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.