Dynamic Light Scattering. Dynamic Light Scattering (DLS) is used to determine the size of a spherical molecule by measuring backscatter after a red laser interacts with the sample. To prepare for DLS, the conjugates were prepared using the protocol described in 3.2.2 Conjugation, but were diluted by a factor of 10. Since the Particle Systems Nanoplus zeta/nano particle analyzer is unable to detect 5 nm conjugates due to their small size, it was not measured; however, information can be inferred from 15 nm and 30 nm conjugates. The samples were loaded in to a DLS microcuvette for this experiment.
Dynamic Light Scattering. From the DLS, it was determined that there is a monolayer of enzymes forming about the AuNPs. The purpose of running DLS is to determine the size of the conjugates in comparison to the free AuNP. By looking at the size and diffusion rates obtained from the DLS, it would be possible to see if a monolayer or multilayer of protein is bound to the AuNPs. Knowing the hydrodynamic diameter would allow us to deduce how many layers of protein are present. This analysis was completed with the 15 nm and 30 nm conjugates. The 5 nm conjugates were too small to be detected, and thus are not included in the analysis because DLS only a lower bound limit of approximately 10 nm. The DLS shows that the conjugates are approximately 32 nm in diameter for 15 nm AuNP conjugates and 44 nm for 30 nm AuNP conjugates. The free AuNPs had hydrodynamic diameters of 18 and 34 nm for the free 15 nm and 30 nm free AuNPs. These hydrodynamic diameters are slightly larger than the actually diameters because of water that binds to the spherical object causing the nanosphere to appear approximately 2-3 nanometers longer than it should. Since DHFR is 4 nm by 2 nm,17 an addition of 8 nm to the diameter of the free AuNP is indicative of a monolayer forming. Since the hydrodynamic diameters are only a few nanometers longer, instead of 16-nm double, it can be concluded that a monolayer of protein, instead of a multilayer, has formed around the AuNPs. Since DLS measures the diffusion of rate of molecules, faster diffusion is observed when the molecules are smaller, hence in both 15 nm and 30 nm AuNP data sets, the free AuNPs have faster diffusion times when compared to that of the conjugates. In the autocorrelation function (ACF) vs time graph, the free AuNP have a faster diffusion rate that that of the respective conjugates (Figure 4.5a,c) . The slower diffusion rate of conjugates is indicative that protein has adequately bound to the AuNPs. Furthermore, intensity distributions show that the conjugates have an increase in diameter when compared to the respective free AuNP (Figure 4.5 b,d).
