borate buffer, bovine serum albumin (BSA), sodium azide and potassium fluoride (KF). A more than 10-fold limit of detection (LOD) decrease compared to the extensively characterized and frequently used donor-acceptor pair of Europium tris(bipyridine) (Eu-TBP) and APC is demonstrated for the FRET system, consisting of the Tb complex and QD. A sub-picomolar LOD for QD is achieved with this system in azide free borate buffer (pH 8.3) containing 2 % BSA and 0.5 M KF. In order to transfer the Strep-Biot model system to a real-life in vitro diagnostic application, two kinds of imunnoassays are investigated using human chorionic gonadotropin (HCG) as analyte. HCG itself, as well as two monoclonal anti-HCG mouse-IgG (immunoglobulin G) antibodies are labeled with the Tb complex and QD, respectively. Although no sufficient evidence for FRET can be found for a sandwich assay, FRET becomes obvious in a direct HCG-IgG assay showing the feasibility of using the Ln-QD donor-acceptor pair as highly sensitive analytical tool for in vitro diagnostics.Förster Resonanzenergietransfer (FRET) spielt eine wichtige Rolle in biochemischen Anwendungen, wie z.B. DNA-Sequenzierung, intrazellulären Protein-Protein-Wechselwirkungen, molekularen Bindungsstudien, in-vitro-Diagnostik und vielen anderen. Zur quantitativen und qualitativen Analyse werden FRET Systeme normalerweise durch molekulare Erkennung von Biomolekülen, die mit Donator- und Acceptorluminophoren markiert sind, ermöglicht. Durch die besonderen photophysikalischen Eigenschaften sowohl von Lanthanidkomplexen (Ln-Komplexen), als auch Halbleiternanokristallen (sog. Quantenpunkten oder Quantumdots - QD), sind diese besonders für FRET Anwendungen geeignet. In der vorliegenden Arbeit wird effizienter FRET zwischen Ln-Komplexen und QD in biochemischen Systemen demonstriert. Die notwendigen theoretischen und praktischen Grundlagen über FRET, Ln-Komplexe, QD und die verwendeten biochemischen Modelle werden dargestellt, und wissenschaftliche als auch kommerzielle Anwendungen werden präsentiert. FRET kann zur Messung von strukturellen Veränderungen und Dynamiken im Bereich von ca. 1 bis 10 nm verwendet werden. Der sehr starke und gut charakterisierte Bindungsprozess zwischen Streptavidin (Strep) und Biotin (Biot) wird als biomolekulares Modellsystem eingesetzt. Ein FRET System wird durch Streptavidinkonjugation mit Ln-Komplexen und QD-Biotinylierung etabliert. Drei Ln-Komplexe (einer mit Tb3+ und zwei mit Eu3+ als Zentralion) werden als Donatoren verwendet, und neben QD werden zwei weitere Acceptoren, das lumineszierende, quervernetzte Protein Allophycocyanin (APC) und ein kommerzieller Fluoreszenzfarbstoff (DY633), untersucht. FRET kann für alle Donator-Acceptor Paare nachgewiesen werden, zum einen durch sensibilisierte Acceptorlumineszenz und zum anderen durch eine über 1000-fach erhöhte Lumineszenzabklingzeit der QD mit über 100 Mikrosekunden. Mittels detailierter photophysikalischer Charakterisierung der Donatoren und Acceptoren können die Biokonjugate analysiert und die FRET Parameter berechnet werden. Für die QD FRET Systeme ergeben sich extrem große Försterradien von über 100 Å, die wesentlich größer sind als für APC und DY633 (ca. 80 bzw. 60 Å). Besondere Aufmerksamkeit gilt der Wechselwirkung mit den Zusatzreagenzien Boratpuffer, Bovines Serumalbumin (BSA), Natriumazid und Kaliumfluorid (KF) in den wässrigen Lösungen. Im Vergleich zum ausgiebig charakterisierten und vielfach verwendeten Donator-Acceptor Paar aus Europium-tris(Bipyridin) (Eu-TBP) und APC wird eine mehr als 10-fache Senkung der Nachweisgrenze für das FRET-System, bestehend aus Tb-Komplex und QD, erreicht. In azidfreiem Boratpuffer (pH 8,3) mit 2 % BSA und 0,5 M KF wird eine subpicomolare QD-Nachweisgrenze für dieses System aufgezeigt. Um den Transfer des Strep-Biot Modellsystems in eine echte in-vitro-diagnostische Anwendung zu demonstrieren, werden zwei Immuntests zum HCG-(Humanes Choriongonadotropin)-Nachweis untersucht. Sowohl HCG als auch monoklonale anti-HCG Maus-IgG-(Immunoglobulin G)-Antikörper werden mit dem Tb-Komplex bzw. mit QD markiert. Obwohl kein ausreichender Nachweis für FRET in einem immunometrischen Assay (oder Sandwichassay) erbracht werden kann, wird FRET in einem direkten HCG-IgG Assay erzielt, wodurch die Realisierbarkeit von Ln-QD Donator-Acceptor Paaren zur hochsensitiven Anwendung in der in-vitro-Diagnostik gezeigt werden kann.
