Preparation Of Immobilized Metal Affinity Magnetic Nanoparticles And Its Application To Purification Of PDNA

Plasmid DNA (pDNA) is the main carrier of the gene vaccine, and the residue of RNA is an important quality evaluation index. It is very difficult to remove RNA for its high content in the intracellular and being similarity with DNA in the composition and the nature. Traditional chromatography is complex and the RNA enzyme degradation method is too costly, and easy to bring secondary pollution. So far, the magnetic separation method was simple, fast, without secondary pollution and has oher advantages that were widely used for DNA separation and purification directly, but there were no reports about the method used for the selective removal of RNA from the DNA/RNA mixture in order to achieve purification of DNA. This work synthesized the Fe3O4@Au-TA-NTA-Zn2+ magnetic nanoparticles (MNPs). Then, based on immobilized metal affinity chromatography principle, use Fe3O4@Au-TA-NTA-Zn2+ MNPs as solid phase carrier, achieved selective removal of RNA from DNA/RNA mixture and the purification of DNA successfully, providing a theoretical basis and new ideas for the medical pDNA purification. The main work and results are as follows:1. Use FeCl3·6H2O, FeCl2·4H2O and NH3·H2O as raw materials to synthetic Fe3O4 magnetic nanoparticles by chemical co-precipitation method, and using sodium citrate to modify the Fe3O4 magnetic nanoparticles. Then, with the reduction of the hydroxylamine hydrochloride, the Au coated the seed Fe3O4 MNPs surface. The results showed that the citric acid modified Fe3O4 nanoparticles were more stable and without coagulation phenomenon at room temperature for several months. The Fe3O4 nanoparticles were superparamagnetic and the saturation magnetization of them was 52.53emu/g by vibrating samplemagnetomete (VSM) measuring. The color of Fe3O4@Au MNPs was reddish-brown, and the characteristic absorption peak of the Fe3O4@Au MNPs after being treated with hydrochloric acid was at 540nm by Ultraviolet-visible spectroscopy (Uv-vis) analysis. Combined with the Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy analysis (XPS) analysis, fully proved the Au-coated Fe3O4 magnetic nanoparticles were successfully synthesized. The size of the Fe3O4@Au MNPs was about 40nm indicated by transmission electron microscopy (TEM). VSM showed the saturation magnetization of the Fe3O4@Au MNPs was 19.30emu/g, and they were still superparamagnetic, dispersion and had strong magnetic response.2. Use lysine as a raw material to synthesis the NTA derivative Lys-NTA through a four-step organic synthesis reaction. The organic molecules could chelate transition metal ions (such as Zn2+) though NTA, connecte other groups by the free amino group. Lipoic acid (TA) as the connecting arm, on end was modified on the surface of Fe3O4@Au though S-Au bond, the terminal carboxyl group on the oher end coupled with Lys-NTA-Zn2+ through the depsipeptide reaction (NHS/EDC-mediated), to prepared Fe3O4@Au-TA-NTA-Zn2+ MNPs. FTIR, hydrogen nuclear magnetic resonance spectroscopy (’HNMR) and mass spectrometry (MS) identification showed that we got the chelating ligand Lys-NTA through four-step organic synthesis reactions. FTIR and XPS analysis showed that Lys-NTA-Zn2+ was conjugated to the surface of Fe3O4@Au successfully. UV-Vis detecation showed the Au characteristic absorption peak red shifted to 562nm. Fe3O4@Au-TA-NTA-Zn2+ MNPs had good properties of magnetic response and dispersibility3. Use Fe3O4@Au-TA-NTA-Zn2+ MNPs as solid-phase adsorbent, studyed the effects of the adsorption time, phosphate concentration, pH and NaCl concentration on the purified of pDNA. The results showed that in 0-2mol/L range, when NaCl concentration was higher, it’s more favorable for the Fe3O4@Au-TA-NTA-Zn2+ MNPs to adsorb RNA. Acidic environment (such as pH 5.8) was conductive to the absorption of RNA, while the alkaline conditions (pH>7.5) inhibit the absorption. There was no adsorption of the nucleic acid of Fe3O4@Au-TA-NTA-Zn2+ MNPs when the concentration of PB was high (for example 100 mmol/L), while the smaller concentration of PB (for example 0.25 mmol/L), the bead not only adsorbed RNA but also pDNA. When the PB concentration of was 0.5 mmol/L, pH5.8, NaCl concentration of 1.5mol/L, the crude extracts was diluted 60-fold, the Fe3O4@Au-TA-NTA-Zn2+ MNPs selectively adsorbed RNA, without pDNA adsorption, that had achieved the purpose of the research and the pDNA was purified.