Preparation And Application Research Of Metal Ion-modified Silica Particles

Adsorption of DNA to solid surfaces has been widely investigated and applied inextraction of DNA during the past decades. The entire DNA molecule behaves as a negativelycharged polyelectrolyte under physiological conditions since it contains multi-phosphategroups. Solid such as silica is usually negative charged due to presence of the silanol groupson the surface. In the majority of reported methods, DNA extraction from biological sampleswas performed by inducing DNA in a high ionic strength buffer in the presence of highconcentration salts and later eluting DNA out in a low ionic strength buffer. Therefore, theadsorption of DNA is very sensitive to salt concentration since both the silica surface andDNA are negatively charged. As a result, the adsorption efficiency of DNA on the silicasurface is limited even under high salt concentration due to the weak feature of the salt bridgebetween the silica surface and DNA. Modification of silica surface with cationic groups hasbeen proven to be an effective way to improve the adsorption efficiency of DNA by directelectrostatic interactions between the silica surface and DNA. The adsorption efficiency isalso sensitive to the salt concentration due to the electrostatic feature of the interactions, inwhich the high efficient adsorption can only be acquired under low salt concentrations. It isstill a challenge to develop an approach which allows the high efficiency adsorption of DNAunder both low and high salt concentrations. In principle, the multi-phosphate groups of DNAcan also act as ligands for metal ions. It is expected that the adsorption of DNA should be lesssensitive to the salt concentration when the interactions between the silica surface and DNAwere dominated by coordination interactions.Immobilized Metal-Affinity Chromatography (IMAC) represents a relatively new separation technique that is primarily appropriate for the purification of proteins with naturalsurface-exposed histidine residues and for recombinant proteins with engineered histidine tagsor histidine clusters. And the method has gained broad popularity in recent years. IMAC is aseparation technique that uses covalently bound chelating compounds on solidchromatographic supports to entrap metal ions, which serve as affinity ligands for variousproteins, making use of coordinative binding of some amino acid residues exposed on thesurface. As with other forms of affinity chromatography, IMAC is used in cases where rapidpurification and substantial purity of the product are necessary, although compared to otheraffinity separation technologies it cannot be classified as highly specific, but only moderatelyso. The benefits of IMAC—ligand stability, high protein loading, mild elution conditions,simple regeneration and low cost—are decisive when developing large-scale purificationprocedures for industrial applications. Even so, there also have some disadvantages andproblems of IMAC. The reported capacities of the commercial IMAC sorbents are usually inthe range of5–10mg/mL or even higher. These values refer to isolated pure proteins orsynthetic mixtures, while capacities for isolation of the target protein from complex sourcesare usually lower, below the allowable limits for clinical-grade proteins. Some matrices, e.g.,those based on IDA chelator, allow a large number of cycles of charging the column withmetal ions and stripping them off. For more strongly bound metals, such as in the case withCo–TALON or Ni–NTA, the suppliers do not recommend stripping off and recharging. This isnot very attractive for commercial applications, due to high cost and frequent labor of packinglarge columns. So we need to improve this approach by introduce a new matrix which issimple and economical.First we used St ber method to prepare200nm SiO₂particles and then modified thesurface with chelating ligand by silane in Chapter2. We established a simple and practicalmethod to research the stability of the chelating ligand on the particles qualitatively andquantitatively. The research showed that these particles were very suitable for biologicalapplication for their good stability and other advantages.In Chapter3, the adsorption of DNA on Fe³⁺immobilized silica particles under differentpH and salt concentration was investigated. At pH lower than4.0, DNA could be adsorbedefficiently (>90%) on the particles surface driven by the coordination interactions between the Fe³⁺ions and phosphate groups of DNA, which was almost not affected by the saltconcentration in the solutions. At pH higher than5.0, the adsorption of DNA became sensitiveto the salt concentration, and high efficient adsorption (⁹0%) of DNA could be acquiredunder high salt concentration driven by the salt bridges. The DNA adsorpted by either wayscan be totally recoverd by increasing the pH or lowering the salt concentration respectively.Compared to pure silica particles, such Fe³⁺immobilized silica particles will be a moreversatile DNA extration agent as providing the possibility for high efficient extraction of DNAby both the coordination interactions and the salt bridges.We applied the silica particles immobilized with metal ion in His-tag protein's isolationand purification process in chapter4. We use the particles to interact with three differentHis-tag proteins, and through the result we found that the content of histidine residue ofprotein can influence the efficiency of extraction. And the maximum extraction efficiency canbe achieved near the protein iso-electric point. We also found that when increasing theconcentration of salt in the solution, the extraction efficiency was significantly reduced. Fromthe investigation, we obtained the optimal extraction conditions: charged with Ni²⁺ions on thesurface of silica particles and in2mM phosphate buffer solution, the pH value of the solutionwas near the iso-electric points of the protein. Compared with traditional IMAC, our particleshave the advantages of small volume, easy concentration, higher load capacity and traceprotein adsorption capacity. Furthermore, we found that the surface charge of these particlescan be changed by the pH value of the solution after loading protein. So we can make use ofthis characteristic for DNA adsorption and desorption experiment.Finally,We use the former route for the preparation of SiO₂particles with immobilizedmetal affinity ligand iminodiacetic acid (IDA) and charged them with Ni²⁺. The particlesshowed highly specific to protein BHb. And they were applied to separate a model proteinmixture of bovine hemoglobin (BHb) and bovine serum albumin (BSA). They could beseparated completely and showed low non-specific adsorption. Then we used these particlesto extract bovine hemoglobin (BHb) from a mixture of BHb and DNA. Furthermore, theparticles could efficiently remove bovine hemoglobin from the mixture of DNA, BSA andBHb. They have potential application in removing abundant protein in proteomic analysis.The particles we prepared were expected to be used for fast and efficient removal of abundant protein bovine hemoglobin (BHb) in bovine blood, and it may be a potentially effective wayto deplete abundant protein in serum. We also use the SiO₂particles charged with Fe³⁺toinvestigate the interaction of DNA and proteins in solution at different pH value. Theseparticles can be used to separate a small amount of DNA impurity from bovine hemoglobinsolution.