Synthesis and characterization of enzyme-Au bioconjugates: HRP and fluorescein-labeled HRP

Enzyme adsorption to Au nanoparticles is important for bioanalytical, catalytic, and biotechnological applications. In this manuscript, adsorption of horseradish peroxidase (HRP) and fluorescein labeled HRP (F-HRP) to colloidal Au nanoparticles is compared. Flocculation analysis indicates some differences in adsorption behavior for the labeled and unlabeled enzymes, particularly at pH 8.0, and to a lesser extent also, at pH 4.0. Zeta potential measurements were quite similar for HRP and F-HRP and leveled off at lower enzyme:Au ratios than observed by flocculation, particularly for pH 6.0 and 8.0. Direct quantification of bound F-HRP was performed by dissolution of Au nanoparticles before fluorescence measurements. We observed F-HRP binding at greater than monolayer coverage on the particles and did not reach saturation even at high solution F-HRP:Au ratios. Indirect quantification of bound unlabeled HRP based on supernatant analysis followed similar trends. More than 50% of the specific enzyme activity was retained for conjugates with a low HRP coverage. With increasing enzyme coverage, the specific activity decreased significantly; however, the total activity per particle remained nearly constant. These findings suggest HRP multilayer formation on the Au nanoparticles, with the outermost layer of HRP contributing to the major part of the conjugate enzyme activity.