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Xu, Jingyue. "Sensitive and mutiplexed microRNA quantification using amplified time-gated Förster resonance energy transfer." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASS137.
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En tant que biomarqueurs de nouvelle génération, les microARNs sont associés à de nombreux cancers et maladies, ce qui a conduit à une forte demande pour le développement de méthodes cliniques de diagnostic des microARNs. Les technologies d'amplification isotherme, telles que l'amplification en cercle roulant et l'assemblage catalytique en épingle à cheveux, sont devenues des méthodes puissantes pour les dosages très rapides, spécifiques et sensibles des microARNs. Cette thèse se concentre sur le développement de biocapteurs de microARNs basés sur les technologies d'amplification isotherme et le transfert d'énergie par résonance de type Förster résolu en temps des complexes de lanthanide aux colorants organiques ou aux points quantiques. Les biocapteurs de microARNs amplifiés proposés ont des limites de détection très basses et sont utilisables dans des échantillons cliniques humains, révélant avec succès leurs pertinence pour le diagnostic du cancer. Comme la détection simultanée de plusieurs microARNs est très demandée, la détection temporelle multiplexée de microARNs est également effectuée basée sur les de durées de vie à l'état excité distinctes des complexes Tb et des colorants. De plus, le nanocapteur microARN amplifié basé sur des points Tb à quantique FRET, a démontré la possibilité d'une détection spectrale multiplexée de microARNs avec une sensibilité et une sélectivité élevéesAs new generation of biomarkers, microRNAs are associated with many cancers and diseases, which has led to a great demand for developing clinical miRNA diagnostic methods. Isothermal amplification technologies, such as rolling circle amplification and catalytic hairpin assembly, have emerged as powerful methods for highly rapid, specific and sensitive microRNA assays. This thesis focuses on developing microRNA biosensors based on isothermal amplification technologies and time-resolved Förster resonance energy transfer from lanthanide complexes to organic dyes or quantum dots. The proposed amplified microRNA biosensors have very low limits of detections, and are applied to human clinical samples, successfully revealing the relevance for cancer diagnostics. As simultaneous detection of multiple microRNAs is highly demanded, temporal multiplexed detection of microRNAs is also realized based on distinguishable excited-state lifetimes of Tb complexes and dyes. Moreover, the amplified microRNA nanosensor based on Tb-to-quantum dots FRET demonstrated the possibility of spectral multiplexed detection of microRNAs with high sensitivity and selectivity
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Herrmann, Janning [Verfasser], Volker [Gutachter] Deckert, and Michael [Gutachter] Börsch. "Plasmon-Controlled resonant energy transfer : influence of optical antennas on the energy transfer rate and efficiency of single molecule FRET-Pairs / Janning Herrmann ; Gutachter: Volker Deckert, Michael Börsch." Jena : Friedrich-Schiller-Universität Jena, 2019. http://d-nb.info/1207273163/34.
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Halpern, Micah. "DEVELOPMENT AND FORENSIC APPLICATION OF DYE PROBE FLUORESCENCE RESONANCE ENERGY TRANSFER FOR IMPROVED DETECTION OF CHANGES IN DN." Master's thesis, University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2871.
