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  • Hildebrandt, Niko  (136)
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  • 1
    Language: English
    In: Journal of the American Chemical Society, 23 January 2013, Vol.135(3), pp.1102-9
    Description: Simultaneous monitoring of multiple molecular interactions and multiplexed detection of several diagnostic biomarkers at very low concentrations have become important issues in advanced biological and chemical sensing. Here we present an optically multiplexed six-color Förster resonance energy transfer (FRET) biosensor for simultaneous monitoring of five different individual binding events. We combined simultaneous FRET from one Tb complex to five different organic dyes measured in a filter-based time-resolved detection format with a sophisticated spectral crosstalk correction, which results in very efficient background suppression. The advantages and robustness of the multiplexed FRET sensor were exemplified by analyzing a 15-component lung cancer immunoassay involving 10 different antibodies and five different tumor markers in a single 50 μL human serum sample. The multiplexed biosensor offers clinically relevant detection limits in the low picomolar (ng/mL) concentration range for all five markers, thus providing an effective early screening tool for lung cancer with the possibility of distinguishing small-cell from non-small-cell lung carcinoma. This novel technology will open new doors for multiple biomarker diagnostics as well as multiplexed real-time imaging and spectroscopy.
    Keywords: Biosensing Techniques ; Color ; Fluorescence Resonance Energy Transfer ; Biomarkers, Tumor -- Blood ; Carcinoma, Non-Small-Cell Lung -- Diagnosis ; Lung Neoplasms -- Diagnosis ; Small Cell Lung Carcinoma -- Diagnosis
    ISSN: 00027863
    E-ISSN: 1520-5126
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  • 2
    Language: English
    In: Analytical and Bioanalytical Chemistry, 2016, Vol.408(17), pp.4475-4483
    Description: The exceptional photophysical properties and the nanometric dimensions of colloidal semiconductor quantum dots (QD) have strongly attracted the bioanalytical community over the last approximately 20 y. In particular, the integration of QDs in the analysis of biological components and interactions, and the related diagnostics using Förster resonance energy transfer (FRET), have allowed researchers to significantly improve and diversify fluorescence-based biosensing. In this TRENDS article, we review some recent developments in QD-FRET biosensing that have implemented this technology in electronic consumer products, multiplexed analysis, and detection without light excitation for diagnostic applications. In selected examples of smartphone-based imaging, single- and multistep FRET, steady-state and time-resolved spectroscopy, and bio/chemiluminescence detection of QDs used as both FRET donors and acceptors, we highlight the advantages of QD-based FRET biosensing for multiplexed and sensitive diagnostics. Graphical Abstract Quantum dots (QDs) can be applied as donors and/or acceptors for Förster resonance energy transfer- (FRET-) based biosensing for multiplexed and sensitive diagnostics in various assay formats
    Keywords: Fluorescence ; Spectroscopy ; Imaging ; Multiplexing ; Nanoparticle ; Immunoassay ; DNA ; RNA ; Smartphone ; BRET ; CRET
    ISSN: 1618-2642
    E-ISSN: 1618-2650
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  • 3
    Language: English
    In: Journal of the American Chemical Society, 25 January 2012, Vol.134(3), pp.1876-91
    Description: The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in Förster resonance energy transfer (FRET)-based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD-A647 donor-acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD.
    Keywords: Quantum Dots ; Biosensing Techniques -- Methods ; DNA -- Chemistry ; Fluorescence Resonance Energy Transfer -- Methods ; Luminescent Agents -- Chemistry ; Peptides -- Chemistry
    ISSN: 00027863
    E-ISSN: 1520-5126
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  • 4
    Language: English
    In: Analytical chemistry, 20 November 2012, Vol.84(22), pp.10136-46
    Description: Semiconductor quantum dots (QDs) are attractive probes for optical sensing and imaging due to their unique photophysical attributes and nanoscale size. In particular, the development of assays and biosensors based on QDs and Förster resonance energy transfer (FRET) continues to be a prominent focus of research. Here, we demonstrate the application of QDs as simultaneous donors and acceptors in a time-gated FRET relay for the multiplexed detection of protease activity. In contrast to the current state-of-the-art, which uses multiple colors of QDs, multiplexing was achieved using only a single color of QD. The other constituents of the FRET relay, a luminescent terbium complex and fluorescent dye, were assembled to QDs via peptides that were selected as substrates for the model proteases trypsin and chymotrypsin. Loss of prompt FRET between the QD and dye signaled the activity of chymotrypsin; loss of time-gated FRET between the terbium and QD signaled the activity of trypsin. We applied the FRET relay in a series of quantitative, real-time kinetic assays of increasing biochemical complexity, including multiplexed sensing, measuring inhibition in a multiplexed format, and tracking the proteolytic activation of an inactive pro-protease to its active form in a coupled, multienzyme system. These capabilities were derived from a ratiometric analysis of the two FRET pathways in the relay and permitted extraction of initial reaction rates, enzyme specificity constants, and apparent inhibition constants. This work adds to the growing body of research on multifunctional nanoparticles and introduces multiplexed sensing as a novel capability for a single nanoparticle vector. Furthermore, the ability to track both enzymes within a coupled biological system using one vector represents a significant advancement for nanoparticle-based biosensing. Prospective applications in biochemical research, applied diagnostics, and drug discovery are discussed.
