Preparation Of Fe₃O₄@Au Nanocomposites And Application For Selective Purification Of Plasmid DNA

Plamid is one of the most important vectors used in the biological and biomedical fields.Currently,magnetic particles are used for separation and purification of pDNA directly from the "RNA-free"cell lysate derived from RNase digestion,which is costly and inevitably introduce second pollution.Till now,there is no report of using magnetic carrier for selective removal of RNA impurities from the mixture of DNA/RNA,to achieve the purification of DNA.Based on immobilized metal affinity chromatography principle,we synthesized the Fe3O4@Au-TA-NTA-Zn2+ magnetic composite nanoparticles,established and optimized the solution for selectivly removal of RNA from DNA/RNA mixture,providing important theoretical basis and new ideas for medical pDNA purification.The main research work and results are as follows:1.Fe3+,Fe2+ and NH3·H2O were used to synthetic Fe3O4 magnetic nanoparticles by chemical co-precipitation method,which were modified with 2,3-dimercaptobutanedioic acid.Then,the resultant magnetic nanoparticles were then coated with gold shells using hydroxylamine hydrochloride reduction mehtod to obtain Fe3O4@Au magnetic composite nanoparticles.Using alkali aging method,lipoic acid(TA)was connected on the surface of Fe3O4@Au.Lys-NTA-Zn2+ was coupled on the surface of Fe3O4@Au magnetic composite nanoparticles through NHS/EDC method forming Fe3O4@Au-TA-NTA-Zn2+ MNPs.The results showed that the Fe3O4 nanoparticles were superparamagnetic and had good stability at room temperature,without coagulation within 10 days.VSM analysis showed the saturation magnetization of Fe3O4 was 53 emu/g.The color of Fe3O4@Au magnetic composite nanoparticles was reddish-brown,with uniform gold coating outside and narrow size distribution of around 35 nm.The saturation magnetization intensity was 17 emu/g.A recovery of 100%can be reached in less than 10 min with the help of external magnetic field.Uv-vis analysis indicated the characteristic absorption peak for the treated Fe3O4@Au magnetic composite nanoparticles was at 555 nm.The surface organic functionalization was verified by FTIR analysis.The final Fe3O4@Au-TA-NTA-Zn2+MNPs still have good magnetic response and dispersibility which are enough for downstream applications.2.Using Fe3O4@Au-TA-NTA-Zn2+ MNPs as solid-phase adsorbent,a method was successfully developed for selective removal of RNA from E.coli lysate by changing the adsorption and elution environmental factors(such as buffer concentration,salt concentration,dosage of magnetic carrier,time,pH,and adding competitive adsorbent etc.).The results showed that the concentration of phosphate buffer inhibited the adsorption of both DNA and RNA,following DNA>RNA.Within a certain range(0-2.0 mol/L),NaCl could promote the adsorption of RNA.pH had little effection on the adsorption of RNA,but when pH<3,it showed prominent inhibitory.When the concentration of phosphate buffer was 0.3 mM(containing 2.0 mol/L NaCl,pH 5.8)and adsorption time 15 min,97%of the RNA from 300μL E.coli lysate could be removed using 0.25 mg Fe3O4@Au-TA-NTA-Zn2+ MNPs.When the concentration of EDTA was 30 mM(containing 0.5 mol/L NaCl)and elution time 15 min,99%of the RNA could be eluted from the Fe3O4@Au-TA-NTA-Zn2+ MNPs.The pDNA remained biological activity as evidenced by enzymatic digestion,which means that the extracted pDNA meet the requirements for biomedical applications.In this study,we selectively remove RNA impurities from the "pDNA/RNA mixture" to achieve the purpose of purifying pDNA.This method is especially suitable for the late purification of medical pDNA and refining of gene vaccine,which also provides a beneficial exploration for the automatic extraction of pDNA.