Accurate and reliable quantification of the protein surface coverage on protein-functionalized nanoparticles.

The ability to accurately quantify the protein coverage on nanoparticles is critical for assessing the quality of the surface chemistry and the success of the functionalization process of protein-nanoparticle conjugates. Surface coverage determination is therefore an integral part in the quality control of protein-modified nanoparticles in industrial nanotechnology. In this work, a novel and conventional method was established for direct quantification of the protein surface coverage on metallic nanoparticles. Different concentrations of pepsin were conjugated to gold nanoparticles (GNPs) by a straightforward adsorptive immobilization process as a model system, and a protein quantitation methodology based on the amino acid analysis of the hydrolysate of the protein-GNP conjugates was established. For this purpose, pepsin functionalized GNPs (pepsin-GNP bioconjugates) were processed via in situ hydrolysis with 6N HCl and subsequent derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC reagent). Direct quantitative amino acid analysis was performed based on measuring the intensity of AQC-glycine derivative by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The method allows for detection of surface coverages as low as 0.1 μg mL-1 pepsin (corresponding to 2.89 × 10-9  mol L-1 ) in the colloidal solution. Method imprecision for replicated surface coverage determinations was <5% RSD and accuracies, as determined by % recoveries, were always in the 98-118% range. This method allows precise and accurate quantification of protein coverages, even when less than 1% of the protein in the reaction mixture is immobilized. It was found that the degree of surface coverage of adsorptively bound pepsin on GNPs correlated with the pepsin concentrations in the conjugation reaction mixtures. Washing with phosphate buffer removed weakly bound proteins, i.e. the soft protein corona. The adsorption behavior could be described by a Freundlich isotherm model. This direct and reliable method promises great potential for the accurate quantification of protein coverages of various protein-nanoparticle bioconjugates.