    Keywords: Fluorescence Resonance Energy Transfer ; Quantum Dots ; Chymotrypsin -- Metabolism ; Enzyme Assays -- Methods ; Trypsin -- Metabolism
    ISSN: 00032700
    E-ISSN: 1520-6882
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  • 5
    Language: English
    In: Angewandte Chemie International Edition, 17 August 2015, Vol.54(34), pp.10024-10029
    Description: The importance of microRNA (miRNA) dysregulation for the development and progression of diseases and the discovery of stable miRNAs in peripheral blood have made these short‐sequence nucleic acids next‐generation biomarkers. Here we present a fully homogeneous multiplexed miRNA FRET assay that combines careful biophotonic design with various RNA hybridization and ligation steps. The single‐step, single‐temperature, and amplification‐free assay provides a unique combination of performance parameters compared to state‐of‐the‐art miRNA detection technologies. Precise multiplexed quantification of miRNA‐20a, ‐20b, and ‐21 at concentrations between 0.05 and 0.5 n in a single 150 μL sample and detection limits between 0.2 and 0.9 n in 7.5 μL serum samples demonstrate the feasibility of both high‐throughput and point‐of‐care clinical diagnostics. : An amplification‐free homogeneous “mix‐and‐measure” assay for the multiplexed detection of up to three microRNAs has been developed. It uses Förster resonance energy transfer from a Tb complex donor to three different dye acceptors and is conducted in one step and at a single temperature.
    Keywords: Clinical Diagnostics ; Fret ; Microrna ; Multiplexing ; Time‐Gated Fluorescence Detection
    ISSN: 1433-7851
    E-ISSN: 1521-3773
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  • 6
    Language: English
    In: The Journal of Physical Chemistry C, 2017, Vol.121(51), pp.28395--28402
    Description: We herein report the first real-space investigation of Förster resonance energy transfer (FRET) in two different types of quantum dot (QD) supramolecular assemblies by observing their three-dimensional (3D) configurations through high-resolution electron tomography. Owing to its critical role in photosynthesis, artificial light-harvesting antennas, and investigation of protein-protein interactions, the mechanism of FRET has been intensively studied by monitoring its excited-state dynamics via various spectroscopic techniques. The utilized electron tomography technique allowed the direct localization of 3D coordinates of individual QDs in self-assembled nanostructures and theoretical estimation of the FRET efficiency of a single fluorophore, domain, or supramolecular assembly. Moreover, the experimental value of the FRET efficiency determined by fluorescence spectroscopy was in good agreement with the magnitude obtained via electron tomography. We believe that the described strategy can be used in single-molecule FRET studies and will help to create a new bridge between material science and molecular/supramolecular photochemistry.
    Keywords: Life Sciences
    ISSN: 19327447
    E-ISSN: 19327455
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  • 7
    Language: English
    In: ACS nano, 26 July 2011, Vol.5(7), pp.5286-90
    Description: Semiconductor quantum dots possess unique photophysical properties such as bright emission with narrow wavelength bandwidth and extremely broad and strong absorption. In combination with their size-dependent color tunability, quantum dots have been proposed as ideal candidates for multiplexed optical bioanalysis for more than a decade. However, the unavailability of stable, reproducible, biocompatible quantum dots with controlled and functional multiple biolabeling has restricted these nanocrystals to research applications. In this issue of ACS Nano, Jennings et al. demonstrate the versatile use of quantum dot antibody conjugates produced by commercially available kits that allow an easy and fast labeling. This Perspective highlights the potential of novel quantum dot bioconjugation approaches in combination with state-of-the-art detection methods and technologies for successful and widely applicable multiplexed biosensing.
    Keywords: Quantum Dots ; Semiconductors ; Staining and Labeling -- Methods
    ISSN: 19360851
    E-ISSN: 1936-086X
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  • 8
    Language: English
    In: Analytical and Bioanalytical Chemistry, Nov, 2010, Vol.398(5), p.2133(8)
    Description: Byline: Chongxiao Tan (1), Nenad Gajovic-Eichelmann (1), Rainer Polzius (2), Niko Hildebrandt (3), Frank F. Bier (1) Keywords: THC; Saliva; Homogeneous; Immunoassay; FITC Abstract: The detection of the major active component of cannabis, I9-tetrahydrocannabinol (THC), becomes increasingly relevant due to its widespread abuse. For control purposes, some easy-to-use, sensitive and inexpensive test methods are needed. We have developed a fluorescence immunoassay utilising THC--fluorescein conjugate as tracer. Fluorescence spectroscopy of the conjugate revealed an unusual property: The relatively weak fluorescence of a dilute tracer solution was increased by a factor of up to 5 after binding of a THC-specific antibody. Fluorescence lifetime measurements in aqueous solutions suggested two different tracer conformations both associated with quenching of fluorescein fluorescence by the intramolecular THC moiety. After antibody binding, the tracer enters a third conformation in which fluorescence quenching of fluorescein is completely suppressed. Utilising this property, we established a homogeneous competitive immunoassay (homogeneous increasing fluorescence immunoassay) with low detection limits. The test requires only two reagents, the new tracer molecule and an anti-THC antibody. A single test takes only 8 min. The dynamic detection range for THC is 0.5 to 20 ng/mL in buffer, with a limit of detection (LOD) of 0.5 ng/mL. The test also works in diluted saliva samples (1:10 dilution with buffer) with an LOD of 2 ng/mL and a dynamic range of 2--50 ng/mL. Author Affiliation: (1) Group Biosensors, Fraunhofer Institute for Biomedical Engineering, Am Muhlenberg 13, 14476, Potsdam-Golm, Germany (2) Research Unit, Dragerwerk AG & Co. KGaA, Moislinger Allee 53-55, 23558, Lubeck, Germany (3) Group NanoPolyPhotonics, Fraunhofer Institute for Applied Polymer Research, Wissenschaftspark Golm, Geiselbergstrasse 69, 14476, Potsdam-Golm, Germany Article History: Registration Date: 07/08/2010 Received Date: 05/05/2010 Accepted Date: 06/08/2010 Online Date: 26/08/2010 Article note: Electronic supplementary material The online version of this article (doi: 10.1007/s00216-010-4109-6) contains supplementary material, which is available to authorized users.
    Keywords: Marijuana ; Detection Equipment ; Fluorescence ; Reagents ; Tracers (Biology) ; Antibodies ; Fluorescein
    ISSN: 1618-2642
    Source: Cengage Learning, Inc.
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  • 9
    In: Chemical Society Reviews, 2015, Vol.44(14), pp.4792-4834
    Description: Semiconductor quantum dots (QDs) have become important fluorescent probes for in vitro and in vivo bioimaging research. Their nanoparticle surfaces for versatile bioconjugation, their adaptable photophysical properties for multiplexed detection, and their superior stability for longer investigation times are the main advantages of QDs compared to other fluorescence imaging agents. Here, we review the recent literature dealing with the design and application of QD-bioconjugates for advanced in vitro and in vivo imaging. After a short summary of QD preparation and their most important properties, different QD-based imaging applications will be discussed from the technological and the biological point of view, ranging from super-resolution microscopy and single-particle tracking over in vitro cell and tissue imaging to in vivo investigations. A substantial part of the review will focus on multifunctional applications, in which the QD fluorescence is combined with drug or gene delivery towards theranostic approaches or with complementary technologies for multimodal imaging. We also briefly discuss QD toxicity issues and give a short outlook on future directions of QD-based bioimaging.
    Keywords: Chemistry;
    ISSN: 0306-0012
    E-ISSN: 1460-4744
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  • 10
    In: Chemical Communications, 2016, Vol.52(29), pp.5080-5095
    Description: Luminescent lanthanide complexes display unrivalled spectroscopic properties, which place them in a special category in the luminescent toolbox. Their long-lived line-like emission spectra are the cornerstones of numerous analytical applications ranging from ultrasensitive homogeneous fluoroimmunoassays to the study of molecular interactions in living cells with multiplexed microscopy. However, achieving such minor miracles is a result of years of synthetic efforts and spectroscopic studies to understand and gather all the necessary requirements for the labels to be efficient. This feature article intends to survey these criteria and to discuss some of the most important examples reported in the literature, before explaining in detail some of the applications of luminescent lanthanide labels to bioanalysis and luminescence microscopy. Finally, the emphasis will be put on some recent applications that hold great potential for future biosensing.
    Keywords: Lanthanoid Series Elements -- Chemistry ; Luminescent Agents -- Chemistry;
    ISSN: 1359-7345
    E-ISSN: 1364-548X
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