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Discovering, screening, and associating changes in DNA sequence are important to a broad range of disciplines and play a central role in Forensic Science. The typical types of changes include sequence variations [single nucleotide polymorphisms (SNP)] and length variations [short tandem repeats (STR)]. The steps for forensic DNA sample processing are similar for both types of changes but diverge at the point of detection. A number of approaches are being explored for SNP genotyping while STR analysis primarily consists of size-based analysis by capillary electrophoresis. Limitations exist for all current detection methods that pose significant impacts to forensic analysis. Bi-allelic SNPs result in three possible genotypes with a minimal amount of information generated per marker. Limitations for SNP analysis are due to the inability to amplify a suitable number of SNP markers from low DNA content samples to provide an appropriate level of discrimination. Multi-allelic STR markers are currently the marker of choice for forensic typing but a variety of experimental artifacts are possible that consist of either biology or technology related causes. Molecular genotyping methods developed across other disciplines have potential to alleviate some of these shortcomings but no current approach is capable of genotyping both SNP and STR loci with a single chemistry. The need for a more effective, efficient, and generalized approach led to development of a unique method called Dye Probe Fluorescence Resonance Energy Transfer (dpFRET) and determination of its suitability for forensic analysis. The development phase of the research consisted of synthetic testing to establish proof of concept for the chemistry followed by polymerase chain reaction (PCR) based assays to demonstrate real world applications. Following successful development, the boundaries and limitations for the technology were established (sensitivity, allelic dropout, mixed samples) and efforts were made to improve the approach. In the process, parallel testing for other fields including molecular pathology and conservation biology were incorporated to explore potential widespread application of this new approach. The overall goal of this project was to develop and explore the limitations for a unique approach to genotyping both SNPs and STRs. A majority of the work involved development of the method itself with the ultimate objective of application for forensic science. The focus of this project was to address and alleviate some of the shortcomings of current approaches that result in potential limitations for forensic analysis. It is expected that future applications of this technology might impact a wide range of disciplines to aid in discovery, screening and association of changes in DNA sequence.M.S.
Department of Chemistry
Sciences
Forensic Science MS
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Altevogt, Andrea [Verfasser], and Willi [Akademischer Betreuer] Bannwarth. "Synthese und Anwendung neuartiger Förster-Resonanz-Energie-Transfer (FRET) Systeme in DNA." Freiburg : Universität, 2011. http://d-nb.info/112346376X/34.
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Hildebrandt, Niko. "Lanthanides and quantum dots time-resolved laser spectroscopy of biochemical Förster resonance energy transfer (FRET) systems /." [S.l.] : [s.n.], 2006. http://opus.kobv.de/ubp/volltexte/2007/1268.
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Olejko, Lydia [Verfasser], and Ilko [Akademischer Betreuer] Bald. "Förster resonance energy transfer (FRET)-based nanophotonics using DNA origami structures / Lydia Olejko ; Betreuer: Ilko Bald." Potsdam : Universität Potsdam, 2017. http://d-nb.info/1218402261/34.
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Pasupulleti, Venkata Kiran. "Characterization of viral proteases from Norwalk virus, poliovirus, and transmissible gastroenteritis virus using a fluorescence resonance energy transfer assay." Thesis, Kansas State University, 2012. http://hdl.handle.net/2097/15068.
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Master of ScienceDepartment of Diagnostic Medicine/Pathobiology
Kyeong-Ok Chang
Positive sense RNA viruses include diverse groups of viruses that cause a wide variety of diseases in humans and animals. Most of these viruses encode proteases that cleave the viral polyprotein into intermediate or mature functional proteins during virus replication. As these proteases play a critical role in virus replication, they represent an attractive target for the development of antiviral drugs. In this study, the main goal was to establish assay systems and characterize the enzymatic activity of related proteases from Norwalk virus (NV), poliovirus, and transmissible gastroenteritis virus (TGEV). These proteases share several common characteristics including a typical chymotrypsin-like fold, a Cys residue as a nucleophile in the catalytic triad (or dyad) composed of Cys, His and Glu (or Asp) residues, and a preference for a Glu or Gln residue at the P1 position on the substrate. We cloned and expressed proteases from these viruses and characterized their enzymatic activities using a fluorescence resonance energy transfer (FRET) assay using a specific FRET substrate corresponding to each viral protease. First, assay conditions of the FRET assay was optimized for each virus protease. Second, inhibition profiles of each virus protein were investigated using five commercially available standard protease inhibitors (chymostatin, leupeptin, antipain, TPCK, and TLCK). The inhibition studies showed that TPCK inhibited NV, poliovirus, and TGEV proteases with varying strength, and chymostatin inhibited only NV protease. All other inhibitors had little effects on the virus proteases. The established FRET assays should facilitate screening potential antivirals.
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Sahoo, Dhananjaya [Verfasser]. "Synthesis of Molecular Rulers to Study Distance and Orientation Dependent Förster Resonance Energy Transfer (FRET) / Dhananjaya Sahoo." Bielefeld : Universitätsbibliothek Bielefeld, 2019. http://d-nb.info/1196640580/34.
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Mitchell, Amanda. "Development of a Novel Genetically Encoded FRET System Using the Unnatural Amino Acid Anap." Thesis, Boston College, 2016. http://hdl.handle.net/2345/bc-ir:107177.
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Thesis advisor: Abhishek ChatterjeeFörster Resonance Energy Transfer (FRET) offers a powerful approach to study biomolecular dynamics in vitro as well as in vivo. The ability to apply FRET imaging to proteins in living cells provides an excellent tool to monitor important dynamic events such as protein conformational changes, protein-protein interactions, and proteolysis reactions. However, selectively incorporating two distinct fluorophores into the target protein(s) that are capable of FRET interaction within the complex cellular milieu is challenging. Consequently, terminal fusion to genetically encoded fluorescent proteins has emerged as the predominant labeling strategy for FRET studies in vivo. However, a major limitation of this strategy stems from the large size of the fluorescent proteins, which may perturb the native properties of the target, and restricted attachment only to the termini of the target. We reasoned that using genetically encoded fluorescent unnatural amino acids would overcome several of these challenges associated with currently available labeling strategies owing to their small size and the ability to introduce them site- specifically and co-translationally. Here, we report the use of the fluorescent unnatural amino acid “Anap” as a FRET donor with green and yellow fluorescent protein acceptors. We demonstrate the utility of this labeling strategy using proteolysis and conformational change models, and step towards in vivo studies by further developing a proteolysis system in cell lysates
Thesis (MS) — Boston College, 2016
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Ellis, Jonathan. "FRET analysis of splicing factors involved in exon and intron definition in living cells." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/4397.
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I have analyzed the interactions between SR proteins and splicing components that are bound at the 5’ or 3’ splice site using fluorescence resonance energy transfer (FRET) microscopy. The SR proteins interact with the U1 snRNP-associated 70 kDa protein (U170K) at the 5’splice site and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) at the 3’ splice site. These interactions have been extensively characterized biochemically in the past, and are proposed to play roles in both intron and exon definition. We employed FRET acceptor photobleaching and fluorescence lifetime imaging microscopy (FLIM) to identify and spatially localise sites of direct interactions of SF2/ASF, and other SR proteins, with U2AF35 and U1-70K in live cell nuclei. These interactions were shown to occur more strongly in interchromatin granule clusters (IGCs). They also occur in the presence of the RNA polymerase II inhibitor, DRB, demonstrating that they are not exclusively co-transcriptional. FLIM data have also revealed a novel interaction between HCC1, a factor highly related to the large subunit of the U2AF splicing factor, with both subunits of U2AF that occur in discrete domains within the nucleoplasm but not within IGCs. These data demonstrate that the interactions defining intron and exon definition do occur in living cells in a transcription-independent manner.
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Clerté, Caroline. "Structure et dynamique globales de la protéine U1A humaine étudiées par spectroscopie de fluorescence." Paris 6, 2001. http://www.theses.fr/2001PA066405.
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Dogra, Navneet. "INVESTIGATING PROTEIN - BILAYER COMPLEXES: A STUDY OF LIGAND - RECEPTOR INTERACTIONS AT MODEL MEMBRANE SURFACE BY USING ELECTRONIC ABSORPTION SPECTROSCOPY AND FLUORESCENCE RESONANCE ENERGY TRANSFER." OpenSIUC, 2014. https://opensiuc.lib.siu.edu/dissertations/812.
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The main aim of work presented here is to design, develop and characterize a colorimetric model membrane (liposome) systems, which can bind with proteins, enzymes, bacteria, virus and other biomolecules. PDA molecules are utilized as a scaffold for the bilayer membrane, and a colorimetric assay is carried out. The holy grail of present work contributes towards the better understanding of protein interactions with the cell bilayer surface. Chapter 1 introduces a brief history on the advent of bilayer systems for cellular research exploration. We presented a literature survey about how liposome systems are used as a complementary technique to understand the fundamental principles of cellular membrane functions. Furthermore, we describe about membrane protein functions and recent findings on how proteins interact with the cell membrane. Finally, we explain conjugated systems and their exploration in bilayer membrane as a colorimetric scaffold. We also touch bases with major fluorescence techniques used in our experiments. Chapter 2 provides details on the preparation protocols of liposome and liposome-protein complexes. We confirmed protein-bilayer interactions by monitoring FRET between PDA and rhodamine molecules. Furthermore, we performed streptavidin-biotin binding studies on the PDA bilayer. Protein binding changed the spectral overlap (J) between PDA and rhodamine, which ultimately increased the fluorescence emission of rhodamine. The goal of performing these studies was to present a complete protocol for the preparation of liposome and protein-liposome complex. In chapter 3, we investigate how proteins bind on the cell membrane. Additionally, we propose a model of protein-bilayer complex. We reported that, by harnessing cell bilayer with specific bio-molecules, we monitored protein--bilayer, protein--protein and enzyme--substrate signal transduction. We have developed a colorimetric system for monitoring vital stimulations occur on the liposomal membrane surface. Bilayer was modified to covalently bind the amino group of lysine residues present on protein molecules. These bio-molecular interactions on bilayer surface provide differential stimulus, which turned out to be the major cause of differential spectroscopic signals depending upon size and shape of the protein bounded to the bilayer. Polydiacetylene (PDA) liposomes are the core of our color based system. These liposomes are used to monitor subtle interactions on the bilayer surface. We have also developed a semi-quantitative method based on the colorimetric response of PDA liposomes; we were able to detect protein molecules at sub-nanomolar concentrations in the solution. It's capability of distinguishing protein molecules based on their chemical and physical interactions to bilayer contributes towards the identity of our system. Interestingly, our mass spectroscopic data suggested non-specific enzymatic cleavage of membrane-bound proteins. These fragments were not present in bulk protein cleavage. We also proposed a model that depicts the covalent binding of protein at the bilayer of liposomes. These studies are intended to investigate protein-bilayer and enzyme-protein interaction occurring on the cell surface. In chapter 4, we focus on the kinetics of protein interaction on bilayer surface and we also attempt to visualize these interactions by exploring fluorescence microscopy. A self-assembled cell membrane is consisted of various lipids, which cluster themselves in their preferred phase separated regions. Lipid clusters are very important for lipid specific protein interactions. We investigated protein binding on such phase separated regions under a fluorescence microscope. Furthermore, we enzymatically catalyzed proteins, which were covalently bonded on the bilayer surface. This catalytic reaction was monitored both spectroscopically and under a fluorescence microscope. These studies were performed to help us in the better understanding of biological interactions at cell surface. Chapter 5, describes the encapsulation and controlled delivery of antimicrobial compounds from liposomes. Use of antimicrobial coatings on food packaging is one of the important technologies of active packaging for improving food safety. There is growing demand for natural antimicrobials because of fear of adverse health effects of synthetic preservatives. The main objective of this study is to compare antimicrobial activity of free versus encapsulated curcumin. Glass surfaces coated with nano-encapsulated curcumin may be used as an active packaging material in preserving liquid foods; however, further study is required to improve antimicrobial activities of polylactic acid PLA surfaces. In chapter 6, we investigate interactions between receptors and ligands at bilayer surface of polydiacetylene (PDA) liposomal nanoparticles using changes in electronic absorption spectroscopy and fluorescence resonance energy transfer (FRET). We study the effect of mode of linkage (covalent versus noncovalent) between the receptor and liposome bilayer. We also examine the effect of size-dependent interactions between liposome and analyte through electronic absorption and FRET responses. Glucose (receptor) molecules were either covalently or noncovalently attached at the bilayer of nanoparticles, and they provided selectivity for molecular interactions between glucose and glycoprotein ligands of E. coli. These interactions induced stress on conjugated PDA chain which resulted in changes (blue to red) in the absorption spectrum of PDA. The changes in electronic absorbance also led to changes in FRET efficiency between conjugated PDA chains (acceptor) and fluorophores (Sulphorhodamine-101) (donor) attached to the bilayer surface. Interestingly, we did not find significant differences in UV−Vis and FRET responses for covalently and noncovalently bound glucose to liposomes following their interactions with E. coli. We attributed these results to close proximity of glucose receptor molecules to the liposome bilayer surface such that induced stress were similar in both the cases. We also found that PDA emission from direct excitation mechanism was ∼2−10 times larger than that of the FRET-based response. These differences in emission signals were attributed to three major reasons: nonspecific interactions between E. coli and liposomes, size differences between analyte and liposomes, and a much higher PDA concentration with respect to sulforhodamine (SR-101). We have proposed a model to explain our experimental observations. Our fundamental studies reported here will help in enhancing our knowledge regarding interactions involved between soft particles at molecular levels. In chapter 7, we conclude the summary of all work carried out in previous chapters.
