Systematic process evaluation of the conjugation of proteins to gold nanoparticles

The present work addresses some fundamental aspects in the preparation of protein-conjugated gold nanoparticles, in order to ensure an appropriate final product. Ten broadly available and/or easy to implement analytical tools were benchmarked and compared in their capacity to provide reliable and conclusive information for each step of the procedure. These techniques included transmission electron microscopy, UV/VIS spectroscopy, dynamic light scattering, zeta-potential, Fourier-transformed infrared spectroscopy, colloidal stability titration, end-point colloidal stability analysis, cyclic voltammetry, agarose gel electrophoresis and size-exclusion chromatography (SEC). Four different proteins widely used as adaptors or blocking agents were tested, together with 13 nm gold nanoparticles containing different surface chemistries. Among all tested techniques, some of the least popular among nanomaterial scientists probed to be the most informative, including colloidal stability, gel electrophoresis and SEC; the latter being also an efficient purification procedure. These three techniques provide low-cost, low time consuming, sensitive and robust ways to assess the success of the nanoparticle bioconjugation steps, especially when used in adequate combinations.