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Aiming at the rational design and the identification of brilliant fluorescent reporters for targeted optical probes and fluorescence assays in biological matrices, we systematically assessed the correlation between dye–protein binding, dye aggregation, and dye hydrophilicity for bioanalytically relevant fluorescent labels. Here, we report on the influence of sulfonic acid groups on dye aggregation and dye–serum protein interactions exemplarily for a family of NIR-emissive cyanine dyes, the DY-67x fluorophores. For highly hydrophobic dyes like DY-675 and DY-676, which show a strong tendency for aggregation in phosphate buffer saline solution, the dye–protein binding constants determined spectroscopically using a 2-state binding model, which considers only protein-bound and unbound dye molecules, can be influenced by the dimerization of the unbound dyes. To consider and quantify this influence, we expanded this common photometric method to a 3-state model that accounts for the presence of dye aggregates in the binding studies. Our results can be exploited for the screening of fluorescent reporters, efficiently providing information on the size of dye–protein interactions and on maximally achievable fluorescence quantum yields in biological systems.
Dye–biomolecule conjugation is frequently accompanied by considerable spectral changes of the dyes absorption spectrum that limit the use of the common photometrical method for the determination of labeling densities. Here, we describe an improvement of this method using the integral absorbance of the dye instead of its absorbance at the long wavelength maximum to determine the concentration of the biomolecule-coupled dye. This approach is illustrated for three different cyanine dyes conjugated to the antibody IgG.
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 (Φf) 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 Φf 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.