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.

The importance of microRNA (miRNA) dysregulation for the development and progression of diseases and the discovery of stable miRNAs in peripheral blood have made these short-sequence nucleic acids next-generation biomarkers. Here we present a fully homogeneous multiplexed miRNA FRET assay that combines careful biophotonic design with various RNA hybridization and ligation steps. The single-step, single-temperature, and amplification-free assay provides a unique combination of performance parameters compared to state-of-the-art miRNA detection technologies. Precise multiplexed quantification of miRNA-20a, -20b, and -21 at concentrations between 0.05 and 0.5 nM in a single 150 μL sample and detection limits between 0.2 and 0.9 nM in 7.5 μL serum samples demonstrate the feasibility of both high-throughput and point-of-care clinical diagnostics.

Luminescent lanthanide labels (LLLs) and semiconductor quantum dots (QDs) are two very special classes of (at least partially) inorganic fluorophores, which provide unique properties for Förster resonance energy transfer (FRET). FRET is an energy-transfer process between an excited donor fluorophore and a ground-state acceptor fluorophore in close proximity (approximately 1-20 nm), and therefore it is extremely well suited for biosensing applications in optical spectroscopy and microscopy. Within this cogent review, we will outline the main photophysical advantages of LLLs and QDs and their special properties for FRET. We will then focus on some recent applications from the FRET biosensing literature using LLLs as donors and QDs as donors and acceptors in combination with several other fluorophores. Recent examples of combining LLLs and QDs for spectral and temporal multiplexing from single-step to multistep FRET demonstrate the versatile and powerful biosensing capabilities of this unique FRET pair. As this review is published in the Forum on Imaging and Sensing, we will also present some new results of our groups concerning LLL-based time-gated cellular imaging with optically trifunctional antibodies and LLL-to-QD FRET-based homogeneous sandwich immunoassays for the detection of carcinoembryonic antigen.

A crucial variable for methodical performance evaluation and comparison of luminescent reporters is the photoluminescence quantum yield (Φ pl). This quantity, defined as the number of emitted photons per number of absorbed photons, is the direct measure of the efficiency of the conversion of absorbed photons into emitted light for small organic dyes, fluorescent proteins, metal-ligand complexes, metal clusters, polymeric nanoparticles, and semiconductor and up-conversion nanocrystals. Φ pl determines the sensitivity for the detection of a specific analyte from the chromophore perspective, together with its molar-absorption coefficient at the excitation wavelength. In this review we discuss different optical and photothermal methods for measuring Φ pl of transparent and scattering systems for the most common classes of luminescent reporters, and critically evaluate their potential and limitations. In addition, reporter-specific effects and sources of uncertainty are addressed. The ultimate objective is to provide users of fluorescence techniques with validated tools for the determination of Φ pl, including a series of Φ pl standards for the ultraviolet, visible, and near-infrared regions, and to enable better judgment of the reliability of literature data.

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, ...

The excitation wavelength (λ) dependence of the photoluminescence (PL) quantum yield (Φ) and decay behavior (τ) of a series of CdSe/CdS quantum dot/quantum rods (QDQRs), consisting of the same spherical CdSe core and rod-shaped CdS shells, with aspect ratios ranging from 2 to 20 was characterized. λ between 400-565 nm were chosen to cover the first excitonic absorption band of the CdSe core material, the onset of absorption of the CdS shell, and the region of predominant shell absorption. A strong λ dependence of relative and absolutely measured Φ and τ was found particularly for the longer QDQRs with higher aspect ratios. This is attributed to combined contributions from a length-dependent shell-to-core exciton localization efficiency, an increasing number of defect states within the shell for the longest QDQRs, and probably also the presence of absorbing, yet non-emitting shell material. Although the Φ values of the QDQRs decrease at shorter wavelength, the extremely high extinction coefficients introduced by the shell outweigh this effect, leading to significantly higher brightness values at wavelengths below the absorption onset of the CdS shell compared with direct excitation of the CdSe cores. Moreover, our results present also an interesting example for the comparability of absolutely measured Φ using an integrating sphere setup and Φ values measured relative to common Φ standards, and underline the need for a correction for particle scattering for QDQRs with high aspect ratios.

Many applications of nanometer- and micrometer-sized particles include their surface functionalization with linkers, sensor molecules, and analyte recognition moieties like (bio)ligands. This requires knowledge of the chemical nature and number of surface groups accessible for subsequent coupling reactions. Particularly attractive for the quantification of these groups are spectrophotometric and fluorometric assays, which can be read out with simple instrumentation. In this respect, we present here a novel family of cleavable spectrophotometric and multimodal reporters for conjugatable amino and carboxyl surface groups on nano- and microparticles. This allows determination of particle-bound labels, unbound reporters in the supernatant, and reporters cleaved off from the particle surface, as well as the remaining thiol groups on particle, by spectrophotometry and inductively coupled optical emission spectrometry (S ICP-OES). Comparison of the performance of these cleavable reporters with conductometry and conventional labels, utilizing changes in intensity or color of absorption or emission, underlines the analytical potential of this versatile concept which elegantly circumvents signal distortions by scattering and encoding dyes and enables straightforward validation by method comparison.