TY - JOUR A1 - Quevedo, Pablo A1 - Behnke, Thomas A1 - Resch-Genger, Ute T1 - Streptavidin conjugation and quantification—a method evaluation for nanoparticles JF - Analytical and Bioanalytical Chemistry N2 - Aiming at the development of validated protocols for protein conjugation of nanomaterials and the determination of protein labeling densities, we systematically assessed the conjugation of the model protein streptavidin (SAv) to 100-, 500-, and 1000-nm-sized polystyrene and silica nanoparticles and dye-encoded polymer particles with two established conjugation chemistries, based upon achievable coupling efficiencies and labeling densities. Bioconjugation reactions compared included EDC/sulfo NHS ester chemistry for direct binding of the SAv to carboxyl groups at the particle surface and maleimide-thiol chemistry in conjunction with heterobifunctional PEG linkers and aminated nanoparticles (NPs). Quantification of the total and functional amounts of SAv on these nanomaterials and unreacted SAv in solution was performed with the BCA assay and the biotin–FITC (BF) titration, relying on different signal generation principles, which are thus prone to different interferences. Our results revealed a clear influence of the conjugation chemistry on the amount of NP crosslinking, yet under optimized reaction conditions, EDC/sulfo NHS ester chemistry and the attachment via heterobifunctional PEG linkers led to comparably efficient SAv coupling and good labeling densities. Particle size can obviously affect protein labeling densities and particularly protein functionality, especially for larger particles. For unstained nanoparticles, direct bioconjugation seems to be the most efficient strategy, whereas for dyeencoded nanoparticles, PEG linkers are to be favored for the prevention of dye–protein interactions which can affect protein functionality specifically in the case of direct SAv binding. Moreover, an influence of particle size on achievable protein labeling densities and protein functionality could be demonstrated. KW - Protein quantification KW - BCA assay KW - Biotin–FITC titration KW - Polystyrene nanoparticles PY - 2016 DO - https://doi.org/10.1007/s00216-016-9510-3 SN - 1618-2642 SN - 1618-2650 VL - 408 IS - 15 SP - 4133 EP - 4149 PB - Springer AN - OPUS4-36618 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -