| Magnetic nanoparticles (MNPs) is a new type of magnetic material developed during recent years, and is a kind of functional composite material consisted of magnetic inorganic particles and organic molecular. Because of the superior physical properties of MNPs, such as superparamagnetic property, high surface/volume ratio, small particle size and long-time suspension in biological environment, these properties render them an important means for nowadays life science research.The preparation of functional MNPs is the foundation of their application of biology and medicine. But most of the present functionalized methods modificating on the surface of MNPs, utilize toxic reagent, which is neither good for researchers’health, nor for enviroment protection. This research reported a method to prepare monodispersed carboxyl-group MNPs (CMNPs) using N, N-DMF (DMF) and methyl methacrylate as reaction media and functional monomer, respectively; and we furtherly investigated the CMNPs as solid carrier and spermine as binding buffer to extract and purify genomic DNA and plasmid DNA. The main conclusions were given as following:A new approach of preparation of monodiperse CMNPs was established. Ferrite magnetic nanoparticles were prepared by co-precipitation method, and dispersed in DMF with the aid of ultrasonic; After methacrylic acid (MAA), bis[2-(methacryloyloxy)ethyl] phosphate (BisMEP), trimethylolpropane triacrylate (TMPTA) and2,2’-Azobisisoheptonitrile (ABVN) as functional monomer, coupling agent, crosslinking agent and inititator, respectively were added, the reaction conditon was controled in order to coat the ferrite magnetic nanoparticles with carboxyl group. The reaction media DMF had two significant advantages, such as (1) low toxicity, which is environment-friendly;(2) high dispersion or dissolve performance to ferrite and organic reagents, which was of great benefit to prepare monodispersed MNPs. The prapared CMNPs were characterized by TEM, XRD, TAG, VSM and FTIR, the results showed that the CMNPs was monodispersed with the particle size for8nm; its main crystalling phase wasy-Fe2O3; the magnetic nuclear content was79.4%; and the saturation magnetization strength was42.9emu/g; the strong absorption at1730cm-1showed that the surface of CMNPs abound in carboxyl groups.The factors such as pH, concentration of salt and concentration of binding buffer were investigated at length when the CMNPs as solid phase carrier and spermine as binding buffer were used to extract and purify genomic DNA. The optimum conditions successfully applied in small scale plasmid DNA extraction. The results showed that:(1) Too low or too high concentrations of spermine were detrimental to the purification of DNA from crude solution when CMNPs used as solid phase carrier; the suitable concentrations of spermine as binding buffer ranged in2-50mM.(2) The effective role of spermine was with wider range in nearly neutral environment; the higher the pH, the narrower the range.(3) The concentration of salt significantly affected the effective range of spermine; The lower the concentration, the wider the range; When the total concentration of salt reached500mM, the spermine would lost its utility.(4) The off-condensed reagent enable the DNA recover double helix by removing the spermine from the DNA; not only does it make DNA disintegrate from CMNPs, but also improve the biocompatibility of DNA.(5) The established method accomplished to extract different size of pDNA ranged in2.7k-9K, and the results of digestion showed that the extracted DNA owned good biology quality. The method does not involve the use of toxic reagents; does not produce amounts of organic wastewater; does not need any centrifugation; and the concentration of spermine and amounts of CMNPs used are very low. It is environment-friendly, effectiye and fast, time and cost-saving, is especially suitable for the construction of the platform for automation, miniaturization and high-throughput DNA separation and purification. |
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