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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.

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.

Semiconductor nanocrystals (quantum dots - QDs) possess unique photophysical properties that make them highly interesting for many biochemical applications. Besides their common use as fluorophores in conventional spectroscopy and microscopy, QDs are well-suited for studying Forster resonance energy transfer (FRET). Size-dependent broadband absorption and narrow emission bands offer several advantages for the use of QDs both as FRET donors and acceptors. QD-based FRET pairs can be efficiently used as biological and chemical sensors for highly sensitive multiplexed detection. In this contribution we present the use of several commercially available QDs (Qdot® Nanocrystals - Invitrogen) as FRET donors in combination with commercial organic dyes as FRET acceptors. In order to investigate the FRET process within our donor-acceptor pairs, we used biotinylated QDs and streptavidin-labeled dyes. The well-known biotinstreptavidin molecular recognition enables effective FRET from QDs to dye molecules and provides defined distances between donor and acceptor. Steady-state and time-resolved fluorescence measurements were performed in order to investigate QD-to-dye FRET. Despite a thick polymer shell around the QDs, our results demonstrate the potential of these QDs as efficient donors both for steady-state and time-resolved FRET applications in nano-biotechnology.