TY  - JOUR
A1  - Sellrie, Frank
A1  - Beck, Michael
A1  - Hildebrandt, Niko
A1  - Micheel, Burkhard
T1  - A homogeneous time-resolved fluoroimmunoassay (TR-FIA) using antibody mediated luminescence quenching
N2  - The determination of low-molecular weight substances (haptens) is demonstrated with a homogeneous time-resolved immunoassay using antibody-induced luminescence quenching. Our novel assay technology uses the newly developed monoclonal antibody (G24-BA9) to quench the luminescence of europium trisbipyridine (EuTBP). We performed a competitive biotin immunoassay including an EuTBP-biotin conjugate, the anti-EuTBP antibody G24-BA9 and streptavidin as assay components. Steric hindrance allows only the binding of either G24-BA9 (to the EuTBP moiety) or streptavidin (to the biotin moiety) to the EuTBP-biotin conjugate. Addition of the analyte biotin resulted in the binding of streptavidin to biotin and a concomitant preferred binding of G24-BA9 to EuTBP-biotin. Since G24-BA9 quenches the luminescence of EuTBP within the conjugate, the luminescence signal could be used to indicate and quantify the presence of free biotin in the system. All experiments were carried out in solution in the presence of 5% serum demonstrating the possibility of using our novel assay for a very fast determination of low molecular weight substances in biological fluids.
Y1  - 2010
UR  - http://www.rsc.org/Publishing/Journals/AY/Index.asp
U6  - https://doi.org/10.1039/C0ay00306a
SN  - 1759-9660
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  - 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  - 
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 Cr<SUP>4+</SUP>: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  - 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  - 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  - 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  - 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  - JOUR
A1  - Geißler, Daniel
A1  - Charbonnière, Loïc J.
A1  - Ziessel, Raymond F.
A1  - Butlin, Nathaniel G.
A1  - Löhmannsröben, Hans-Gerd
A1  - Hildebrandt, Niko
T1  - Quantum dot biosensors for ultrasensitive multiplexed diagnostics
N2  - Time- and color-resolved detection of Foerster resonance energy transfer (FRET) from luminescent terbium complexes to different semiconductor quantum dots results in a fivefold multiplexed bioassay with sub-picomolar detection limits for all five bioanalytes (see picture). The detection of up to five biomarkers occurs with a sensitivity that is 40-240-fold higher than one of the best-established single-analyte reference assays.
Y1  - 2010
UR  - http://www3.interscience.wiley.com/cgi-bin/jhome/26737/
U6  - https://doi.org/10.1002/anie.200906399
SN  - 1433-7851
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 Eu<SUP>3+</SUP>-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  - 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  - JOUR
A1  - Hildebrandt, Niko
A1  - Geissler, Daniel
ED  - Zahavy, E
ED  - Ordentlich, A
ED  - Yitzhaki, S
ED  - Shafferman, A
T1  - Semiconductor quantum dots as FRET acceptors for multiplexed diagnostics and molecular ruler application
JF  - Advances in Experimental Medicine and Biology
N2  - Applications based on Forster resonance energy transfer (FRET) play an important role for the determination of concentrations and distances within nanometer-scale systems in vitro and in vivo in many fields of biotechnology. Semiconductor nanocrystals (Quantum dots - QDs) possess ideal properties for their application as FRET acceptors when the donors have long excited state lifetimes and when direct excitation of QDs can be efficiently suppressed. Therefore, luminescent terbium complexes (LTCs) with excited state lifetimes of more than 2 ms are ideal FRET donor candidates for QD-acceptors. This chapter will give a short overview of theoretical and practical background of FRET, QDs and LTCs, and present some recent applications of LTC-QD FRET pairs for multiplexed ultra-sensitive in vitro diagnostics and nanometer-resolution molecular distance measurements.
KW  - Diagnostics
KW  - FRET
KW  - Imaging
KW  - Quantum dots
KW  - Terbium
Y1  - 2012
SN  - 978-94-007-2554-6
SN  - 978-94-007-2555-3
U6  - https://doi.org/10.1007/978-94-007-2555-3_8
SN  - 0065-2598
VL  - 733
SP  - 75
EP  - 86
PB  - Springer
CY  - Dordrecht
ER  - 
TY  - JOUR
A1  - Cywinski, Piotr J.
A1  - Moro, Artur J.
A1  - Ritschel, Thomas
A1  - Hildebrandt, Niko
A1  - Löhmannsröben, Hans-Gerd
T1  - Sensitive and selective fluorescence detection of guanosine nucleotides by nanoparticles conjugated with a naphthyridine receptor
JF  - Analytical & bioanalytical chemistry
N2  - Novel fluorescent nanosensors, based on a naphthyridine receptor, have been developed for the detection of guanosine nucleotides, and both their sensitivity and selectivity to various nucleotides were evaluated. The nanosensors were constructed from polystyrene nanoparticles functionalized by (N-(7-((3-aminophenyl) ethynyl)-1,8-naphthyridin- 2-yl) acetamide) via carbodiimide ester activation. We show that this naphthyridine nanosensor binds guanosine nucleotides preferentially over adenine, cytosine, and thymidine nucleotides. Upon interaction with nucleotides, the fluorescence of the nanosensor is gradually quenched yielding Stern-Volmer constants in the range of 2.1 to 35.9mM(-1). For all the studied quenchers, limits of detection (LOD) and tolerance levels for the nanosensors were also determined. The lowest (3 sigma) LOD was found for guanosine 3',5'-cyclic monophosphate (cGMP) and it was as low as 150 ng/ml. In addition, we demonstrated that the spatial arrangement of bound analytes on the nanosensors' surfaces is what is responsible for their selectivity to different guanosine nucleotides. We found a correlation between the changes of the fluorescence signal and the number of phosphate groups of a nucleotide. Results of molecular modeling and zeta-potential measurements confirm that the arrangement of analytes on the surface provides for the selectivity of the nanosensors. These fluorescent nanosensors have the potential to be applied in multi-analyte, array-based detection platforms, as well as in multiplexed microfluidic systems.
