Functionalized Graphene-based Composites In The Separation And Purification Of Proteins

Graphene, a novel two-dimentional carbon nanomaterial, has attracted extensive attentions in the fields of science and engineering owing to its unique and outstanding physical and chemical properties. The specific and huge surface area of graphene makes it as a potential candidate in the development of solid-phase adsorbent. In the present work, a series of functionlized graphene composites are prepared to achieve highly selective isolation of protein species from complex sample matrixes. The practical performances of these composites for selective isolation of proteins are carefully investigated, and new approaches are established for higly selective isolation of targeted protein species from various complex sample matrixes, i.e., human whole blood, chicken egg white and cell lysate.In chapter 1, a short review is given to the history of graphene. In addition, its structure, property and preparation schemes are discussed. Moreover, the developments of surface functionalization of graphene, graphene-based composites as well as their applications in vaious fields, especially for sample preparation applications, are summarized.In chapter 2, a novel functionalized graphene-based composite is prepared by successive modification of graphene oxide (GO) with epichlorohydrin (ECH), iminodiacetic acid (IDA) and 1-phenylboronic acid (1-PBA) through covalent functionalization and then chelated with nickel ions. Characterizations by FT-IR, XRD, SEM, TGA and ICP-MS demonstrate the successful modifications of the graphene surface, deriving a GO-PBA-IDA-Ni composite with a Ni2+immobilization amount of 3.01×10-3 mol g-1. The composite shows favorable selectivity for the adsorption of lysozyme (Lys). In practice, a homogeneous GO-PBA-IDA-Ni film with a thickness of ca.1.0 μm is fabricated by filtering the composite through a cellulose membrane with a pore aperture of 1.2 μmn. The GO-PBA-IDA-Ni film is subsequently fastened into a rounded membrane cartridge and incorporated into a sequential injection system for on-line selective isolation of Lys. An adsorption efficiency of ca.96%is achieved for 25 μg mL-1 Lys in 500 μL of sample solution at a loading rate of 5 μL s-1. Metal affinity and electrostatic interactions are the main driving forces for governing protein adsorption behaviors. The retained Lys is readily recovered by a borate buffer (pH 10) containing 1.0 mol L-1 NaCl and 20 mmol L-1 imidazole, giving rise to a recovery of ca.90%. The practical applicability of the composite is demonstrated by selective isolation of Lys from chicken egg white and SDS-PAGE assay indicates that Lys is selectively isolated with high purity from complex matrices.In chapter 3, nickel nanoparticle decorated graphene (GP-Ni) is prepared by one-pot hydrothermal reduction of graphene oxide and nickel cations by hydrazine hydrate in the presence of poly(sodium-p-styrenesulfonate) (PSS). The GP-Ni hybrid is characterized by XRD, TEM, SEM, XPS, Raman and FT-IR spectra, demonstrating the formation of poly-dispersed nickel nanoparticles with an average size of 83 nm attached on the surface of graphene sheets. The GP-Ni hybrid exhibits ferromagnetic behavior with a magnetization saturation of 31.1 emu g-1 at 10000 Oersted (Oe). The GP-Ni also possesses favorable stability in aqueous medium and rapid magnetic response to an external magnetic field. These make it a novel magnetic adsorbent for the separation/isolation of His6-tagged recombinant proteins from a complex sample matrix (cell lysate). The targeted protein species is captured onto the surface of the GP-Ni hybrid via specific metal affinity force between polyhistidine groups and nickel nanoparticles. The SDS-PAGE assay indicates highly selective separation of His6-tagged SmtA from cell lysate. The GP-Ni hybrid displays favorable performance on the separation/isolation of His6-tagged recombinant proteins with respect to the commercial NTA-Ni2+column.In chapter 4, graphene oxide-La(BTC)(H2O)6 (H3BTC=1,3,5-benzenetricarboxylic acid) metal organic framework composites (LaMOF-GOn, n=1-6, corresponding to the percentage of GO at 1%,2%,3%,4%,5% and 10%) are prepared through a simple and large-scale method at room temperature. The obtained composites are characterized by ATR-FTIR spectra, SEM, XRD, TGA and N2 adsorption-desorption isotherm. The presence of GO significantly changes the morphologies of the composites from spindly rectangular rods to irregular thick blocks and increases their surface area from 14.8 cm2 g-1 (LaMOFs) to 26.6 cm2 g-1 (LaMOF-GO3), while at the same time the crystalline structure of La(BTC)(H2O)6 is maintained. As a novel solid-phase adsorbent the LaMOF-GO composite exhibits outstanding adsorption properties for proteins. The strong hydrophobic interaction, especially π-π interaction between protein and the composite, is the main driving force for protein adsorption. In particular, highly selective isolation of hemoglobin (Hb) is achieved by using LaMOF-GO3 composite as sorbent in 4 mM B-R buffer containing 0.05 mol L-1 NaCl at pH 8. The retained Hb could be effectively recovered with a 1 mM B-R buffer at pH 10, giving rise to a recovery of 63%. The practical applicability of the LaMOF-GO3 composite is demonstrated by selective adsorption of Hb from human whole blood. SDS-PAGE assays indicate that Hb could be selectively isolated with high purity from biological samples of complex matrixes.In chapter 5, polymeric ionic liquid (PIL), poly(1-vinyl-3-ethylimidazolium bromide) (P(ViEtIm+Br-)), modified reduced graphene oxide (rGO) nanosheets (PIL-rGO) are prepared during the reduction process by hydrazine hydrate. The PIL-rGO is further self-assembled onto the surface of SiO2 nanoparticles through electrostatic interactions as demonstrated by TEM & SEM images. The obtained PIL-rGO@SiO2 nano-hybrid exhibits highly selective adsorption toward acidic protein, i.e., ovalbumin (Ova) as a model in the present case. The strong electrostatic attractions and π-π interactions between Ova and the nano-hybrid are the main driving forces for protein adsorption. An adsorption efficiency of ca.95% is achieved for 150 mg L-1 Ova in a 4 mM B-R buffer at pH 5, along with an ultra-high adsorption capacity of ca.917.4 mg g-1 as compared to those adsorbents for similar purposes. The adsorbed Ova could be effectively recovered from the surface of the nano-hybrid by using a 0.4%(w/v) SDS solution, giving rise to a recovery of ca.70%. The practical applicability of PIL-rGO@SiO2 nano-hybrid is demonstrated by selective isolation and removal of Ova from a complex biological sample matrix, i.e., chicken egg white.Chapter 6 summarizes the above works and on this basis perspectives for future development of functionalized graphene-based materials are given.