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Sarca, Anamaria Daniela. "FRET-based detection and quantification of HIV-1 Virion Maturation." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263567.
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付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム京都大学
新制・課程博士
博士(医学)
甲第23106号
医博第4733号
京都大学大学院医学研究科医学専攻
(主査)教授 小柳 義夫, 教授 松田 道行, 教授 朝長 啓造
学位規則第4条第1項該当
Doctor of Medical Science
Kyoto University
DFAM
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Schuler, Benjamin, Everett A. Lipman, Peter J. Steinbach, Michael Kumke, and William A. Eaton. "Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence." Universität Potsdam, 2005. http://opus.kobv.de/ubp/volltexte/2007/1222/.
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To determine whether Förster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Förster theory, with polyproline treated as a rigid rod. At donor–acceptor distances much less than the Förster radius R0, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Förster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor–acceptor distances.
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Wegner, David Karl. "Förster Resonance Energy Transfer from Terbium Complexes to Quantum Dots for Multiplexed Homogeneous Immunoassays and Molecular Rulers." Thesis, Paris 11, 2015. http://www.theses.fr/2015PA112109/document.
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Le transfert d'énergie par résonance de type Förster (FRET) est un transfert d'énergie non radiatif d'un donneur à un accepteur à proximité. En raison de sa dépendance de la distance extrêmement sensible entre env. 1 et 20 nm, FRET joue un rôle important dans la nanobiotechnologie. Ainsi FRET peut être utilisé comme système de transduction du signal, mais aussi pour l'estimation de la distance entre le donneur et l'accepteur.Les accepteurs de FRET utilisés dans ce travail étaient des nanocristaux semi-conducteurs (quantum dots, QD). Ce type de luminophore est bien connu pour ses propriétés photophysiques supérieures. Leur absorption forte et spectralement large, et leur photoluminescence (PL) brillante et spectralement fine et de l'accordabilité spectrale de la PL sont idéalement adaptés aux applications de FRET. La combinaison des QDs comme accepteurs de FRET avec des complexes luminescents de terbium (CLT) comme donneurs permet des grandes distances de FRET (> 10 nm). La distance de FRET est caractéristique d'une paire FRET et décrit la distance à laquelle l'efficacité de FRET est égale à 50%. CLT sont idéal comme donneurs de FRET parce qu'ils fournissent des longues durées de vie des états excités à l'ordre de la milliseconde. Cette longue période de décroissance de PL permet de mesurer en temps décalé pour une répression d’autofluorescence et la PL des QDs directement excités, ce qui augmente fortement la sensibilité de détection. Les bandes d'émission de PL structurés de CLT et la PL accordable de QDs sont idéales pour l'application dans le diagnostic multiplexé.La thèse se compose de deux parties. Dans la première le couple FRET CLT-QD a été utilisé dans des immunodosages de FRET homogènes pour la détection de marqueurs biologiques antigène prostatique spécifique (TPSA), énolase specifique des neurones (NSE), antigène carcino-embryonnaire (CEA), et le récepteur du facteur de croissance épidermique (EGFR). La sensibilité du dosage immunologique a été optimisé en utilisant des différents types d'anticorps IgG, F (ab')2, F (ab), et pour EGFR un anticorps de chaîne lourde unique. Des limites de détection picomolaires étaient réalisées en utilisant des échantillons de sérum de petit volume et des mesures sur des lecteurs de microplaques cliniques. Une étude détaillée des différents systèmes de FRET en utilisant la spectroscopie résolue en temps a été réalisée pour étudier l'influence des différents anticorps sur la distance, la fonctionnalité, et la sensibilité des immunodosages. L'étude a été complétée par la mesure de NSE et CEA dans un format duplexé et des échantillons réels de patients.Dans la deuxième partie le FRET pour des mesures de distance nanométriques (réglette moléculaire ou spectroscopique) étaient étudiés. FRET en résolution temporelle a permis de calculer la distance entre le donneur et l’accepteur. Par conséquent, deux stratégies de liaisons différentes ont été étudiées pour établir une proximité entre le CLT et le QD : la reconnaissance biotine-streptavidine et l’auto-assemblage médié par polyhistidine. Une étude en résolution temporelle détaillée a été effectuée avec des QDs de différentes tailles, formes et revêtements de surface