## 4 Analysis of Mouse Serum and Interaction with Nanoparticles  
This section presents an application example of 3D-ccDLS and CORENN method to study the interaction of nanoparticles with a more complex biofluid. Recently we have shown that CONTIN method can be used to distinguish translational and rotational motion of the components of mouse-serum and to investigate changes of their hydrodynamic radii upon interaction with gold nanoparticles of different shape [10] at a mouse serum-concentration of 33%. As a further application example for particle monitoring with 3D-ccDLS and CORENN method, we present here recent results from a study of the interaction of gold nanospheres and a low concentration of mouse serum. The chosen mouse serum is the same product of sterile, pre-filtered mouse serum (BioWest, Nuaille, France) as used in [10]. We filtered the purchased serum through a 1.2 um micropore membrane in order to remove large agglomerates and thus to improve the signal stability for DLS measurements [10]. Figure 2 shows the result of the additional filtering. Figure 2: DLS data of mouse serum before and after filtering through a 1.2 μm micropore membrane: (**a**) Correlation functions; (**b**) Example of relative scattered intensity as a function of hydrodynamic radius based on CONTIN analysis. Upon filtering the correlation function decays faster, and shows less noise at large lag times, which is consistent with the removal of larger particles. The analyzed PDI decreased from 2.72 to 1.61. Figure 2b furthermore illustrates an example that the presence of large agglomerates before filtering might affect calculated results of hydrodynamic radii also for smaller particles. Here, an example is shown based on CONTIN analysis. The observation of a shift towards smaller apparent hydrodynamic radii may indicate a change in particle-particle interactions or a possible change of the effective viscosity upon removal of bigger agglomerates, however, it also illustrates the high sensitivity of the CONTIN algorithm to noise at large lag time, when the autocorrelation function approaches zero. Using the CORENN method, we have performed further analysis of our 3D-ccDLS data recorded at various volume concentration between 9% and 100% (with respect to the filtered mouse serum), using DNAse-free distilled water for dilution. Figure 3 shows the Cumulant and CORENN analysis, as well as optical absorbance of the filtered mouse serum at various concentration. The optical UV-vis-NIR absorbance spectra of mouse serum (Figure 3c), show absorbance in the ultraviolet range, which is characteristic for proteins. In agreement with the Beer-Lambert law, the absorption peak intensity at 410 nm, which can be indicative for enzymatic reactions or other components of the mouse serum, is proportional to the serum concentration (Figure 3c) [10]. As main components we consider proteins in the mouse serum. Mouse serum was recently studied by CONTIN analysis [10]. Rotational and translational motion were distinguished as well as the presence of agglomerates [10]. Figure 3b shows results from CORENN analysis of mouse-serum DLS data, samples were prepared and data recorded as described in [10]. The analysis using the CORENN method shows consistency with the CONTIN analysis presented in [10]. It clearly distinguishes the rotational and translation motion of proteins in the mouse-serum, as well as the formation of agglomerates. The analyzed mode of the rotational peak, with a hydrodynamic radius between 2 and 5 nm, stays stable over the full range of concentration (Figure 2b), and can be related to the particles lengths [23]. The mode with a hydrodynamic radius around 14 nm (Figure 3b) is related to translation; its obtained positions show similar values over the entire range of concentrations within the error (Figure 3b).