2.6. Inhibition and/or Dissociation of Protein Aggregation in the Presence of Nanocomposites

The aggregation-inhibiting activity of nanocomposites was investigated by adding 100 μL of SC-NCs and PR-NCs (1, 2, 5, and 8 mg/mL in Tris-HCl, pH-7.0) separately to the HSA + G-fibril during the aggregation process. The respective nanocomposites with different concentrations were abbreviated as SC-NC-100, SC-NC-200, SC-NC-500, SC-NC-800, PR-NC-100, PR-NC-200, PR-NC-500, and PR-NC-800. For dilution of samples, Tris-HCl buffer (20 mM; pH-7.0) was used. For dissociation studies, 100 μL of both the nanocomposites (at the same concentrations) were added to the protein solution after 24 h and the solutions were further incubated for 24 h under similar conditions.

Both the inhibition and dissociation process was monitored by different spectroscopy and microscopy techniques, viz., Thioflavin T (ThT),³⁹ Congo Red,⁴⁰ transmission electron microscopy (TEM),⁴¹ Fluorescence microscopy imaging,⁴² protein intrinsic fluorescence,⁴³ ANS fluorescence assay,⁴⁴ and CD spectroscopy.¹¹ Native polyacrylamide gel electrophoresis (native PAGE) of nanocomposite-treated protein aggregates and the supernatant obtained after the magnetic separation of protein–nanocomposite complexes was also carried out.⁴⁵ All these methods are explained in detail in the Supporting Information (Methods).