CN101728040B - Preparing method of pomegranate-shaped magnetic nanometer particle congery and applications thereof in DNA separation and purification - Google Patents
Preparing method of pomegranate-shaped magnetic nanometer particle congery and applications thereof in DNA separation and purification Download PDFInfo
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- CN101728040B CN101728040B CN2010101017637A CN201010101763A CN101728040B CN 101728040 B CN101728040 B CN 101728040B CN 2010101017637 A CN2010101017637 A CN 2010101017637A CN 201010101763 A CN201010101763 A CN 201010101763A CN 101728040 B CN101728040 B CN 101728040B
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Abstract
The invention relates to a preparing method of a pomegranate-shaped magnetic nanometer particle congery and applications thereof in DNA separation and purification, which belongs to the technical fields of nanometer material and molecular biology. The invention comprises a preparing method of a superparamagnetic pomegranate-shaped magnetic nanometer particle congery, and applications thereof in DNA separation and purification of K562 cells and genetically engineered soybeans. The invention provides a convenient and easy method for preparing the pomegranate-shaped magnetic nanometer particle congery by an organic solution high-temperature reaction process, the spherical particles with large diameter are obtained, the magnetic response is strong, the water solubility is good, and the reaction steps are reduced. The prepared magnetic nanometer particle congery can be used for high-efficiency DNA separation and purification of animals and plants, K562 cells and genetically engineered soybeans, greatly enhances the detection sensitivity, and has convenient operation.
Description
Technical field
A kind of preparation method of superparamagnetism pomegranate-shaped magnetic nanometer particle congery and the application in the DNA separation and purification thereof belong to nano material and technical field of molecular biology.
Background technology
Ferroferric oxide magnetic nano-particles toxicity is low, has magnetic responsiveness and superparamagnetism, and stronger magnetic is promptly arranged in externally-applied magnetic field, and when withdrawing magnetic field, magnetic disappears very soon, and remanent magnetism is zero, can be by permanent magnetization.Being flocked together by a lot of little magnetic nanoparticles forms bigger spherical cluster magnetic nano-particle, and the seed gathering situation of its shape and ripe pomegranate is similar, and we are referred to as pomegranate-shaped magnetic nanometer particle congery, and it has stronger magnetic responsiveness.
Use the Trisodium Citrate coated ferroferric oxide magnetic nano-particles, can make it both have superparamagnetism and water-soluble preferably, have 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 separation efficiency.In addition, had big specific surface area, provide the foundation, demonstrate wide application prospect in fields such as cellular segregation, protein purification and DNA separation detection for modifying multiple High Density Molecular probe by the magnetic nano-particle after coating.
Generally speaking, the preparation method of magnetic nano-particle can be divided into physics method, biological process and chemical method.Wherein the physics method serves as main representative with the mechanical ball milling method; Ball milled is that the particle with micron or submicron grinds for a long time; Be distributed to then in the oil-based media and get, the 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 biological process preparation to compare homogeneous and pattern rule, shortcoming is comparatively difficulty of microbial 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 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 method and high-temperature decomposition, and heterogeneous preparation method has microemulsion method, sol-gel method, 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 ferroferric oxide magnetic nano-particles that magnetic responsiveness is stronger, yet form ripe unified preparation method so far.
The present invention adopts simple organic solvent pyroreaction method development uniform particle diameter, the biocompatibility pomegranate-shaped magnetic nanometer particle congery that magnetic responsiveness is strong, and functional group-COOH is rich on the magnetic nanometer particle congery surface of this Trisodium Citrate parcel.Under certain salt concn, DNA can be incorporated into the surface have-the terminal magnetic grain of COOH on, in case combine, available 70% ethanol cleans DNA-magnetic grain mixture, uses the Tris-EDTA damping fluid with the DNA wash-out then.Prepared pomegranate-shaped magnetic nanometer particle congery can be applicable to the separation and purification of plant-animal DNA, and is easy and simple to handle, time saving and energy saving.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of pomegranate-shaped magnetic nanometer particle congery; Particle diameter homogeneous, the magnetic responsiveness of preparation are strong; Have excellent biological compatibility, can be used for the separation and purification of DNA in the plant-animal DNA detection process, working method is simple and easy to do.
