Morphology analysis using AFM
To analyze the morphology of the samples, AFM imaging was performed as shown in Figure 5. The native BSA molecules were in globular shape with an average size of 30-40 nm (Figure 5(a)). This size may be overestimated due to the finite radius (~10 nm) of AFM tip. For the samples sheared at constant 300 s-1 for 15 min, particles sizes were in the range of 50 to 70 nm (Figure 5(b)) i.e. slightly larger than native BSA. However, at higher temperatures 60 and 65°C, particle sizes were much increased due to the aggregation as observed through enhanced Th-T fluorescence and loss of helicity. The aggregate dimension ranges were 70-100 nm and 100-200 nm for the samples sheared at 60°C and 65°C, respectively (Figures 5(c) and (d)). Here mixtures of globular as well as elongated clumps were observed. These sizes and shapes of aggregates agreed with the reported data (Dubey et al. 2017). The size of aggregates obtained after shearing at 65°C were found to be comparable with thermal treated BSA at 75 °C (Figure 5(e)).