KW  - Naphthyridine receptor
KW  - cGMP
KW  - Base pairing
KW  - Nucleotide nanosensor
KW  - Fluorescence spectroscopy
Y1  - 2011
U6  - https://doi.org/10.1007/s00216-010-4420-2
SN  - 1618-2642
VL  - 399
IS  - 3
SP  - 1215
EP  - 1222
PB  - Springer
CY  - Heidelberg
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  - Morgner, Frank
A1  - Stufler, Stefan
A1  - Geissler, Daniel
A1  - Medintz, Igor L.
A1  - Algar, W. Russ
A1  - Susumu, Kimihiro
A1  - Stewart, Michael H.
A1  - Blanco-Canosa, Juan B.
A1  - Dawson, Philip E.
A1  - Hildebrandt, Niko
T1  - Terbium to quantum dot FRET Bioconjugates for clinical diagnostics  influence of human plasma on optical and assembly properties
JF  - Sensors
N2  - Forster resonance energy transfer (FRET) from luminescent terbium complexes (LTC) as donors to semiconductor quantum dots (QDs) as acceptors allows extraordinary large FRET efficiencies due to the long Forster distances afforded. Moreover, time-gated detection permits an efficient suppression of autofluorescent background leading to sub-picomolar detection limits even within multiplexed detection formats. These characteristics make FRET-systems with LTC and QDs excellent candidates for clinical diagnostics. So far, such proofs of principle for highly sensitive multiplexed biosensing have only been performed under optimized buffer conditions and interactions between real-life clinical media such as human serum or plasma and LTC-QD-FRET-systems have not yet been taken into account. Here we present an extensive spectroscopic analysis of absorption, excitation and emission spectra along with the luminescence decay times of both the single components as well as the assembled FRET-systems in TRIS-buffer, TRIS-buffer with 2% bovine serum albumin, and fresh human plasma. Moreover, we evaluated homogeneous LTC-QD FRET assays in QD conjugates assembled with either the well-known, specific biotin-streptavidin biological interaction or, alternatively, the metal-affinity coordination of histidine to zinc. In the case of conjugates assembled with biotin-streptavidin no significant interference with the optical and binding properties occurs whereas the histidine-zinc system appears to be affected by human plasma.
KW  - FRET
KW  - quantum dots
KW  - terbium
KW  - luminescence lifetime
KW  - blood
KW  - plasma
KW  - clinical diagnostics
KW  - biotin
KW  - streptavidin
KW  - histidin
KW  - immunoassay
Y1  - 2011
U6  - https://doi.org/10.3390/s111009667
SN  - 1424-8220
VL  - 11
IS  - 10
SP  - 9667
EP  - 9684
PB  - MDPI
CY  - Basel
ER  - 
TY  - JOUR
A1  - Kupstat, Annette
A1  - Kumke, Michael Uwe
A1  - Hildebrandt, Niko
T1  - Toward sensitive, quantitative point-of-care testing (POCT) of protein markers miniaturization of a homogeneous time-resolved fluoroimmunoassay for prostate-specific antigen detection
JF  - The analyst : the analytical journal of the Royal Society of Chemistry
N2  - Point-of-care testing (POCT) systems which allow for a sensitive, quantitative detection of protein markers are extremely useful for the early detection and therapy progress monitoring of cancer. However, currently commercially available POCT devices are mainly limited to the qualitative detection of protein markers. In this study we demonstrate the successive miniaturization of a sensitive and fast assay for the quantitative detection of prostate-specific antigen (PSA) using a well established and clinically approved homogeneous time-resolved fluoroimmunoassay technology (TRACE (R)) on a commercial plate-reader system (KRYPTOR (R)). Regarding the initial requirements for the development of POCT devices we applied a 30-fold assay volume reduction (150 mu L to 5 mu L) to achieve a reasonable lab-on-a-chip volume and a 24-fold and 120-fold excitation pulse energy reduction to achieve reasonable pulse energies for low-cost miniature excitation sources. Due to highly efficient optimization of key POCT parameters our miniaturized PSA assay achieved a 30% increased sensitivity and a 2-fold improved limit of detection compared to the standard plate-reader method. Our results demonstrate the successful implementation of key parameters for a significant miniaturization and for cost reduction in the clinically approved KRYPTOR (R) platform for protein detection. The technological alterations required are easy-to-implement and can be immediately adapted for more than 30 diagnostic protein markers already available for the KRYPTOR (R) platform. These features strongly recommend our assay format to be utilized in innovative, sensitive, quantitative POCT of protein markers.
Y1  - 2011
U6  - https://doi.org/10.1039/c0an00684j
SN  - 0003-2654
VL  - 136
IS  - 5
SP  - 1029
EP  - 1035
PB  - Royal Society of Chemistry
CY  - Cambridge
ER  -