Analytische Chemie
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The design of bright and functional dye–protein conjugates requires hydrophilic and stable fluorophores with high molar absorption coefficients and high fluorescence quantum yields,which must not be prone to dimerization, as well as conservation of protein function and suppression of protein association. Although many synthetic dyes meet these needs, the influence of dye charge on bioconjugate performance is commonly neglected. This encouraged us to assess the spectroscopic properties, antibody functionality, binding behavior, folding, and association of conjugates of the therapeutic antibodies trastuzumab and cetuximab with the red cyanine dyes S0586, S2381,and 6SIDCC (bearing two, three, and six sulfonate Groups respectively.
Our results demonstrate a negligible effect of dye labeling on antibody folding, yet a strong influence of Label charge and density on antibody isoelectric points and association.Especially 6SIDCC decreased strongly the isoelectric points of both antibodies and their heavy or light chains even at low labeling degrees, thus favoring protein association. Although an increasingly negative dye charge reduces Antigen affinity as shown in a competitive immunoassay, all conjugates still bound to cells overexpressing the target of the respective antibody. Obviously, dyes that cause minimum dimerization with a small number of charged groups are best for conjugate brightness, minimum protein association, and strong target binding. This underlines the need to consider dye Charge for the rational design of conjugates with Optimum performance.
Dye-stained micrometer-sized polymer beads are important tools in the life sciences with applications in biomedical, biochemical, and clinical research. Here, bead-based assays are increasingly used, for example, in DNA sequencing and the detection of autoimmune diseases or pathogenic microorganisms. Moreover, stained beads are employed as calibration tools for fluorescence microscopy and flow cytometry methods with increasing complexity. To address the requirements concerning the relevant fluorescence features, the spectroscopic properties of representative polymer beads with diameters ranging from about 1 to 10 μm stained with varying concentrations of rhodamine 6G were systematically assessed. The observed dependence of the spectral properties, fluorescence decay kinetics, and fluorescence quantum yields on bead size and dye loading concentration is attributed to different fluorescence characteristics of fluorophores located in the particle core and near-surface dye molecules. Supported by the fluorescence anisotropy measurements, the origin of the observed alteration of fluorescence features is ascribed to a combination of excitation energy transfer and polarity-related effects that are especially pronounced at the interface of the bead and the surrounding medium. The results of our studies underline the need to carefully control and optimize all Parameters that can affect the fluorescence properties of the dye-stained beads.