The Development And Application Of Four Types Of Magnetic Nano-particles Applied In Nucleic Acid Extraction

Objective To develop four types of iron oxide nanoparticles such as PDA@Fe₃O₄,SiO₂@Fe₃O₄,CTA@Fe₃O_4.To inspect the physicochemical properties,and to optimize and evaluate the extraction and purification system for DNA in four genomes based on paramagnetic particle method.Meanwhile,to evaluate the influence of genomic DNA extracted from different modified groups of magnetic nanoparticles on the High-resolution melting（HRM）for SNP genotyping capacity.Method The core of ferric tetroxide（Fe₃O₄）nanoparticles,is prepared in the hydrothermal method.Polydopamine（PDA）,silica（SiO₂）and chitosan（CTA）are coated on the surface of nanometer ferric oxide beads by chemical modification.The micro-morphology and physical and chemical properties of magnetic particles such as Fe₃O₄ and PDA@Fe₃O_4,SiO₂@Fe₃O_4,CTA@Fe₃O₄ are characterized by analytical techniques of Transmission Electron Microscopy（TEM）,X-Ray Diffraction（XRD）,Fourier infrared spectroscopy（FT-IR）,Vibration Sample Magnetometer（VSM）and Zeta potentiometer.With the centrifugal column extraction as a reference,the independent sample t-test is used for statistical processing to evaluate the gaining rate and purity of DNA extracted by modified magnetic particles and naked nuclear magnetic particles,and to calculate the maximum extraction capacity.For 4 SNP-points of rs12641369 G>A,rs2725252 G>T,rs3114018 C>A,rs2302515 C>G,the extracted DNA is taken in the above method for a template to detect PCR-HRM;Taking Sanger sequencing as the gold standard,the sensitivity and specificity in the detection is evaluated.The repeatability and reproducibility are evaluated by repeatability experiments.Result（1）It’s observed by transmission electron microscopy that four types of nanometer ferric oxide particles are spherical in shape,and the size are basically the same as that of the particle at about 100 nm,and the layout are general;The XRD test results show that the position of diffraction peaks is consistent with the standard parameters.The index of the corresponding crystal surface is（220）,（311）,（400）,（422）,（511）and（533）,and there are no other impurity peaks showing.All products are mono-phase cubic crystals;The infrared spectrum shows that the feature absorption peak of PDA@Fe₃O₄ is at 3418cm^-1,2922cm^-1,1466cm^-1 and 876cm^-1;The feature absorption peak for SiO₂@Fe₃O₄ is at 1648 cm^-1;The feature absorption peak of CTA@Fe₃O₄ is at 1648 cm^-1,1603 cm^-1,and 1385 cm^-1,indicating that dopamine,silica,and chitosan are successfully modified on the surface of the magnetic particles and combined by means of electrostatic,hydrogen bonds,and Van der Waals forces.The result of hysteresis loops has shown that the magnetization intensity of four types of magnetic particles begins to increase when the intensity of the field is-2500 Oe,the intensity is the most obvious ranging from the-2000 Oe to 2000 Oe,and reaches the saturation state basically at 3000 Oe.The saturated magnetic values of Fe₃O₄,PDA@Fe₃O₄,SiO₂@Fe₃O₄,and CTA@Fe₃O₄ are 71.5 emug^-1,40.7emug^-1,46.5emug_-¹ and58.9emug^-1,respectively;Zeta potential result shows that with pH of the solution increasing,Zeta potential values gradually decrease,and the isoelectric points of Fe₃O₄,PDA@Fe₃O₄,SiO₂@Fe₃O₄,and CTA@Fe₃O₄ in the system are 5.0,5.5,7.2,and 6.5,respectively.（2）From the result of extraction quality of the five optical nucleic acid extraction systems it has shown that the nucleic acid gaining rate of PDA@Fe₃O₄ is higher than that of the centrifugal column method（P<0.05）;The extraction rate in the method of SiO₂@Fe₃O₄ is comparable to that in centrifugal column,and the differences are not statistically significant（P<0.05）;The gaining rate of DNA extraction in CTA@Fe₃O₄method and Fe₃O₄ method is lower than that of the column formulation and there are significant differences（P<0.01）;Comparing with methods of PDA@Fe₃O₄,SiO₂@Fe₃O₄,CTA@Fe₃O₄,Fe₃O₄ with the column formulation,there is no statistical difference in A260/A280 ratio（P>0.05）;Ratio of A260/A230 in PDA@Fe₃O₄,SiO₂@Fe₃O₄,CTA@Fe₃O₄ and Fe₃O₄（1.60±0.14）is lower than that in the column extraction,and differences are statistically significant（P<0.01）.The capabilities of DNA maximum adsorption in Fe₃O₄,PDA@Fe₃O₄,SiO₂@Fe₃O₄ and CTA@Fe₃O₄ are 23.3mg/g,116.7 mg/g,96.4 mg/g,and 48.3 mg/g,respectively.（3）From the result of the test performance evaluation,it is shown that except for the 2 samples,the melting curves have drifted and got reviewed,and the remained samples could be directly genotyped.The sensitivity and specificity of the final typing in the method of four nucleic acid extraction reached 100%.In repetitive experiments,CV of the melting curve Tm corresponding to the four nucleic acid extraction methods and the SNP sites is ranging from 0.04%to 0.15%;In the reproduction experiment,the CVs of the melting curve Tm corresponding to the SNP-site of the four magnetic particles nucleic acid extraction methods are ranging from 0.07%to 0.32%.Although there are slight fluctuations in the melting curve between batches,that does not affect the correct typing of genes.There are good repetition and reproducibility in 10 inter-batches and 10 tests at 4 positions among patches.Statistical analysis has shown that except for rs2302515 C>G,there are significant differences in Tm value of purification and wild of rs12641369 G>A,rs12641369 G>A;rs2725252 G>T and rs3114018 C>A（P<0.01）,which can clearly distinguish different pure genotypes.Conclusion In this study,four magnetic nanoparticles used for nucleic acid extraction were successfully prepared.The self-built nucleic acid extraction systems of four magnetic particles have little influence on the result of PCR-HRM detection.Four SNPs of rs12641369 G>A,rs2725252 G>T;rs3114018 C>A,rs2302515 C>G can be regularly genotyped and have a good detection performance,which has a certain clinical application value.