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Chapter 6 – Production of Few Layer Graphene in Aqueous Media for Biological Studies or G band broadening), meaning that the basal plane of graphene contained mostly sp2 carbons facilitating 𝜋 → 𝜋 interactions of Tyr and Trp.415, 416 The as-prepared G-HSA had a pH value of ~6.8 in PBS buffer. The isoelectric point (IEP), the pH value at which a molecule does not have any electrical charge, of HSA is around 4.7.236 Consequently, in the G/HSA adsorbate, the anionic charge density would become dominant due to the carboxylate ions, which would induce electrostatic repulsion between the graphene sheets and, with excess HSA, enabling a stable dispersion (𝜁 value of G/HSA is -27.87 mV).228 The 𝜁 value of G/HSA-wash and its supernatant was -13.4 mV and -22.87 mV, respectively. One possible explanation for the observed reduced stability of G/HSA-wash upon removing excess HSA could be that the relative effect of surface charge screening by Na+ of PBS buffer was slightly higher than the repulsion between the graphene sheets. However, as the protein is net charged and has positively-charged regions on its surface, the electrostatic attractions cannot be fully excluded.236 From the above results, it is clear that direct exfoliation of graphite in HSA resulted in HSA-coated graphene sheets. It has been reported that serum proteins, which are normally present in the media during cell growth has strong adsorption on GO, mitigating cytotoxicity.232 In order to understand the interaction of pristine graphene in the presence of neat HSA, 0.1 v/v% dialysed G/DMF dispersion was added slowly to the neat HSA solution (5 mg/ml filtered in 20 nm filter) and characterised by DLS and fluorescence spectroscopy. The HSA control had a size of ~3.8 nm similar to reported.406, 407 A clear evolution of the size differences could be observed from Figure 6.18a. Gradual addition of graphene reduced the light scattering intensity of HSA and the IPSD spectra became dominated by its hydrodynamic size around 100 nm. However, one cannot neglect the possibility of observing the interaction of HSA and graphene by DLS. Fluorescence spectroscopy confirmed HSA and graphene interaction by means of fluorescence quenching (Figure 6.18b,c). As mentioned earlier, graphene acted as a fluorescence quencher by interacting with the Tyr and Trp residues of HSA.236, 415, 416 Upon gradual addition of graphene to HSA control solutions, the Tyr and Trp fluorescence reduces rapidly (~ 86 % quenching with 0.36 μg of 0.1 v/v% G/DMF addition) confirming its strong interaction. 190PDF Image | PRODUCTION AND APPLICATIONS OF GRAPHENE AND ITS COMPOSITES
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