Technical scheme of the present invention: a kind of preparation method of pomegranate-shaped magnetic nanometer particle congery and the application in the plant-animal DNA detection comprise the preparation of pomegranate-shaped magnetic nanometer particle congery, the separation and purification of plant-animal DNA.
(1) preparation of pomegranate-shaped magnetic nanometer particle congery adopts organic solvent pyroreaction method synthetic in reactor drum, and concrete steps are following:
(1) take by weighing the 0.65g FERRIC CHLORIDE ANHYDROUS and the 0.4g trisodium citrate is put into reactor drum, add 20mL terepthaloyl moietie as solvent, in solvent phase, feed nitrogen 10min to drive away oxygen, magnetic agitation makes solid all be dissolved in the solution;
(2) take by weighing the 1.2g anhydrous sodium acetate and join in the reaction system, continue to stir, place oil bath to be heated to 220 ℃ reactor drum, magnetic agitation is reacted 10h and is stopped heating simultaneously, continues magnetic agitation and is cooled to room temperature;
(3) reacted solution adds the 20mL absolute ethyl alcohol, ultrasonic concussion 10min, and the centrifugal 10min of 6000rpm abandons supernatant;
(4) the deposition part continues to add the 20mL absolute ethyl alcohol, ultrasonic concussion 10min, and the centrifugal 10min of 6000rpm abandons supernatant;
(5) the deposition part adds the 20mL deionized water, ultrasonic concussion 10min, and the centrifugal 10min of 6000rpm abandons supernatant;
(6) deposition is scattered in the 10mL deionized water, is the pomegranate-shaped magnetic nanometer particle congery dispersion liquid, and room temperature preservation is subsequent use.
The pomegranate-shaped magnetic nanometer particle congery of method for preparing is spherical, and between 100～500nm, preparation condition is controlled for aggregate particle size (diameter of a plurality of particle clusters bunch macroparticle that forms afterwards), and magnetic responsiveness is strong, good dispersivity in water and saline water.Can be used for the separation and purification of animal-plant gene group DNA.
(2) be used for the separation and purification of K562 cell (leukemia cell) DNA, step is following:
(1) the K562 cell of getting the 1mL cultivation is in the 1.5mL centrifuge tube, and the centrifugal 5min of 1000rpm abandons supernatant;
(2) sedimentation cell is with 1mL saline water 1000rpm centrifuge washing, centrifuge washing 2 times;
(3) cell behind the centrifuge washing adds 1mL cell pyrolysis liquid (3M NaI, 5M urea, 40g/L triton x-100,10mM EDTA Disodium, 25mM Tris-HCl); Add the Proteinase K 15 μ L of 25 μ g/ μ L, the RNA enzyme 5 μ L of 40 μ g/ μ L behind the mixing, fully 30min is hatched in 37 ℃ of water-baths behind the mixing;
(4) add the pomegranate-shaped magnetic nanometer particle congery dispersion liquid 10 μ L that (one) method makes, fully mixing leaves standstill 5min, adds 40 μ L Virahols, leaves standstill 5min;
(5) magnetic separates, and abandons supernatant, adds mass concentration 70% ethanol 1mL washing, and magnetic separates, and abandons supernatant;
(6) add 50 μ L TE damping fluids (10mM Tris-HCl, 1mM EDTA Disodium, pH8.0), 5min is hatched in 37 ℃ of water-baths, magnetic separates behind the mixing, gets supernatant, is K562 cell DNA elutriant.
