Three-dimensional Graphene-based Composites For Isolation/Purification Of Proteins And Nucleic Acids

Graphene is one rising member of the carbon-based materials family and has attracted wide attention from scientists for its unique optical,thermal,electrical,chemical and mechanical properties.Its inherent π-electron system,good biocompatibility,large specific surface area and other properties make it a potential for solid phase adsorption,applying in sample pretreatment.Graphene is a plane of carbon atoms arranged in a sp2 hybrid honeycomb conjugated structure,and exhibits strong affinity for the molecules contain an aromatic ring structure,such as proteins,nucleic acids etc.However,graphene shows a poor specificial adsorption to a certain protein or a certain type of protein when it was used for separation/purification of biological samples.Therefore,the modification and functionalization of graphene is necessary to increase its adsorption specificity of the target molecules.The phosphate backbone exists in the structure of DNA not protein,favors interact with metal oxides nanoparticles.Based on the above background,this thesis prepared a series of graphene composites,and applied them or NiO nanoparticle to the pretreatment of biological samples,in order to achieve efficient selective isolation of target proteins or DNA in complex matrix samples and establish the new separation/purification system.In the first chapter,the types and properties of graphene,the preparation methods of graphene and three-dimensional graphene are briefly summarized.The methods of graphene materials in the separation of organic molecules,heavy metals,proteins and nucleic acids is summarized.In the second chapter,a nickel-doped three-dimensional reduced graphene oxide composite was synthesized.In the process of forming the three-dimensional structure of graphene,nickel was incorporated.The obtained composite material had an adsorption efficiency of hemoglobin of 98.5%.Hydrophobic interaction and nickel metal affinity mainly drove Hb adsorption.The adsorbed Hb was stripped by cetyl ammonium bromide,giving to the recovery of 93.6%.The secondary structure of the recovered Hb showed no change,and the original protein activity was maintained after the adsorption/desorption process.The composite was used to separate Hb from human whole blood.It verified by gel electrophoresis that the obtained three-dimensional graphene composite can indeed separate/purify Hb.In the third chapter,the composites were prepared by in situ self-assembly with the nickel oxide nanoparticle packaged.Using triethylenetetramine as a crosslinking agent and a reducing agent providing a basic situation.The graphene oxide solution self-assembled into a three-dimensional graphene hydrogel,and the forming of nickel oxide nanoparticles were encapsulated into the interior structure of the hydrogel as well.Graphene hydrogel formed only in an hour.Using ovalbumin and lysozyme as protein adsorption models to study the adsorption behavior,the adsorption and recovery efficiencies of ovalbumin were 90.6%and 90.7%,respectively.Ovalbumin contains the number of oligosaccharide chains,and hydrogen bonds might exist between the hydroxyl groups of the chain and the amino groups on the surface of the composite.In addition,chemical bonds and metal affinity also contributed to the OVA adsorption.Ovalbumin was successfully isolated from egg white by the obtained composite.In the fourth chapter,NiO nanoparticles were screened from six nanoparticles of NiO,CoO,ZnO,TiO2,CeO2 and Fe3O4 due to its high stable adsorption for short single-stranded DNA.NiO nanoparticles can still show a good adsorption to DNA even the DNA solution mixed with BSA or serum.In order to further study the adsorption process,the functions of pH,salt concentration,DNA length and DNA sequence on the adsorption process were investigated.The adsorption mechanism was studied by adding chemical reagents or denaturing agents.Polyphosphoric acid molecules were the most effective agent for displacing the adsorbed DNA,while simple inorganic phosphate molecules just had little effect on DNA desorption.NiO nanoparticles had a potential to be a solid phase extraction agent for enriching DNA from complex matrix samples.In the fifth chapter,a simple chemical reduction method was used to prepare reduced graphene oxide composite(TiO2-rGOBA).The interior of the composite exhibited a porous structure,and the conjugated hydrophobic region increased.The internally inserted TiO2 nano-aggregates cause nano-scale protrusions on the surface of the sheet to facilitate DNA attachment.The electrostatic attraction between TiO2-rGOBA composite and DNA was the main driving force for DNA adsorption.The adsorption of DNA was investigated by using the salmon DNA as the model.The adsorption efficiency of DNA is 98.4%(B-R buffer,pH=3.0),and the DNA could be stripped by buffer with pH=10,giving to the recovery of 96.3%.In order to verify the feasibility and accuracy of the method,the composite was used for the separation of DNA from human whole blood.The purity of the obtained DNA by above method was almost no different from that of the commercial kit.In the sixth chapter,the research contents of this thesis are summarized,and the future development and application trends of graphene-based materials are prospected.