@misc{BeckHildebrandtLoehmannsroeben2006, author = {Beck, Michael and Hildebrandt, Niko and L{\"o}hmannsr{\"o}ben, Hans-Gerd}, title = {Quantum dots as acceptors in FRET-assays containing serum}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-9504}, year = {2006}, abstract = {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 F{\"o}rster 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 F{\"o}rster-radius, estimated from the absorption and emission bands, was ca. 77 {\AA}. 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.}, subject = {Quantenpunkt}, language = {en} } @article{CharbonniereHildebrandt2008, author = {Charbonni{\`e}re, Loic J. and Hildebrandt, Niko}, title = {Lanthanide complexes and quantum dots : a bright wedding for resonance energy transfer}, year = {2008}, abstract = {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).}, language = {en} } @article{CywinskiHammannHuehnetal.2014, author = {Cywinski, Piotr J. and Hammann, Tommy and Huehn, Dominik and Parak, Wolfgang J. and Hildebrandt, Niko and L{\"o}hmannsr{\"o}ben, Hans-Gerd}, title = {Europium-quantum dot nanobioconjugates as luminescent probes for time-gated biosensing}, series = {Journal of biomedical optics}, volume = {19}, journal = {Journal of biomedical optics}, number = {10}, publisher = {SPIE}, address = {Bellingham}, issn = {1083-3668}, doi = {10.1117/1.JBO.19.10.101506}, pages = {8}, year = {2014}, abstract = {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)}, language = {en} } @article{CywinskiMoroRitscheletal.2011, author = {Cywinski, Piotr J. and Moro, Artur J. and Ritschel, Thomas and Hildebrandt, Niko and L{\"o}hmannsr{\"o}ben, Hans-Gerd}, title = {Sensitive and selective fluorescence detection of guanosine nucleotides by nanoparticles conjugated with a naphthyridine receptor}, series = {Analytical \& bioanalytical chemistry}, volume = {399}, journal = {Analytical \& bioanalytical chemistry}, number = {3}, publisher = {Springer}, address = {Heidelberg}, issn = {1618-2642}, doi = {10.1007/s00216-010-4420-2}, pages = {1215 -- 1222}, year = {2011}, abstract = {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.}, language = {en} } @article{GeisslerStuflerLoehmannsroebenetal.2013, author = {Geissler, Daniel and Stufler, Stefan and L{\"o}hmannsr{\"o}ben, Hans-Gerd and Hildebrandt, Niko}, title = {Six-color time-resolved forster resonance energy transfer for ultrasensitive multiplexed biosensing}, series = {Journal of the American Chemical Society}, volume = {135}, journal = {Journal of the American Chemical Society}, number = {3}, publisher = {American Chemical Society}, address = {Washington}, issn = {0002-7863}, doi = {10.1021/ja310317n}, pages = {1102 -- 1109}, year = {2013}, abstract = {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.}, language = {en} } @article{GeisslerButlinHilletal.2008, author = {Geißler, Daniel and Butlin, Nathaniel G. and Hill, Diana and L{\"o}hmannsr{\"o}ben, Hans-Gerd and Hildebrandt, Niko}, title = {Multiplexed diagnostics and spectroscopic ruler applications with terbium to quantum dots FRET}, issn = {1605-7422}, year = {2008}, language = {en} } @article{GeisslerCharbonniereZiesseletal.2010, author = {Geißler, Daniel and Charbonni{\`e}re, Lo{\"i}c J. and Ziessel, Raymond F. and Butlin, Nathaniel G. and L{\"o}hmannsr{\"o}ben, Hans-Gerd and Hildebrandt, Niko}, title = {Quantum dot biosensors for ultrasensitive multiplexed diagnostics}, issn = {1433-7851}, doi = {10.1002/anie.200906399}, year = {2010}, abstract = {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.}, language = {en} } @phdthesis{Hildebrandt2006, author = {Hildebrandt, Niko}, title = {Lanthanides and quantum dots : time-resolved laser spectroscopy of biochemical F{\"o}rster Resonance Energy Transfer (FRET) systems}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-12686}, school = {Universit{\"a}t Potsdam}, year = {2006}, abstract = {F{\"o}rster 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 F{\"o}rster radii of more than 100 {\AA} are achieved for QD as acceptors, considerably larger than for APC and DY633 (ca. 80 and 60 {\AA}). 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.}, language = {en} } @article{HildebrandtCharbonniereBecketal.2005, author = {Hildebrandt, Niko and Charbonniere, Lo{\"i}c J. and Beck, Michael and Ziessel, Raymond F. and L{\"o}hmannsr{\"o}ben, Hans-Gerd}, title = {Quantum dots as efficient energy acceptors in a time-resolved fluoroimmunoassay}, issn = {1433-7851}, year = {2005}, language = {en} } @article{HildebrandtGeissler2012, author = {Hildebrandt, Niko and Geissler, Daniel}, title = {Semiconductor quantum dots as FRET acceptors for multiplexed diagnostics and molecular ruler application}, series = {Advances in Experimental Medicine and Biology}, volume = {733}, journal = {Advances in Experimental Medicine and Biology}, editor = {Zahavy, E and Ordentlich, A and Yitzhaki, S and Shafferman, A}, publisher = {Springer}, address = {Dordrecht}, isbn = {978-94-007-2554-6}, issn = {0065-2598}, doi = {10.1007/978-94-007-2555-3_8}, pages = {75 -- 86}, year = {2012}, abstract = {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.}, language = {en} }