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MDH (light spheres) and CS (dark spheres) were adsorbed either sequentially or simultaneously with Au nanoparticles. The amount of each enzyme measured for each bioconjugate is depicted as accurately as possible by estimating the number of layers of each enzyme present from their sizes. The diameter of each enzyme was determined by averaging measurements made in the x, y, and z dimensions of each enzymes crystal structure. This diameter was then used to calculate the area a single enzyme molecule would occupy on the surface area of the 30.7 nm diameter Au nanoparticle. The number of MDH or CS molecules necessary to cover the entire surface area of the nanoparticle was considered "monolayer coverage". To determine the number of MDH or CS necessary to form a complete layer on an existing MDH:Au or CS:Au bioconjugate, 2 times the average diameter of the previously immobilized enzyme was added to the diameter of the nanoparticle, and the surface area was recalculated. Note that, while some degree of enzyme unfolding is likely, we have not attempted to depict this in the scheme.
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