Chelating Ligands and Nanomaterials Based on Graphene Oxide for the Reduction and Sequestration of Radiometals and Protein Purification

Nuclear waste remediation and protein purification using immobilized metals can benefit enormously from the design and implementation of novel chelating nanomaterials. Available commercial resins capable of sequestering metals are based on bulk materials such as organic polymers, ceramics, and their composites. The use of two-dimensional nanoplatforms with large surface areas expands the horizon for the research and development of new systems with higher efficiency and lower cost. Fundamental science challenges remain in the synthesis and characterization of these nanomaterials and their integration with known or novel coordinating ligands. Chemical exfoliation of graphite under strong oxidizing conditions yields graphene oxide (GO) nanosheets with oxy functional groups on the carbon basal plane and edges. The structure of GO is still not well understood due to its non-stoichiometric nature and strong dependence on synthetic conditions. Herein, GO was investigated to better understand its mechanism of formation, the nature of its surface functional groups, and its suitability as a reduction and sequestration platform for radiometals and protein purification. It was found that ozonolysis of graphite in the widely-used Hummers’ method, hitherto largely overlooked in the literature, leads to the formation of secondary epoxy and peroxymonosulfate ester functional groups, arising from a rotationally hindered Criegee intermediate and sulfuric acid in a strongly favored thermodynamic process. This mechanistic step is followed by the formation of adjacent epoxy and hydroxy groups as major functionalities on GO via a radical process, and leaves a significant amount of unreacted peroxides above levels previously recognized. The thermal decomposition of GO releases sulfur oxygen species (SO2 and SO), but does not appear to release ozone even if this reaction pathway is moderately favored thermodynamically. A new structural model is proposed for GO based on detailed chemical and spectroscopic data that provides insight into observed chemical reactivity and physical properties, including the antimicrobial activity and protein deactivation. Titanium dioxide (TiO2) is a widely studied semi-conductor photoreductant with applications in water splitting and decomposition of organic compounds. It has been previously demonstrated that TiO2 can reduce the radioactive waste metal 99Tc under UV irradiation. Monodispersed TiO2 anatase nanoparticles were synthesized on the GO surface, showing excellent coverage and strong binding to the carbon basal plane, yielding TiO2/GO nanocomposites (TGO). TGO is characterized by an absorption window shifted to the visible region of the spectrum compared to pristine TiO2. Results show that TGO can reduce up to 40% of 99Tc from its oxidized form pertechnetate (TcO4-) under UV irradiation, but photocatalyzes little reduction of 99Tc under visible light conditions. The chemistry of chelates for radiometals has important impacts on radioactive waste cleanup and radiopharmaceutical design. It is well known that within Group 7 congeners Re and Tc are characterized by similar physical-chemical properties. Thus, natural Re (37.4% 185Re and 62.6% 187Re) is commonly used as a non-radioactive analogue of 99Tc. Further, the radioisotopes 186Re and 188Re are important medical isotopes for radiotherapy. In this investigation, the binding stability of natural Re was compared with that of 188Re using two classes of chelates. Novel compounds with two nitrogen and two sulfur binding sites (N2S2) and known tripeptides with three nitrogen and one sulfur binding sites (N3S) were evaluated. It was shown that 188Re complexed with N3S ligands at tracer levels (≤ nanomolar concentration) proved to be unstable after a few hours, in contrast to previous studies with macroscopic amounts of natural Re which demonstrated greater stability. The N2S2 chelates appeared to stably bind 188Re at tracer levels, indicating a more resilient behavior towards the radiometal at the tracer level compared to N3S chelates. GO has been recently proposed as a support for immobilized metal affinity chromatography (IMAC) resins which are widely used adsorbent materials for protein purification processes. The typical IMAC resin is based on three-dimensional polysaccharide beads containing metal-coordinating ligands. The two-dimensional nature of GO has the potential for minimizing the amount of supporting material with respect to the metal-binding ligands and maximizing the capture of target proteins. Here, four new IMAC resins were synthesized and characterized based on GO and carboxylated GO (CGO) functionalized with mono- and bis-nitriloacetic acid sites (NTA and bis-NTA). The ligand bis-NTA is proposed here for the first time. Morphological, structural, vibrational, and spectroscopic studies were conducted on these IMAC resins revealing their different level of suitability for protein purification applications. Elemental analysis revealed a nickel content as high as 58.6 mg/mL of resin, at least one or two orders of magnitude higher than common commercially available resins (0.35 to 1.06 mg/mL). The GO and CGO-based resins were employed in the purification of e-green fluorescence protein (eGFP) in batch mode. The protein loading was as high as 50.3 mg/mL of resin with the CGO-based bis-NiNTA resins. These results indicated that the increase in nickel content has already translated to an amount of eluted protein at least on par with commercial resins in the purification of eGFP. It should be pointed out that the comparison between performance parameters of 2D-nanomaterials and macroscopic spherical beads is generally underestimated because it does not consider the differences in packing factors and swelling properties. It is expected that future resin optimization of the chemical conditions for protein purification, especially reduction of non-specific binding by use of surfactants, will increase protein loading to levels much superior to commercially available IMAC resins. The possibility of using our IMAC resins in radioisotope waste remediation was also tested. Initial results indicate that CGO, and to a lesser extent GO-NTA and CGO-NTA, can be used to effectively capture reduced 99Tc (63%, 44%, and 37% of control sample, respectively). In this thesis, the following major results were achieved: First, an in-depth characterization of GO and CGO is presented along with their mechanisms of formation. Second, GO was combined with TiO2 to form TGO, a platform for the reduction of 99Tc. Third, the tracer level binding of 188Re by N3S ligands was shown to be much less stable than at macroscopic levels, while it is stable with the N2S2 ligands. Fourth, GO and CGO-based resins with NTA and bis-NTA ligands were synthesized, characterized, and used for the purification of eGFP. The protein loading capacity was at least as high as commercial resins, but was affected by a high non-specific binding. Fifth, CGO exhibited an impressive binding affinity for reduced 99Tc. Overall, GO and CGO based nanomaterials have been shown to have tremendous potential for radiometal reduction and sequestration as well as protein purification