title: New fluorescent labels with tunable hydrophilicity for the rational design of bright optical probes for molecular imaging.
abstract: 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.
authors.affiliation: BAM Federal Institute for Materials Research and Testing, Division 1.10 Biophotonics, Richard-Willstaetter-Strasse 11, D-12489 Berlin, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Pauli
authors.forename: Kai
authors.initials: K
authors.lastname: Licha
authors.forename: Janis
authors.initials: J
authors.lastname: Berkemeyer
authors.forename: Markus
authors.initials: M
authors.lastname: Grabolle
authors.forename: Monika
authors.initials: M
authors.lastname: Spieles
authors.forename: Nicole
authors.initials: N
authors.lastname: Wegner
authors.forename: Pia
authors.initials: P
authors.lastname: Welker
authors.forename: Ute
authors.initials: U
authors.lastname: Resch-Genger
mesh_terms: D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D057054:Molecular Imaging; D015335:Molecular Probes; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D015335:Molecular Probes