TY - JOUR A1 - Hildebrandt, Niko A1 - Charbonniere, Loïc J. A1 - Beck, Michael A1 - Ziessel, Raymond F. A1 - Löhmannsröben, Hans-Gerd T1 - Quantum dots as efficient energy acceptors in a time-resolved fluoroimmunoassay Y1 - 2005 SN - 1433-7851 ER - TY - THES A1 - Hildebrandt, Niko T1 - Lanthanides and quantum dots : time-resolved laser spectroscopy of biochemical Förster Resonance Energy Transfer (FRET) systems T1 - Lanthanide und Quantenpunkte : zeitaufgelöste Laserspektroskopie an biochemischen Förster-Resonanzenergietransfer (FRET) Systemen N2 - 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. N2 - 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. KW - FRET KW - Lanthanide KW - Quantenpunkte KW - Zeitaufgelöster Immunoassay KW - Spektroskopie KW - FRET KW - Lanthanides KW - Quantum Dots KW - Time-resolved Immunoassay KW - Spectroscopy Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-12686 ER - TY - GEN A1 - Löhmannsröben, Hans-Gerd A1 - Beck, Michael A1 - Hildebrandt, Niko A1 - Schmälzlin, Elmar A1 - van Dongen, Joost T. T1 - New challenges in biophotonics : laser-based fluoroimmuno analysis and in-vivo optical oxygen monitoring N2 - Two examples of our biophotonic research utilizing nanoparticles are presented, namely laser-based fluoroimmuno analysis and in-vivo optical oxygen monitoring. Results of the work include significantly enhanced sensitivity of a homogeneous fluorescence immunoassay and markedly improved spatial resolution of oxygen gradients in root nodules of a legume species. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 018 KW - Sauerstoff KW - Quantenpunkt KW - Lumineszenz KW - Immunoassay KW - Energietransfer KW - Fluoreszenz-Resonanz-Energie-Transfer KW - Nanopartikel KW - Lanthanoide KW - Optode KW - Förster Resonanz Energie Transfer KW - Biophotonik KW - biophotonics KW - nanoparticles KW - immunoassay KW - oxygen KW - optode Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-10120 ER - TY - GEN A1 - Niederkrüger, Matthias A1 - Salb, Christian A1 - Beck, Michael A1 - Hildebrandt, Niko A1 - Löhmannsröben, Hans-Gerd A1 - Marowsky, Gerd T1 - Improvement of a fluorescence immunoassay with a compact diode-pumped solid state laser at 315 nm N2 - We demonstrate the improvement of fluorescence immunoassay (FIA) diagnostics in deploying a newly developed compact diode-pumped solid state (DPSS) laser with emission at 315 nm. The laser is based on the quasi-three-level transition in Nd:YAG at 946 nm. The pulsed operation is either realized by an active Q-switch using an electro-optical device or by introduction of a Cr4+:YAG saturable absorber as passive Q-switch element. By extra-cavity second harmonic generation in different nonlinear crystal media we obtained blue light at 473 nm. Subsequent mixing of the fundamental and the second harmonic in a β-barium-borate crystal provided pulsed emission at 315 nm with up to 20 μJ maximum pulse energy and 17 ns pulse duration. Substitution of a nitrogen laser in a FIA diagnostics system by the DPSS laser succeeded in considerable improvement of the detection limit. Despite significantly lower pulse energies (7 μJ DPSS laser versus 150 μJ nitrogen laser), in preliminary investigations the limit of detection was reduced by a factor of three for a typical FIA. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 016 KW - Immunoassay KW - Fluoreszenz-Resonanz-Energie-Transfer KW - Neodym-YAG-Laser KW - 946 nm KW - 473 nm KW - 315 nm KW - gepulster DPSS Laser KW - sättigbarer Absorber KW - fluorescence immunoassay KW - 946 nm KW - 473 nm KW - 315 nm KW - pulsed DPSS laser Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-10150 ER - TY - GEN A1 - Beck, Michael A1 - Hildebrandt, Niko A1 - Löhmannsröben, Hans-Gerd T1 - Quantum dots as acceptors in FRET-assays containing serum N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 019 KW - Quantenpunkt KW - Lumineszenz KW - Serum KW - Europium KW - Immunoassay KW - Energietransfer KW - Fluoreszenz-Resonanz-Energie-Transfer KW - Förster-Resonanz-Energie-Transfer KW - Quantum Dot KW - Luminescence KW - Serum KW - Europium KW - Immunoassay KW - Energy Transfer KW - FRET Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-9504 ER - TY - JOUR A1 - Geissler, Daniel A1 - Stufler, Stefan A1 - Löhmannsröben, Hans-Gerd A1 - Hildebrandt, Niko T1 - Six-color time-resolved forster resonance energy transfer for ultrasensitive multiplexed biosensing JF - Journal of the American Chemical Society N2 - 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 Forster 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 mu L 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. Y1 - 2013 U6 - https://doi.org/10.1021/ja310317n SN - 0002-7863 VL - 135 IS - 3 SP - 1102 EP - 1109 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Charbonnière, Loic J. A1 - Hildebrandt, Niko T1 - Lanthanide complexes and quantum dots : a bright wedding for resonance energy transfer N2 - In this microreview we describe the principle of Forster resonance energy transfer (FRET) occurring between closely spaced energy-donor and -acceptor molecules. The theoretical treatment is depicted in relation with the data extractable from spectroscopic measurements. We present the specific case of semiconductor nanocrystals (or quantum dots QDs) as energy donors in FRET experiments and a particular emphasis is put on the specific advantages of these fluorophores with regard to both their exceptional photophysical properties and their nanoscopic morphology. In a following section, the special attributes of luminescent lanthanide complexes (LLCs) are outlined with illustrations of properties such as their characteristic emission spectra, long-lived luminescence, and large "Stokes shift". Finally, the successful combination of LLCs and QDs in FRET experiments is demonstrated, showing the unrivaled benefits of this singular marriage, opening doors for energy transfer at very large distances and excellent sensitivity of detection within the frame of time-resolved fluoroimmunoassays. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008). Y1 - 2008 ER - TY - JOUR A1 - Kadjane, Pascal A1 - Starck, Matthieu A1 - Camerel, Franck A1 - Hill, Diana A1 - Hildebrandt, Niko A1 - Ziessel, Raymond A1 - Charbonnière, Loïc J. T1 - Divergent approach to a large variety of versatile luminescent lanthanide complexes N2 - Using a regioselective strategy for nucleophilic aromatic substitution on polyfluoropyridines, a nonacoordinating precursor was designed that is adequately suited for complexation of lanthanide cations. Further functionalizations afforded numerous applications for near-IR emission, two-photon absorption spectroscopy, or the formation of luminescent gels. Y1 - 2009 UR - http://pubs.acs.org/journal/inocaj U6 - https://doi.org/10.1021/Ic9001169 SN - 0020-1669 ER - TY - JOUR A1 - Geißler, Daniel A1 - Butlin, Nathaniel G. A1 - Hill, Diana A1 - Löhmannsröben, Hans-Gerd A1 - Hildebrandt, Niko T1 - Multiplexed diagnostics and spectroscopic ruler applications with terbium to quantum dots FRET Y1 - 2008 SN - 1605-7422 ER - TY - JOUR A1 - Cywinski, Piotr J. A1 - Hammann, Tommy A1 - Huehn, Dominik A1 - Parak, Wolfgang J. A1 - Hildebrandt, Niko A1 - Löhmannsröben, Hans-Gerd T1 - Europium-quantum dot nanobioconjugates as luminescent probes for time-gated biosensing JF - Journal of biomedical optics N2 - Nanobioconjugates have been synthesized using cadmium selenide quantum dots (QDs), europium complexes (EuCs), and biotin. In those conjugates, long-lived photoluminescence (PL) is provided by the europium complexes, which efficiently transfer energy via Forster resonance energy transfer (FRET) to the QDs in close spatial proximity. As a result, the conjugates have a PL emission spectrum characteristic for QDs combined with the long PL decay time characteristic for EuCs. The nanobioconjugates synthesis strategy and photo-physical properties are described as well as their performance in a time-resolved streptavidin-biotin PL assay. In order to prepare the QD-EuC-biotin conjugates, first an amphiphilic polymer has been functionalized with the EuC and biotin. Then, the polymer has been brought onto the surface of the QDs (either QD655 or QD705) to provide functionality and to make the QDs water dispersible. Due to a short distance between EuC and QD, an efficient FRET can be observed. Additionally, the QD-EuC-biotin conjugates' functionality has been demonstrated in a PL assay yielding good signal discrimination, both from autofluorescence and directly excited QDs. These newly designed QD-EuC-biotin conjugates expand the class of highly sensitive tools for bioanalytical optical detection methods for diagnostic and imaging applications. (C) 2014 Society of Photo-Optical Instrumentation Engineers (SPIE) KW - quantum dots KW - europium complex KW - amphiphilic polymer assembly KW - nanobioconjugate KW - biosensor KW - time-resolved fluorescence Y1 - 2014 U6 - https://doi.org/10.1117/1.JBO.19.10.101506 SN - 1083-3668 SN - 1560-2281 VL - 19 IS - 10 PB - SPIE CY - Bellingham ER -