TY - CONF A1 - Pauli, Jutta A1 - Spieles, Monika A1 - Resch-Genger, Ute A1 - Licha, K. T1 - Analytical and Spctroscopic tools for the Design of Highly Flourescent Conjugates T2 - ANAKON 2013 CY - Essen, Germany DA - 2013-03-04 PY - 2013 AN - OPUS4-28471 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Pauli, Jutta A1 - Grabolle, Markus A1 - Resch-Genger, Ute A1 - Licha, K. A1 - Welker, P. T1 - Sharpening the fluorescence properties of cyanine bioconjugates: a novel series of highly sulfonated pentamethine cyanines T2 - ESMI 2013 CY - Turin, Italy DA - 2013-05-27 PY - 2013 AN - OPUS4-28955 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Pauli, Jutta A1 - Licha, K. A1 - Berkemeyer, Janis Manuel A1 - Grabolle, Markus A1 - Spieles, Monika A1 - Wegner, N. A1 - Welker, P. A1 - Resch-Genger, Ute T1 - New fluorescent labels with tunable hydrophilicity for the rational design of bright optical probes for molecular imaging N2 - The rational design of bright optical probes and dye–biomolecule conjugates in the NIR-region requires fluorescent labels that retain their high fluorescence quantum yields when bound to a recognition unit or upon interaction with a target. Because hydrophilicity-controlled dye aggregation in conjunction with homo-FRET presents one of the major fluorescence deactivation pathways in dye–protein conjugates, fluorescent labels are required that enable higher labeling degrees with minimum dye aggregation. Aiming at a better understanding of the factors governing dye–dye interactions, we systematically studied the signal-relevant spectroscopic properties, hydrophilicity, and aggregation behavior of the novel xS-IDCC series of symmetric pentamethines equipped with two, four, and six sulfonic acid groups and selected conjugates of these dyes with IgG and the antibody cetuximab (ctx) directed against the cancer-related epidermal growth factor (EGF) receptor in comparison to the gold standard Cy5.5. With 6S-IDCC, which displays a molar absorption coefficient of 190 000 M–1 cm–1 and a fluorescence quantum yield (Φf) of 0.18 in aqueous media like PBS and nearly no aggregation, we could identify a fluorophore with a similarly good performance as Cy5.5. Bioconjugation of 6S-IDCC and Cy5.5 yielded highly emissive targeted probes with comparable Φf values of 0.29 for a dye-to-protein (D/P) ratio <1 and a reduced number of protein-bound dye aggregates in the case of 6S-IDCC. Binding studies of the ctx conjugates of both dyes performed by fluorescence microscopy and FACS revealed that the binding strength between the targeted probes and the EGF receptor at the cell membrane is independent of D/P ratio. These results underline the importance of an application-specific tuning of dye hydrophilicity for the design of bright fluorescent reporters and efficient optical probes. Moreover, we could demonstrate the potential of fluorescence spectroscopy to predict the size of fluorescence signals resulting for other fluorescence techniques such as FACS. KW - Cyanine KW - Cetuximab KW - IgG KW - Protein labeling KW - Fluorescence quantum yield KW - Hydrophilicity KW - Dimerization constant PY - 2013 DO - https://doi.org/10.1021/bc4000349 SN - 1043-1802 SN - 1520-4812 VL - 24 IS - 7 SP - 1174 EP - 1185 CY - Washington, DC AN - OPUS4-29076 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Pauli, Jutta A1 - Napp, J. A1 - Streng, Julia A1 - Alves, F. A1 - Resch-Genger, Ute T1 - Brilliant and Functional Herceptin Conjugates with a Remarkably High Number of Bound Fluorophores T2 - MOBI 2013 CY - Heidelberg, Germany DA - 2013-10-28 PY - 2013 AN - OPUS4-29576 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Pauli, Jutta A1 - Spieles, Monika A1 - Licha, K. A1 - Resch-Genger, Ute T1 - Design of Highly Fluorescent Conjugates T2 - MOBI 2013 CY - Heidelberg, Germany DA - 2013-10-28 PY - 2013 AN - OPUS4-29577 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Würth, Christian A1 - Grabolle, Markus A1 - Pauli, Jutta A1 - Spieles, Monika A1 - Resch-Genger, Ute T1 - Relative and absolute determination of fluorescence quantum yields of transparent samples N2 - Luminescence techniques are among the most widely used detection methods in the life and material sciences. At the core of these methods is an ever-increasing variety of fluorescent reporters (i.e., simple dyes, fluorescent labels, probes, sensors and switches) from different fluorophore classes ranging from small organic dyes and metal ion complexes, quantum dots and upconversion nanocrystals to differently sized fluorophore-doped or fluorophore-labeled polymeric particles. A key parameter for fluorophore comparison is the fluorescence quantum yield (Φf), which is the direct measure for the efficiency of the conversion of absorbed light into emitted light. In this protocol, we describe procedures for relative and absolute determinations of Φf values of fluorophores in transparent solution using optical methods, and we address typical sources of uncertainty and fluorophore class-specific challenges. For relative determinations of Φf, the sample is analyzed using a conventional fluorescence spectrometer. For absolute determinations of Φf, a calibrated stand-alone integrating sphere setup is used. To reduce standard-related uncertainties for relative measurements, we introduce a series of eight candidate quantum yield standards for the wavelength region of ~350–950 nm, which we have assessed with commercial and custom-designed instrumentation. With these protocols and standards, uncertainties of 5–10% can be achieved within 2 h. PY - 2013 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-294202 DO - https://doi.org/10.1038/nprot.2013.087 SN - 1754-2189 SN - 1750-2799 VL - 8 IS - 8 SP - 1535 EP - 1550 PB - Nature Publishing Group CY - Basingstoke AN - OPUS4-29420 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -