Colorful bioassays: Time‐ and color‐resolved detection of Förster 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.

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.

In the present study, the photophysical properties of the perylene dye PBI [N,N@-bis(1-hexylheptyl)-3,4 : 9,10perylenebis(dicarboximide)] and the quenching of PBI Ñuorescence by organic quencher molecules in acetonitrile was investigated with stationary and time-resolved Ñuorescence and absorption measurements. On the basis of a simpliÐed reaction scheme, a comprehensive analysis of the Ñuorescence quenching process was achieved for 8 PBIÈquencher molecular pairs for which the formation of free PBI ions was observed. It is notable that both the anionic and cationic species (2PBI~, 2PBI`) were detected as primary products of the intermolecular ElT processes. Together with other data from our group and by Mataga et al. (Chem. Phys., 1988, 127, 249) an evaluation of the results within the framework of the Marcus theory of nonÈadiabatic electron transfer was performed. It was found that with an overall variation of the standard free energy changes for the charge recombination (CR) reaction to the PBI ground and triplet states of more (*G CR G ) ( *G CR T ) than 2 eV the CR reaction rate constants cover the whole energetic range of the non-adiabatic electron transfer, i.e. from the normal to the Marcus inverted regime.

A time-resolved fluoro-immunoassay (TR-FIA) format is presented based on resonance energy transfer from visible emitting lanthanide complexes of europium and terbium, as energy donors, to semiconductor CdSe/ZnS core/shell nanocrystals (quantum dots, QD), as energy acceptors. The spatial proximity of the donor-acceptor pairs is obtained through the biological recognition process of biotin, coated at the surface of the dots (Biot-QD), and streptavidin labeled with the lanthanide markers (Ln-strep). The energy transfer phenomenon is evident from simultaneous lanthanide emission quenching and QD emission sensitization with a 1000-fold increase of the QD luminescence decay time reaching the hundred mus regime. Delayed emission detection allows for quantification of the recognition process and demonstrated a nearly quantitative association of the biotins to streptavidin with sensitivity limits reaching 1.2 pM of QD. Spectral characterization permits calculation of the energy transfer parameters. Extremely large Förster radii (R(0)) values were obtained for Tb (104 A) and Eu (96 A) as a result of the relevant spectral overlap of donor emission and acceptor absorption. Special attention was paid to interactions with the varying constituents of the buffer for sensitivity and transfer efficiency optimization. The energy transfer phenomenon was also monitored by time-resolved luminescence microscopy experiments. At elevated concentration (>10(-)(5) M), Tb-strep precipitated in the form of pellets with long-lived green luminescence, whereas addition of Biot-QD led to red emitting pellets, with long excited-state decay times. The Ln-QD donor-acceptor hybrids appear as highly sensitive analytical tools both for TR-FIA and time-resolved luminescence microscopy experiments.

Dedicated to Professor Herbert Dreeskamp on the occasion of his 76th birthdayThe last decade has witnessed the emergence of semiconductor nanoparticles as very attractive building blocks for nanotechnology. Spherically or ellipsoidally shaped nanoparticles, also called quantum dots (qdots), display sizedependent optical properties with extremely large absorption cross sections.[1] When appropriately protected from the surrounding media by a covering shell, they display narrow and symmetric emission bands with quantum yields approaching unity in some cases.[2] The inorganic cores of qdots are also more robust than organic dyes or luminescent proteins toward photobleaching. [3,4] Recent accounts have reported on the possibility of derivatizing qdot surfaces, [5] thereby offering a broad scope of opportunities for chemical and biological interactions.[6] These combined features make qdots excellent tools in analytical techniques associated with fluorescence spectroscopy, reaching sensitivity limits going down to the single particle.[4] Qdots display extremely high extinction coefficients over a wide range of wavelengths as a result of the absorption of photons with energies higher than the band gap of the semiconductor material. The resulting excitons (electron-hole pairs) recombine to generate photons with narrow emission bands. These properties make qdots excellent energy donors in fluorescence (or Förster) resonance energy transfer (FRET) experiments, [7] a technique that is ideally suited for events occurring at the nanometer scale, such as numerous biological processes. For instance, a homogeneous maltose sensor has been described in which the maltose concentration can be monitored by the recovery of qdot luminescence concomitant to the displacement of a quencher dye. [8] Importantly, if qdots are to be used as energy acceptors, significant direct excitation may be undesirable as it limits the extent of resonance transfer and results in spurious fluorescence emission. The use of qdots as energy acceptors has proven possible in a few solid-state devices based on semiconductor quantum wells, [9] with smaller-sized qdots, [10] and with a blue-emitting polymer [11] as the energy donor, but very rarely in discrete molecular systems in solution. [12,13] Mattoussi and co-workers explained low energy-transfer efficiency from organic or inorganic donors to the qdot acceptors by the fact that the rate of FRET is much slower than the fast radiative decay rates of the donors. [12] In the present work, we report that CdSe/ZnS core shell qdots are excellent energy acceptors in a time-resolved fluoroimmunoassay system in which a terbium chelate with long-lived luminescence lifetime is the energy donor and the strong biotin-streptavidin interaction is used for the recognition process.The TbL chelate consisted of a terbium cation embedded in a ligand made of two 6-carboxybipyridyl arms organized on a glutamate framework (Figure 1). The terminal carboxylate function of the glutamic residue was activated with the sodium salt of ...

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, ...

A long-standing and profound problem in astronomy is the diffi culty in obtaining deep nearinfrared observations due to the extreme brightness and variability of the night sky at these wavelengths. A solution to this problem is crucial if we are to obtain the deepest possible observations of the early Universe, as redshifted starlight from distant galaxies appears at these wavelengths. The atmospheric emission between 1,000 and 1,800 nm arises almost entirely from a forest of extremely bright, very narrow hydroxyl emission lines that varies on timescales of minutes. The astronomical community has long envisaged the prospect of selectively removing these lines, while retaining high throughput between them. Here we demonstrate such a fi lter for the fi rst time, presenting results from the fi rst on-sky tests. Its use on current 8 m telescopes and future 30 m telescopes will open up many new research avenues in the years to come.

A technique has been developed to measure absolute intracellular oxygen concentrations in green plants. Oxygen-sensitive phosphorescent microbeads were injected into the cells and an optical multifrequency phase-modulation technique was used to discriminate the sensor signal from the strong autofluorescence of the plant tissue. The method was established using photosynthesis-competent cells of the giant algae Chara corallina L., and was validated by application to various cell types of other plant species.