In memory of Theodor Förster on the centenary of his birth on May 15th 2010Applications based on Förster resonance energy transfer (FRET) play an important role in the determination of concentrations and distances within nanometer-scale systems in vitro and in vivo in the fields of biology, biochemistry, medicine, and other life sciences. [1][2][3] Due to the r À6 distance dependence of FRET, structural changes of molecular systems in the 1-10 nm range can be measured with high accuracy far below the light diffraction limit. Stryer et al. [4,5] demonstrated the spectroscopic ruler FRET technique more than 40 years ago, and it is still frequently used for in-and exvivo studies of inter-and intramolecular interactions by spectroscopy and microscopy down to the single-molecule level.[6-9] Several FRET-based biosensors for functional intracellular investigations have been developed. [10][11][12][13][14] Although most of these applications use single sensors, there have been some recent developments of dual FRET pairs for cellular imaging using fluorescent proteins, [15][16][17] and even with a single excitation wavelength.[18] Using a multiplexed FRET technique allows the simultaneous measurement of multiple distances or conformational changes, thereby decreasing time and effort whilst increasing bioanalytical information due to the possible correlation of simultaneous events.The FRET pair combination of luminescent terbium complexes (LTCs) as donors and semiconductor quantum dots (QDs) as acceptors holds significant advantages concerning sensitivity, distance, and multiparametric analysis compared to other donor-acceptor pairs. [19,20] Due to large overlap integral values, exceptionally long Förster radii (R 0 , the donor-acceptor distance at which the FRET efficiency is 50 %) of up to 11 nm can be achieved, [21][22][23] whereas conventional donor-acceptor pairs have much smaller R 0 values that rarely exceed 6 nm.[24] Although nanoplasmonic molecular rulers have been developed for which distances of up to about 70 nm can be measured, [25,26] these applications use relatively large noble metal nanoparticles (up to 40 nm) and are restricted in their multiplexed use of simultaneously measuring variable distances of different systems (for example, several different intracellular functional events within one measurement). The pioneering work of Weiss et al. demonstrated multiplexed optical rulers using quantum dots and ultrahigh-resolution colocalization (UHRC).[27] Although FRET has advantages concerning resolution accuracy and dynamic measurements, [28] UHRC is well-suited to measuring distances in the range of few nanometers to tens of micrometers. [29] Two very important aspects for intracellular studies with QDs are the shape and the size of these nanosensors, which can be crucial, for example, for cell penetration and for evaluation of the nanoparticle impact on the targeted biomolecules. Measuring the core/shell dimensions of the semiconductor material with TEM is possible with relatively good accuracy. However, ...

Luminescent semiconductor quantum dots (QDs) play an important role in optical biosensing and, in particular, in FRET (Förster resonance energy transfer)-based luminescent probes. The QD materials that form the basis for these probes are in actuality quite heterogeneous and consist of different types of QDs with variations in material compositions, surface coatings, and available biofunctionalization strategies. To optimize their role in active sensors that rely on FRET, extensive physicochemical characterization is required. A technique that can provide precise information about size, shape, and bioconjugation properties of different QD–biomolecule conjugates from a single sample and measurement under actual experimental biosensing conditions would therefore be highly important for advancing QDs to a next generation nanobiosensing tool. Here, we present a detailed FRET study on a large set of QD–biomolecule conjugates, which allows for a homogeneous solution-phase size, shape, and bioconjugation analysis of peptide and protein self-assembled QDs at subnanomolar concentrations and with subnanometer resolution. Direct incorporation of luminescent Tb-complexes (Tb) in the peptides or proteins leads to Tb-to-QD FRET upon assembly to the different QD surfaces. Luminescence decay times and time-gated intensities, which precisely decode the FRET interactions, provide a wealth of useful information on the underlying composite structure and even biochemical functionality. In contrast to other high-resolution techniques, which require rather sophisticated instrumentation, well-defined experimental conditions, and low sample throughput, our technique uses a commercial time-resolved fluorescence plate reader for very fast and simple data acquisition of many aqueous samples in a standard microtiter plate.

Förster 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 Förster 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.

Hemolysis, the rupturing of red blood cells, can result from numerous medical conditions (in vivo) or occur after collecting blood specimen or extracting plasma and serum out of whole blood (in vitro). In clinical laboratory practice, hemolysis can be a serious problem due to its potential to bias detection of various analytes or biomarkers. Here we present the first ''mix-and-measure'' method to assess the degree of hemolysis in biosamples using luminescence spectroscopy. Luminescent terbium complexes (LTC) were studied in the presence of free hemoglobin (Hb) as indicators for hemolysis in TRIS-buffer, and in fresh human plasma with absorption, excitation and emission measurements. Our findings indicate dynamic as well as resonance energy transfer (FRET) between the LTC and the porphyrin ligand of hemoglobin. This transfer leads to a decrease in luminescence intensity and decay time even at nanomolar hemoglobin concentrations either in buffer or plasma. Luminescent terbium complexes are very sensitive to free hemoglobin in buffer and blood plasma. Due to the instant change in luminescence properties of the LTC in presence of Hb it is possible to access the concentration of hemoglobin via spectroscopic methods without incubation time or further treatment of the sample thus enabling a rapid and sensitive detection of hemolysis in clinical diagnostics.

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Contactless pressure monitoring based on Förster resonance energy transfer between donor–acceptors pairs immobilized within a thermoplastic elastomer is demonstrated for novel stretchable opto-electronics and opto-mechanical sensors.

Anwendungen auf der Grundlage des Förster-Resonanzenergietransfers (FRET) spielen eine wichtige Rolle bei der Bestimmung von Konzentrationen und Abständen in nanoskaligen Systemen in der Biologie, Biochemie und Medizin. [1][2][3] Wegen der r À6 -Abstandsabhängigkeit von FRET können Strukturveränderungen molekularer Systeme im Bereich 1-10 nm mit sehr hoher Genauigkeit weit unterhalb der Beugungsbegrenzung des Lichts gemessen werden. Stryer et al. [4,5] beschrieben das "spektroskopische Lineal" bereits vor über 40 Jahren, und es wird nach wie vor häufig für Inund Ex-vivo-Studien inter-und intramolekularer Wechselwirkungen mittels Spektroskopie und Mikroskopie teilweise bis auf Einzelmolekülniveau verwendet. [6][7][8][9] Zahlreiche FRET-basierte Biosensoren für intrazelluläre Untersuchungen wurden bis heute entwickelt. [10][11][12][13][14] Obwohl die meisten dieser Anwendungen einzelne Sensoren verwenden, gibt es einige neuere Entwicklungen dualer FRET-Paare für ZellImaging mit fluoreszierenden Proteinen [15][16][17] und hier sogar mit nur einer Anregungswellenlänge.[18] Die Verwendung der Multiplexing-FRET-Technik erlaubt eine simultane Messung mehrerer Abstände oder Konformationsänderungen, womit Zeit und Aufwand verringert und die bioanalytische Information durch eine mögliche Korrelation der simultanen Vorgänge erhöht werden kann.Die FRET-Kombination von lumineszierenden TerbiumKomplexen (luminescent terbium complexes, LTCs) als Donoren und Halbleiterquantenpunkten (quantum dots, QDs) als Akzeptoren bietet signifikante Vorteile bezüglich Empfindlichkeit, Abstand und multiparametrischer Analyse ("Multiplexing") im Vergleich zu anderen Donor-AkzeptorPaaren. [19,20] Wegen der großen Überlappungsintegralwerte können außergewöhnlich große Förster-Radien (R 0 , der Donor-Akzeptor-Abstand, bei dem die FRET-Effizienz 50 % beträgt) bis zu 11 nm erreicht werden, [21][22][23] wogegen konventionelle Donor-Akzeptor-Paare selten R 0 -Werte von mehr als 6 nm erreichen.[24] Es wurden nanoplasmonische Lineale entwickelt, bei denen Abstände von bis zu ca. 70 nm gemessen werden können, [25,26] allerdings verwenden diese Anwendungen relativ große Edelmetall-Nanopartikel (bis zu 40 nm) und sind beim Multiplexing zur simultanen Messung mehrerer variabler Abstände (z. B. bei vielen intrazellulären Vorgängen innerhalb einer Messung) eingeschränkt. Die Pionierarbeit von Weiss et al. demonstrierte Quantenpunktbasierte optische Multiplexlineale mittels ultrahochauflösen-der Kolokalisation (ultrahigh-resolution colocalization, UHRC).[27] Obwohl FRET Vorteile bezüglich Auflösungsge-nauigkeit und für dynamische Messungen hat, [28] ist UHRC gut für Abstandsmessung im Bereich einiger nm bis zu einigen 10 mm geeignet. [29] Zwei sehr wichtige Aspekte für intrazelluläre Studien mit QDs sind die Form und die Größe dieser Nanosensoren, die beide entscheidend sein können z. B. für die Zellaufnahme und den Einfluss der QDs auf die zu untersuchenden Biomoleküle. Kern und Schale der QDs können durch Transmissionselektronenmikroskopie (TEM) relativ gen...