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Auswahl der wissenschaftlichen Literatur zum Thema „Transfert d'énergie résonant de Förster (FRET)“
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Dissertationen zum Thema "Transfert d'énergie résonant de Förster (FRET)"
Qiu, Xue. „Transfert d'énergie par résonance de type Förster pour les diagnostics multiplexés des récepteurs du facteur de croissance épidermique et microARNs“. Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS153/document.
Der volle Inhalt der QuelleThe new mission of clinical diagnostics and therapeutics, especially in point-of-care testing and precision medicine, has led to an increasing demand for multiplex and high throughput analyses of large numbers of biomolecules within a single sample. The thesis focuses on developing multiplexed biosensors based on time-resolved Förster resonance energy transfer from lanthanide complexes to organic dyes or quantum dots. I present several new techniques to simultaneously and multiplexed detect cancer related protein biomarkers (human epidermal growth factor receptor) or microRNAs (hsa- miR-20a-5p, hsa-miR-20b-5p and hsa-miR-21-5p) with very low limits of detections. I have used different strategies to achieve multiplexed detections such as spectral multiplexed detection based on different emission spectra of different luminophores, and temporal multiplexed detection based on distinguishable excited-state lifetimes of luminophores. The work is not only an applied research that can be used in clinical diagnostics but also a fundamental research of time-resolved FRET, which opens a new dimension of detection and greatly increases the number of biomarkers that can be detected
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.
Der volle Inhalt der QuelleNucleic 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
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.
Der volle Inhalt der QuelleFörster 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
Jana, Subha. „Biodetection using fluorescence energy transfer from Quantum dot excited whispering gallery modes to fluorescent acceptors“. Electronic Thesis or Diss., Université Paris sciences et lettres, 2021. http://www.theses.fr/2021UPSLS081.
Der volle Inhalt der QuelleQuantification of specific biomarkers is an important diagnostic tool. Standard immunoassays such as ELISA require extensive washing steps and signal amplification, in particular when the biomarker of interest is only present at very low concentrations. On the other hand, non-radiative Förster resonance energy transfer (FRET) has been used to design one-step homogenous bioassays which do not require any washing steps, where the biomarker enables the formation of a sandwich complex involving donor-labeled and acceptor-labeled antibodies. FRET from the donor to the acceptor then provides an optical signature of the complex formation, hence of the biomarker of interest. However, FRET which is highly sensitive to the donor-acceptor distance, only occurs in a significant rate when the distance between the donor and acceptor is less than 10 nanometers; thus the large size of many biological complexes limits the efficiency of energy transfer, preventing sensitive detection. Here I propose a novel energy transfer modality that uses solution-phase optical microcavities to enhance energy transfer. Following that, I describe a bio-sensing scheme designed to detect a cancer biomarker DNA in solution.To this aim, I have designed microcavity structures in which fluorescent colloidal quantum dots are located inside dielectric polymer microspheres to enable strong coupling of their fluorescence emission with the cavity resonance modes or whispering gallery modes (WGMs) of the microspheres. A detailed study was carried out to comprehend the structural and optical properties of these optical microcavities. I also characterized the energy transfer between these modes and acceptor dye-loaded nanoparticles present in the evanescent field, within a few tens of nanometers above the microsphere surface. An analytical model was constructed to provide insights into the WGM mediated energy transfer (WGET) mechanisms. Moreover, a comparison between WGET and FRET revealed the superiority of WGET in the context of building sensors with improved sensitivity and longer range of detection. In the last part of the thesis, a strategy is discussed in detail to provide biological functionalities to these optical microcavities which would enable them to interact with target analytes such as DNA, RNA, and proteins with high specificity, and moreover to reduce non-specific interactions. This strategy then was adapted to attach DNA capture probes onto the WGM enabled microcavities. Using the DNA attached microspheres as optical donor in combination with probe-DNA functionalized dye nanoparticles as optical acceptors, a biosensing assay has been successfully demonstrated to detect a cancer biomarker DNA called survivin in the solution phase. This assay did not only show good sensitivity towards the target, but also it has proven to be highly specific. The detection scheme has been demonstrated in a sophisticated confocal microscope at the single microsphere level, then successfully translated to a much simpler spectrofluorometer that measures fluorescence from the whole sample solution; the signature of the sandwich complex formation was also effectively detected.In conclusion, I demonstrated that microcavity-assisted energy transfer has several advantages over regular FRET assays. A real bio-sensing assay based on the WGET principle has also been successfully designed to detect cancer biomarkers with high sensitivity and specificity. This study thus opens up many possibilities to design high-performing and more accurate assays to detect varieties of biological entities
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.
Der volle Inhalt der QuelleThis 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
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.
Der volle Inhalt der QuelleAs 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
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.
Der volle Inhalt der QuelleEngineered 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