combiné avec des CLT liés à trois différentes biomolécules. L'analyse des courbes de décroissance multiexponentielle des donneurs et accepteurs permettait à obtenir des informations sur la taille, la forme et la biofonctionnalité des bioconjugués CLT-QD. Les résultats étaient en accord avec d'autres méthodes d'analyse de structure, telles que la microscopie électronique à transmission (MET) ou la diffusion de lumière dynamique (DLS), mais avec l'avantage d'une mesure homogène à la résolution 3-dimensionelle (impossible pour le MET), sans l'inclusion d'une couche d'hydratation (l’inconvénient de DLS) et en faible concentration dans le même environnement que celui utilisé pour l'application biologiqueFörster resonance energy transfer (FRET) is a non-radiative energy transfer from a donor to an acceptor in close proximity. Due to its extremely sensitive distance dependence in the 1 – 20 nm range, FRET plays an important role in nanobiotechnology. Thereby FRET can be used as signal transduction system but also for the distance estimation between donor and acceptor. The selected FRET acceptors in this work were semiconductor nanocrystals (quantum dots, QDs). This type of luminophore is well known for its superior photophysical properties. Their strong and broad absorption and their bright, narrow-band, and size-tunable photoluminescence (PL) emission make QDs ideally suited for FRET application. Combing QDs as FRET acceptors with luminescent terbium complexes (LTC) as FRET donors offers exceptionally large Förster distances of more than 10 nm. The Förster distance is characteristic of a FRET pair and is the distance at which the FRET efficiency equals 50 %. A large Förster distance is desirable as it offers the detection of biological interactions over large distances. LTC are suitable FRET donors for QDs because they provide long excited-state lifetimes in the millisecond range. This long PL decay time enables time-gated measurements for the suppression of autofluorescence and PL of directly excited QDs, which strongly increases the detection sensitivity. Additionally, the structured PL emission bands of LTCs together with the size-tunable PL emission bands of QDs make this FRET pair ideal for the application in multiplexed diagnostics, which is the measurement of multiple biomarkers in a single sample.The PhD thesis consists of two parts. In the first part the LTC-QD FRET pair was used within homogeneous FRET immunoassays for the detection of the biomarkers prostate specific antigen (TPSA), neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), and epidermal growth factor receptor (EGFR). The immunoassay sensitivity was optimized using different types of antibodies IgG, F(ab’)2,F(ab), and for EGFR single heavy chain antibodies, which differ largely in their size. The use of small-volume serum samples and measurements on clinical as well customized fluorescence plate readers result in picomolar detection limits for all measured biomarkers. In addition to these QD-based in vitro diagnostic tests, a detailed study of the different FRET-systems using time-resolved spectroscopy was performed. The investigation revealed the influence of the different antibodies on distance, functionality, and sensitivity of the FRET immunoassays. The study was completed by the measurement of NSE and CEA in a duplexed format and real patient samples were investigated.The second part was to use FRET for nanometric distance measurements as molecular or spectroscopic ruler. Time-resolved FRET measurements enabled the calculation of the distance between donor and acceptor. Therefore two different binding strategies were investigated to establish a close proximity between the LTC-donor to the QD-acceptor, namely biotin-streptavidin recognition and polyhistidine mediated self-assembly. A detailed time-resolved study was performed of QDs with different sizes, shapes, and surface coatings in combination with LTC bound to three different host biomolecules, which also possessed different sizes, shapes, orientations, and binding conditions. The analysis of the multi-exponential decay curves of donor and acceptor allowed to obtain information about the size, shape, and biofunctionality of the investigated QD bioconjugates. The results were in agreement with other structural analysis methods, such as transmission electron microscopy (TEM) or dynamic light scattering (DLS), but with the advantage of a homogeneous measurement with three-dimensional resolution (not possible for TEM), without the inclusion of a hydration shell (drawback for DLS), and at low concentration in the same environment as used for the biological application