TY - JOUR A1 - Linck, Lena A1 - Kapusta, P. A1 - Resch-Genger, Ute T1 - Spectroscopic and photophysical properties of dUTP and internally DNA bound fluorophores for optimized signal detection in biological formats N2 - Efficient signal generation in DNA-based assays requires understanding of the influence of fluorophore’s interactions on the spectroscopic properties. The resulting changes in fluorescence intensity, quantum yield, emission anisotropy, and fluorescence lifetime provide straightforward tools for the study of molecular dynamics and interaction between labels and nucleic acids. Searching for bright fluorescent reporters for rolling circle amplification (RCA) as efficient signal enhancement strategy for biological formats, we investigated the spectroscopic properties of seven dyes: cyanines, rhodamines, and BODIPYs. They spectrally resemble Cy3, the most frequently used fluorophore in biodetection formats, and are measured in six samples (free dye, dye-dUTP, internally labeled ssDNA and dsDNA—single- and triple-labeled) using steady-state and time-resolved fluorometry. Special emphasis was dedicated to characterizing the nature of the interaction of these fluorophores differing in dye class, charge, and rigidity. Our results suggest dye charge and structure as main factors governing the dye’s interactions, with DY-555 and Cy3B presenting the best candidates for our envisaged signal amplification strategy. This label comparison underlines the importance of a proper understanding of structure-property relations and dye–biomolecule interactions for reporter choice and presents a road map towards the design and interpretation of experiments using these labels on DNA of known sequence. KW - DNA KW - Fluorescent dye KW - Fluorescence quantum yield KW - Cyanine KW - Rhodamine KW - Fluorescence lifetime KW - Guanosine quenching PY - 2012 DO - https://doi.org/10.1111/j.1751-1097.2012.01119.x SN - 0031-8655 SN - 1751-1097 VL - 88 IS - 4 SP - 867 EP - 875 PB - American Society for Photobiology CY - Augusta, Ga. AN - OPUS4-27080 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Grabolle, Markus A1 - Kapusta, P. A1 - Nann, T. A1 - Shu, X. A1 - Ziegler, J. A1 - Resch-Genger, Ute T1 - Fluorescence lifetime multiplexing with nanocrystals and organic labels N2 - The potential of semiconducting nanocrystals or so-called quantum dots (QDs) for lifetime multiplexing has not been investigated yet, despite the increasing use of QDs in (bio)analytical detection, biosensing, and fluorescence imaging and the obvious need for simple and cost-effective tools and strategies for the simultaneous detection of multiple analytes or events. This is most likely related to their multiexponential decay behavior as for multiplex chromophores, typically monoexponential decay kinetics are requested. The fluorescence decay kinetics of various mixtures of a long-lived, multiexponentially decaying CdSe QD and a short-lived organic dye were analyzed, and a model was developed for the quantification of these labels from the measured complex decay kinetics as a first proof-of-concept for the huge potential of these labels for lifetime multiplexing. In a second step, we evaluated the potential of mixtures of two types of QDs, varying in constituent material to realize distinguishable, yet multiexponential decay kinetics and similar absorption and emission spectra. Strategies for lifetime multiplexing with nanocrystalline labels were derived on the basis of these measurements. KW - Multiplexing KW - Lifetime KW - Fluorescence KW - Photoluminescence KW - TCSPC KW - Quantum dot KW - Nanocrystal KW - CdSe KW - InP KW - Organic dye KW - DCM PY - 2009 DO - https://doi.org/10.1021/ac900934a SN - 0003-2700 SN - 1520-6882 VL - 81 IS - 18 SP - 7807 EP - 7813 PB - American Chemical Society CY - Washington, DC AN - OPUS4-20279 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -