CN101640088A - Precipitation-oxidation method for preparing glucan ferroferric oxide magnetic nano particle and application thereof - Google Patents

Precipitation-oxidation method for preparing glucan ferroferric oxide magnetic nano particle and application thereof Download PDF

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CN101640088A
CN101640088A CN200910181247A CN200910181247A CN101640088A CN 101640088 A CN101640088 A CN 101640088A CN 200910181247 A CN200910181247 A CN 200910181247A CN 200910181247 A CN200910181247 A CN 200910181247A CN 101640088 A CN101640088 A CN 101640088A
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magnetic nano
ferroferric oxide
glucan
particle
oxide magnetic
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CN101640088B (en
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胥传来
刘微波
陈伟
赵媛
彭池方
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Jiangnan University
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Jiangnan University
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Abstract

The invention relates to a precipitation-oxidation method for preparing aglucan ferroferric oxide magnetic nano particle and an application thereof, belonging to the technical fields of nano materialsand bioengineering. The invention comprises a method for preparing a glucan ferroferric oxide magnetic nano particle used for extraction and purification of DNA and the purification of PCR products in the process of transgenic soy detection. The invention provides a simple, convenient and easy precipitation-oxidation one-step method for preparing the glucan ferroferric oxide magnetic nano particle, simplifies the reaction condition, obtains spherical particle with larger particle diameter, high magnetic response and favorable water solubility; the prepared magnetic nano particle is used for the efficient extraction and purification of the DNA and the purification of PCR products in the process of transgenic soy detection, greatly improves the sensitivity of detection and is simpler and more convenient to operate.

Description

Precipitation-oxidation method for preparing glucan ferroferric oxide magnetic nano-particle and application thereof
Technical field
A kind of superparamagnetism glucan ferroferric oxide magnetic nanometer particle process method and the application in transgenosis detects thereof belong to nano material and technical field of bioengineering.
Background technology
Ferroferric oxide magnetic nano-particles toxicity is low, has magnetic responsiveness, when enough hour of particle diameter, has superparamagnetism, and stronger magnetic is promptly arranged in externally-applied magnetic field, when withdrawing magnetic field, and the very fast disappearance of magnetic, remanent magnetism is zero, can be by permanent magnetization.
Glucan has another name called dextran, is the homopolysaccharide that is polymerized by several glucose molecules, is mainly connected itself and catabolite (CO in vivo with α-(1,6) glycosidic bond by the D-glucopyanosyl 2, H 2O) nontoxic to human body, glucan is soluble in water, can replace a part of whole blood in the blood transfusion process, and the expansion agent (being called dextran) as the blood plasma volume is widely used clinically.
Ferroferric oxide magnetic nano-particles through glucan coats can make it both have magnetic and water-soluble, has surface active groups again, can be further and various biomolecules couplings such as cell, enzyme, protein, antibody and nucleic acid.Under the effect of externally-applied magnetic field, magnetic nano-particle can separate with end liquid easily, has easy and simple to handle and the high advantage of separative efficiency.In addition, the glucan ferroferric oxide magnetic nano particle has big specific area, provides the foundation for modifying multiple High Density Molecular probe, demonstrates wide application prospect in fields such as cell separation, protein purification and DNA separation detection.
Generally speaking, the preparation method of magnetic nano-particle can be divided into physics method, bioanalysis and chemical method.Wherein the physics method serves as main representative with the mechanical ball milling method, ball-milling method is that the particle with micron or sub-micron grinds for a long time, be distributed to then in the oil-based media and get, the particle size distribution of the particle that this preparation method obtains is than broad, and the time that this preparation method consumed simultaneously is also long.The biological preparation method of magnetic nano-particle is meant that mainly magnetic nano-particle is present in various organisms such as bacterium widely, in the bodies such as pigeon, utilize the particle diameter of bioanalysis preparation to compare homogeneous and pattern rule, shortcoming is comparatively difficulty of Bacteria Culture, and the process that particle extracts is also comparatively loaded down with trivial details.So it is present, the synthetic magnetic nano-particle that mainly also is limited to the chemical method preparation for the research magnetic nano-particle, the chemical synthesis of magnetic Nano material can be divided into homogeneous phase preparation method and heterogeneous preparation method again, wherein the homogeneous phase preparation method comprises coprecipitation and high-temperature decomposition, and heterogeneous preparation method has microemulsion method, sol-gel process, sonochemical method, laser decomposition method and electrochemical deposition method etc.
Since the last century the eighties, people constantly attempt prepared in various methods uniform particle diameter, biocompatibility glucan ferroferric oxide magnetic nano particle that magnetic responsiveness is stronger, yet form ripe unified preparation method so far.
The present invention adopts precipitation simple, with low cost-oxidizing process development uniform particle diameter, biocompatibility glucan ferroferric oxide magnetic nano particle that magnetic responsiveness is strong, is applied to the extraction purifying of DNA in the testing process of genetically engineered soybean and the purifying of PCR product.
High-quality DNA extractive technique is the prerequisite of PCR, Southern hybridization and gene clone equimolecular biological test, is that molecular biology is the most basic, one of most important work.For the extraction of genetically engineered soybean seed DNA, generally adopt some traditional DNA isolation and purification methods such as CTAB method, not only need contact multiple organic solvent and toxic reagent, and control numerous and diverse being difficult to of step.The glucan ferroferric oxide magnetic nano particle can combine with dna molecular by electrostatic adsorption, can obtain the higher dna molecular of purifying rate with buffer solution elution again.In addition, through such purifying, can remove the unnecessary primer that is mingled in the PCR product, the product that obtains can be directly used in order-checking.
Summary of the invention
The object of the present invention is to provide a kind of glucan ferroferric oxide magnetic nanometer particle process method, particle diameter homogeneous, the magnetic responsiveness of preparation are strong, has excellent biological compatibility, can be used for the extraction purifying of DNA in the genetically engineered soybean testing process and the purifying of PCR product, method of operation is simple and easy to do.
Technical scheme of the present invention: a kind of superparamagnetism glucan ferroferric oxide nanometer particle process method and the application in the genetically engineered soybean testing process comprise the preparation of superparamagnetic nanoparticle, the extraction purifying of transgenic soybean DNA, the purifying of PCR product.
The preparation of glucan ferroferric oxide magnetic nano particle adopts precipitation-oxidation one-step method synthetic in dextran solution, and its chemical equation is:
Fe 2++2OH -＝Fe(OH) 2，6Fe(OH) 2+O 2＝2Fe 3O 4+6H 2O
Concrete steps are as follows:
(1) 5g Dextran T-40 is under nitrogen protection, and mechanical agitation makes it to be dissolved in fully in the 10mL deionized water.
(2) 4mmol FeCl 24H 2O is dissolved under nitrogen protection in the 10mL deionized water, slowly splashes into to continue in the dextran solution to stir.
(3) behind warming-in-water to 60～80 ℃, 350rpm stirred for several minute, slowly drip the ammoniacal liquor 10mL of mass concentration 25%～28%, reactant liquor becomes blueness, removes nitrogen protection, makes the reaction system ingress of air.
(4) after reaction a period of time, reactant liquor becomes black by blueness gradually, finally obtains the black colloidal solution, stops reaction, leaves standstill cooling.
(5) with glacial acetic acid black colloidal solution pH is transferred to neutrality, centrifugal, abandon precipitation, supernatant promptly obtains the glucan ferroferric oxide magnetic nano particle with 0.22 μ m membrane filtration.Be scattered in the carbonate buffer solution of pH 9.6.
Described step (2), (3) generate the ferrous hydroxide precipitation earlier, and then ingress of air is oxidized to tri-iron tetroxide.
The glucan ferroferric oxide magnetic nano particle of method for preparing is spherical, average aggregate particle size (diameter of the macroparticle that forms after a plurality of particle clusters) is about 100nm, magnetic responsiveness is strong, good dispersion in water and physiological saline, the aggregate and precipitate of dewatering in absolute ethyl alcohol.Can be used for the separation and purification of DNA in the genetically engineered soybean testing process.
Be used for the extraction purifying of transgenic soybean DNA, step is as follows:
(1) 3 genetically engineered soybean seed is cleaned, and soaks imbibition 24h in the pure water, and manual peeling adds a small amount of quartz sand, grinds to form paste under the ice bath, changes in the 10mL centrifuge tube.
(2) add the extract of 65 ℃ of preheatings fast to the 9mL place, extract is: the mixed liquor that contains 0.1M Tris-HCl, 0.02M EDTA, 1.5M NaCl, mass concentration 2%PVP 40,2%CTAB and 3% beta-mercaptoethanol, fluctuate, 65 ℃ of water-bath 1h, every therebetween 5min puts upside down three times, the centrifugal 10min of 10000rpm gets supernatant.
(3) add and the isopyknic chloroform of supernatant-isoamyl alcohol mixed solution in the supernatant, chloroform: the isoamyl alcohol volume ratio is 24: 1, slowly turns upside down 30 times, and the centrifugal 10min of 10000rpm gets supernatant 1mL in the 1.5mL centrifuge tube.
(4) the glucan ferroferric oxide magnetic nano particle of adding 10 μ L 10mg/mL, the glucan ferroferric oxide magnetic nano particle combines with the transgenic soybean DNA molecule, make it obtain separation and purification, Magnetic Isolation, precipitation is washed 3 times with absolute ethyl alcohol 20 μ L, naturally volatilization 30min, what be dissolved into 50 μ L contains 10mM Tris, 1mMEDTA, in the TE buffer solution of pH7.4.
Be used for the purifying of the PCR product of transgenic soybean DNA, step is as follows:
Solid content is the ultrasonic dispersion of the magnetic nano-particle of 10mg/mL 5min, getting 15 μ L magnetic nano-particle dispersion liquids adds in the PCR reaction tube, the hybridization solution 20 μ L that add mass concentration 10%PEG-8000,0.2M NaCl, hatch 5min for 37 ℃, magnetic separates, and abandoning supernatant is with the ethanol 20 μ L cleaning magnetic grain 2～3 times and the air-dry 2min of mass concentration 70%, Tris-EDTA eluent 20 μ L eluted dnas, ultraviolet records OD 260/ OD 280Ratio, and get 2 μ L and carry out agarose gel electrophoresis, measure dna sequence dna.
Beneficial effect of the present invention: the invention provides a kind of simple and easy to do precipitation-oxidation method for preparing glucan ferroferric oxide magnetic nano-particle, simplified the condition of reaction.The magnetic nano-particle of preparation is used for genetically engineered soybean testing process DNA efficiently extracting and purifying, and the purifying of PCR product, improve the sensitivity that detects greatly, and operation is easier.
Description of drawings
The transmission electron microscope picture of Fig. 1 glucan ferroferric oxide magnetic nano particle.
Embodiment
The preparation of embodiment 1. glucan ferroferric oxide magnetic nano particles, concrete steps are as follows:
(1) 5g Dextran T-40 is under nitrogen protection, and the 200rpm mechanical agitation makes it to be dissolved in fully in the 10mL deionized water.
(2) 4mmol FeCl 24H 2O is dissolved under nitrogen protection in the 10mL deionized water, slowly splashes into to continue in the dextran solution to stir.
(3) warming-in-water to 60 ℃ after 350rpm stirs 5min, slowly drips 25%～28% ammoniacal liquor 10mL, and reactant liquor becomes blueness, removes nitrogen protection, makes the reaction system ingress of air.
(4) behind the 20min, reactant liquor becomes black by blueness gradually, finally obtains the black colloidal solution, stops reaction, cooling 30min.
(5) with glacial acetic acid black colloidal solution pH is transferred to 7.0, the centrifugal 10min of 8000rpm abandons precipitation, and supernatant promptly obtains the glucan ferroferric oxide magnetic nano particle with 0.22 μ m membrane filtration.Be scattered in the carbonate buffer solution of pH 9.6.
The extraction purifying of embodiment 2. transgenic soybean DNAs, step is as follows:
(1) 3 genetically engineered soybean seed is cleaned, and soaks imbibition 24h in the pure water, and manual peeling adds a small amount of quartz sand, grinds to form paste under the ice bath, changes in the 10mL centrifuge tube.
(2) add the extract (0.1M Tris-HCl pH8.0,0.02M EDTA pH8.0,1.5M NaCl, 2%PVP 40,2%CTAB, 3% beta-mercaptoethanol) of 65 ℃ of preheatings fast to the 9mL place, fluctuate, 65 ℃ of water-bath 1h, every therebetween 5min puts upside down three times, the centrifugal 10min of 10000rpm gets supernatant.
(3) supernatant add equal-volume chloroform-isoamyl alcohol (24: 1, V/V) slowly turn upside down 30 times, the centrifugal 10min of 10000rpm gets supernatant 1mL and joins in the 1.5mL centrifuge tube.
(4) the glucan ferroferric oxide magnetic nano particle of adding 10 μ L 10mg/mL, Magnetic Isolation, precipitation is washed 3 times with absolute ethyl alcohol 20 μ L, the 30min that volatilizees naturally, (10mM Tris, 1mMEDTA are pH7.4) in the buffer solution to be dissolved into 50 μ L TE.
The purifying of embodiment 3.PCR product, step is as follows:
Solid content is the ultrasonic dispersion of the magnetic nano-particle of 10mg/mL 5min, getting 15 μ L magnetic nano-particle dispersion liquids adds in the PCR reaction tube, add hybridization solution (10%PEG-8000,0.2M NaCl) 20 μ L are hatched 5min for 37 ℃, magnetic separates, abandoning supernatant, ethanol 20 μ L with mass concentration 70% clean the magnetic grain 2～3 times and air-dry 2min, Tris-EDTA eluent 20 μ L eluted dnas, and ultraviolet records OD 260/ OD 280Ratio, and get 2 μ L and carry out agarose gel electrophoresis, measure dna sequence dna.

Claims (4)

1, a kind of glucan ferroferric oxide magnetic nanometer particle process method is characterized in that adopting precipitation-oxidation one-step method synthetic in dextran solution, and its chemical equation is:
Fe 2++2OH -＝＝Fe(OH) 2，6Fe(OH) 2+O 2＝＝2Fe 3O 4+6H 2O
Step is as follows:
(1) 5g Dextran T-40 is under nitrogen protection, and mechanical agitation makes it to be dissolved in fully in the 10mL deionized water;
(2) 4mmol FeCl 24H 2O is dissolved under nitrogen protection in the 10mL deionized water, slowly splashes into to continue in the dextran solution to stir;
(3) behind warming-in-water to 60～80 ℃, 350rpm stirred for several minute, slowly drip the ammoniacal liquor 10mL of mass concentration 25%～28%, reactant liquor becomes blueness, removes nitrogen protection, makes the reaction system ingress of air;
(4) after reaction a period of time, reactant liquor becomes black by blueness gradually, finally obtains the black colloidal solution, stops reaction, leaves standstill cooling;
(5) with glacial acetic acid black colloidal solution pH is transferred to neutrality, centrifugal, abandon precipitation, supernatant promptly obtains the glucan ferroferric oxide magnetic nano particle with 0.22 μ m membrane filtration, is scattered in the carbonate buffer solution of pH 9.6;
Described step (2), (3) generate the ferrous hydroxide precipitation earlier, and then ingress of air is oxidized to tri-iron tetroxide.
2, preparation method according to claim 1 is characterized in that prepared glucan ferroferric oxide magnetic nano particle, and its particle diameter is the 100nm grade.
3, the application of the prepared glucan ferroferric oxide magnetic nano particle of the described preparation method of claim 1 is characterized in that being used for the extraction purifying of transgenic soybean DNA, and step is as follows:
(1) 3 genetically engineered soybean seed is cleaned, and soaks imbibition 24h in the pure water, and manual peeling adds a small amount of quartz sand, grinds to form paste under the ice bath, changes in the 10mL centrifuge tube;
(2) add the extract of 65 ℃ of preheatings fast to the 9mL place, extract is: the mixed liquor that contains 0.1M Tris-HCl, 0.02M EDTA, 1.5M NaCl, mass concentration 2%PVP 40,2%CTAB and 3% beta-mercaptoethanol, fluctuate, 65 ℃ of water-bath 1h, every therebetween 5min puts upside down three times, the centrifugal 10min of 10000rpm gets supernatant;
(3) add and the isopyknic chloroform of supernatant-isoamyl alcohol mixed solution in the supernatant, chloroform: the isoamyl alcohol volume ratio is 24: 1, slowly turns upside down 30 times, and the centrifugal 10min of 10000rpm gets supernatant 1mL and joins in the 1.5mL centrifuge tube;
(4) the glucan ferroferric oxide magnetic nano particle of adding 10 μ L 10mg/mL, the glucan ferroferric oxide magnetic nano particle combines with the transgenic soybean DNA molecule, make it obtain separation and purification, Magnetic Isolation, precipitation is washed 3 times with absolute ethyl alcohol 20 μ L, naturally volatilization 30min, what be dissolved into 50 μ L contains 10mM Tris, 1mMEDTA, in the TE buffer solution of pH7.4.
4, the application of the prepared glucan ferroferric oxide magnetic nano particle of the described preparation method of claim 1 is characterized in that being used for the purifying of the PCR product of transgenic soybean DNA, and step is as follows:
Solid content is the ultrasonic dispersion of the magnetic nano-particle of 10mg/mL 5min, getting 15 μ L magnetic nano-particle dispersion liquids adds in the PCR reaction tube, the hybridization solution 20 μ L that add mass concentration 10%PEG-8000,0.2M NaCl, hatch 5min for 37 ℃, magnetic separates, and abandoning supernatant is with the ethanol 20 μ L cleaning magnetic grain 2～3 times and the air-dry 2min of mass concentration 70%, Tris-EDTA eluent 20 μ L eluted dnas, ultraviolet records OD 260/ OD 280Ratio, and get 2 μ L and carry out agarose gel electrophoresis, measure dna sequence dna.
CN2009101812477A 2009-07-21 2009-07-21 Precipitation-oxidation method for preparing glucan ferroferric oxide magnetic nano particle and application thereof Expired - Fee Related CN101640088B (en)

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