title: Suitable labels for molecular imaging--influence of dye structure and hydrophilicity on the spectroscopic properties of IgG conjugates.
abstract: Aiming at the design of highly brilliant NIR emissive optical probes, e.g., for in vivo near-infrared fluorescence imaging (NIRF), we studied the absorption and fluorescence properties of the asymmetric cyanines Dy678, Dy681, Dy682, and Dy676 conjugated to the model antibody IgG. The ultimate goal was here to derive general structure-property relationships for suitable NIR fluorescent labels. These Dy dyes that spectrally match Cy5 and Cy5.5, respectively, were chosen to differ in chromophore structure, i.e., in the substitution pattern of the benzopyrylium end group and in the number of sulfonic acid groups. Spectroscopic studies of the free and IgG-bound fluorophores revealed a dependence of the obtained dye-to-protein ratios on dye hydrophilicity and control of the fluorescence quantum yields (Φ(f)) of the IgG conjugates by the interplay of different fluorescence reduction pathways like dye aggregation and fluorescence resonance energy transfer (FRET). Based upon aggregation studies with these dyes, the amount of dye dimers in the IgG conjugates was determined pointing to dye hydrophilicity as major parameter controlling aggregation. To gain further insight into the exact mechanism of dye dimerization at the protein, labeling experiments at different reaction conditions but constant dye-to-protein ratios in the reaction solution were performed. With Dy682 that displays a Φ(f) of 0.20 in PBS and 0.10 for moderate dye-to-protein ratio of 2.5, a low aggregation tendency, and a superior reactivity in IgG labeling, we identified a promising diagnostic tool for the design of NIR fluorescent probes and protein conjugates.
authors.affiliation: BAM Bundesanstalt für Materialforschung und -prüfung, OE I.5, Richard-Willstaetter-Str. 11, D-12489 Berlin, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Pauli
authors.forename: Markus
authors.initials: M
authors.lastname: Grabolle
authors.forename: Robert
authors.initials: R
authors.lastname: Brehm
authors.forename: Monika
authors.initials: M
authors.lastname: Spieles
authors.forename: Franziska M
authors.initials: FM
authors.lastname: Hamann
authors.forename: Matthias
authors.initials: M
authors.lastname: Wenzel
authors.forename: Ingrid
authors.initials: I
authors.lastname: Hilger
authors.forename: Ute
authors.initials: U
authors.lastname: Resch-Genger
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000737:chemistry*; D002417:Cattle; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D018796:Immunoconjugates / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D057054:Molecular Imaging / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D018796:Immunoconjugates; D007074:Immunoglobulin G; D012710:Serum Albumin, Bovine