%0 Journal Article %1 Buschmann2003 %A Buschmann, V %A Weston, KD %A Sauer, M %C 1155 16TH ST, NW, WASHINGTON, DC 20036 USA %D 2003 %I AMER CHEMICAL SOC %J BIOCONJUGATE CHEMISTRY %K sauer %N 1 %P 195-204 %T Spectroscopic study and evaluation of red-absorbing fluorescent dyes %U http://dx.doi.org/10.1021/bc025600x %V 14 %X The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATT0655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATT0655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.

@article{Buschmann2003, abstract = {The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATT0655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATT0655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.}, added-at = {2011-03-01T10:57:13.000+0100}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, affiliation = {Sauer, M (Reprint Author), Univ Heidelberg, Inst Chem Phys, Neuenheimer Feld 253, D-69120 Heidelberg, Germany. Univ Heidelberg, Inst Chem Phys, D-69120 Heidelberg, Germany. Florida State Univ, Dept Chem \& Biochem, Tallahassee, FL 32306 USA.}, author = {Buschmann, V and Weston, KD and Sauer, M}, biburl = {https://www.bibsonomy.org/bibtex/24d768c7c2e4ad70b54bb9fa30a0de624/reichert}, cited-references = {AFFLECK RL, 1996, ANAL CHEM, V68, P2270. ARDENJACOB J, 1997, J FLUORESC S, V7, S91. AUBIN JE, 1979, J HISTOCHEM CYTOCHEM, V27, P35. BAYLISS NS, 1950, J CHEM PHYS, V18, P292. CHENG PC, 1995, HDB BIOL CONFOCAL MI, P327. CHEVALIER J, 1997, J HISTOCHEM CYTOCHEM, V45, P481. CZERNEY P, 2001, BIOL CHEM, V382, P495. DAEHNE S, 1998, EAR INFRARED DYES HI. DREXHAGE DK, 1973, DYE LASERS, P144. EDMAN L, 1996, P NATL ACAD SCI USA, V93, P6710. EGGELING C, 1998, P NATL ACAD SCI USA, V95, P1556. EMANS N, 1995, BIOPHYS J, V69, P716. FLANAGAN JH, 1997, BIOCONJUGATE CHEM, V8, P751. FOSTER T, 1949, Z NATURFORSCH A, V4, P321. GORDON GW, 1998, BIOPHYS J, V74, P2702. GRUBER HJ, 2000, BIOCONJUGATE CHEM, V11, P696. HAUGLAND RP, 1996, HDB FLUORESCENT PROB. 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WILLIAMS DC, 1995, ANAL CHEM, V67, P3427.}, doc-delivery-number = {637YY}, groups = {public}, interhash = {0efbb050be7a712a7bf38fe1a000a7c2}, intrahash = {4d768c7c2e4ad70b54bb9fa30a0de624}, issn = {1043-1802}, journal = {BIOCONJUGATE CHEMISTRY}, journal-iso = {Bioconjugate Chem.}, keywords = {sauer}, keywords-plus = {CAPILLARY GEL-ELECTROPHORESIS; PULSED SEMICONDUCTOR-LASERS; RESONANCE ENERGY-TRANSFER; SINGLE-MOLECULE LEVEL; LABELING REAGENTS; SUCCINIMIDYL ESTERS; ELECTRON-TRANSFER; BIOTIN BINDING; DNA; DYNAMICS}, language = {English}, month = {JAN-FEB}, number = 1, number-of-cited-references = {52}, pages = {195-204}, publisher = {AMER CHEMICAL SOC}, subject-category = {Biochemical Research Methods; Biochemistry \& Molecular Biology; Chemistry, Multidisciplinary; Chemistry, Organic}, times-cited = {86}, timestamp = {2011-03-09T14:16:47.000+0100}, title = {Spectroscopic study and evaluation of red-absorbing fluorescent dyes}, type = {Article}, unique-id = {ISI:000180542400023}, url = {http://dx.doi.org/10.1021/bc025600x}, username = {reichert}, volume = 14, year = 2003 }