(3) be used for the separation and purification of transgenic soybean DNA, step is following:
(1) 3 genetically engineered soybean seed is cleaned, and soaks imbibition 24h in the pure water, and manual peeling adds 0.2g silica sand, grinds to form paste under the ice bath, changes in the 10mL centrifuge tube;
(2) add extracting solution (pH8.0,0.1M Tris-HCl, pH8.0,0.02M EDTA Disodium, the 1.5M NaCl of 65 ℃ of preheatings fast; Mass concentration 2% Vinylpyrrolidone polymer 40, mass concentration 2% cetyl trimethylammonium bromide and mass concentration 3% beta-mercaptoethanol) to the 9mL place, fluctuate; 65 ℃ of water-bath 30min; Every therebetween 5min puts upside down three times, and the centrifugal 10min of 10000rpm gets supernatant;
(3) supernatant add equal-volume chloroform-primary isoamyl alcohol mixed solution (24: 1, V/V), slowly turn upside down 30 times, the centrifugal 10min of 10000rpm gets supernatant 1mL and is added in the 1.5mL centrifuge tube;
(4) add the pomegranate-shaped magnetic nanometer particle congery dispersion liquid 10 μ L that (one) method makes, fully mixing leaves standstill 5min, magnetic resolution, and deposition is washed 3 times with absolute ethyl alcohol, naturally volatilization 30min;
(5) add 100 μ L TE damping fluids (10mM Tris-HCl, 1mM EDTA Disodium, pH8.0), 5min is hatched in 37 ℃ of water-baths, magnetic separates behind the mixing, gets supernatant, is the transgenic soybean DNA elutriant.
Prepared pomegranate-shaped magnetic nanometer particle congery can be applicable to the separation and purification of plant-animal DNA.
Beneficial effect of the present invention: the invention provides the method that a kind of simple and easy to do organic solvent pyroreaction legal system is equipped with pomegranate-shaped magnetic nanometer particle congery, obtain the spherical particle of greater particle size, magnetic responsiveness is strong, and good water solubility has been simplified the step of reacting.The pomegranate-shaped magnetic nanometer particle congery of preparation can be used for the high efficiency separation purifying of plant-animal DNA, is used for the separation and purification of K562 cell DNA and the separation and purification of transgenic soybean DNA, improve the sensitivity that detects greatly, and operation is easier.
Description of drawings
The TEM of Fig. 1 pomegranate-shaped magnetic nanometer particle (transmission electron microscope) figure.
Sepharose (0.8%) electrophorogram of the K562 cell DNA that Fig. 2 extracts.
The SEM of Fig. 3 pomegranate-shaped magnetic nanometer particle (ESEM) figure.
Embodiment
The preparation of embodiment 1. pomegranate-shaped magnetic nanometer particle congeries
(1) take by weighing the 0.65g FERRIC CHLORIDE ANHYDROUS and the 0.4g trisodium citrate is put into reactor drum, add 20mL terepthaloyl moietie as solvent, in solvent phase, feed nitrogen 10min to drive away oxygen, magnetic agitation makes solid all be dissolved in the solution;
(2) take by weighing the 1.2g anhydrous sodium acetate and join in the reaction system, continue to stir, place oil bath to be heated to 220 ℃ reactor drum, magnetic agitation is reacted 10h and is stopped heating simultaneously, continues magnetic agitation and is cooled to room temperature;
(3) reacted solution adds the 20mL absolute ethyl alcohol, ultrasonic concussion 10min, and the centrifugal 10min of 6000rpm abandons supernatant;
(4) the deposition part continues to add the 20mL absolute ethyl alcohol, ultrasonic concussion 10min, and the centrifugal 10min of 6000rpm abandons supernatant;
(5) the deposition part adds the 20mL deionized water, ultrasonic concussion 10min, and the centrifugal 10min of 6000rpm abandons supernatant;
(6) deposition is scattered in the 10mL deionized water, is the pomegranate-shaped magnetic nanometer particle congery dispersion liquid, and room temperature preservation is subsequent use.
The separation and purification of embodiment 2.K562 cell (leukemia cell) DNA, step is following:
(1) the K562 cell of getting the 1mL cultivation is in the 1.5mL centrifuge tube, and the centrifugal 5min of 1000rpm abandons supernatant;
(2) sedimentation cell is with 1mL saline water 1000rpm centrifuge washing, centrifuge washing 2 times;
(3) add 1mL cell pyrolysis liquid (3M NaI, 5M Urea, 40g/L Triton-X-100,10mMEDTA, 25mM Tris-HCl), add behind the mixing 15 μ L Proteinase Ks (25 μ g/ μ L), 5 μ L RNA enzymes (40 μ g/ μ L) fully behind the mixing 37 ℃ of water-baths hatch 30min;
(4) add the pomegranate-shaped magnetic nanometer particle congery dispersion liquid 10 μ L that embodiment 1 makes, fully mixing leaves standstill 5min, adds 40 μ L Virahols, leaves standstill 5min;
(5) magnetic separates, and abandons supernatant, adds mass concentration 70% ethanol 1mL washing, and magnetic separates, and abandons supernatant;
(6) add 50 μ L TE damping fluids (10mM Tris-HCl, 1mM EDTA, pH8.0), 5min is hatched in 37 ℃ of water-baths, magnetic separates behind the mixing, gets supernatant, is K562 cell DNA elutriant.
3. the extraction purifying of transgenic soybean DNA, step is following:
(1) 3 genetically engineered soybean seed is cleaned, and soaks imbibition 24h in the pure water, and manual peeling adds 0.2g silica sand, grinds to form paste under the ice bath, changes in the 10mL centrifuge tube;
(2) add extracting solution (the 0.1M Tris-HCl of 65 ℃ of preheatings fast; PH8.0,0.02M EDTA, pH8.0,1.5MNaCl, 2%PVP40,2%CTAB, 3% beta-mercaptoethanol) to the 9mL place, fluctuate; 65 ℃ of water-bath 30min; Every therebetween 5min puts upside down three times, and the centrifugal 10min of 10000rpm gets supernatant;
(3) supernatant add equal-volume chloroform-primary isoamyl alcohol mixing solutions (24: 1, V/V), slowly turn upside down 30 times, the centrifugal 10min of 10000rpm gets supernatant 1mL and is added in the 1.5mL centrifuge tube;
(4) add the pomegranate-shaped magnetic nanometer particle congery dispersion liquid 10 μ L that embodiment 1 makes, magnetic resolution, deposition is washed 3 times with absolute ethyl alcohol, naturally volatilization 30min;
(5) add 100 μ L TE damping fluids (10mM Tris-HCl, 1mM EDTA, pH8.0), 5min is hatched in 37 ℃ of water-baths, magnetic separates behind the mixing, gets supernatant, is the transgenic soybean DNA elutriant.
Claims (5)
1. the preparation method of a pomegranate-shaped magnetic nanometer particle congery is characterized in that adopting organic solvent pyroreaction method synthetic in reactor drum, and its step is following:
(1) take by weighing the 0.65g FERRIC CHLORIDE ANHYDROUS and the 0.4g trisodium citrate is put into reactor drum, add 20mL terepthaloyl moietie as solvent, in solvent phase, feed nitrogen 10min to drive away oxygen, magnetic agitation makes solid all be dissolved in the solution;
(2) take by weighing the 1.2g anhydrous sodium acetate and join in the reaction system, continue to stir, place oil bath to be heated to 220 ℃ reactor drum, magnetic agitation is reacted 10h and is stopped heating simultaneously, continues magnetic agitation and is cooled to room temperature;
(3) reacted solution adds the 20mL absolute ethyl alcohol, ultrasonic concussion 10min, and the centrifugal 10min of 6000rpm abandons supernatant;
(4) the deposition part continues to add the 20mL absolute ethyl alcohol, ultrasonic concussion 10min, and the centrifugal 10min of 6000rpm abandons supernatant;
(5) the deposition part adds the 20mL deionized water, ultrasonic concussion 10min, and the centrifugal 10min of 6000rpm abandons supernatant;
(6) deposition is scattered in the 10mL deionized water, is the pomegranate-shaped magnetic nanometer particle congery dispersion liquid, and room temperature preservation is subsequent use.
2. preparation method according to claim 1 is characterized in that prepared pomegranate-shaped magnetic nanometer particle congery, and its particle diameter is 100～500nm.
3. the application of the prepared pomegranate-shaped magnetic nanometer particle congery of the described preparation method of claim 1 is characterized in that being used for the separation and purification of K562 cell DNA, and step is following:
(1) the K562 cell of getting the 1mL cultivation is in the 1.5mL centrifuge tube, and the centrifugal 5min of 1000rpm abandons supernatant;
(2) sedimentation cell is with 1mL saline water 1000rpm centrifuge washing, centrifuge washing 2 times;
(3) cell behind the centrifuge washing adds the 1mL cell pyrolysis liquid, adds the Proteinase K 15 μ L of 25 μ g/ μ L, the RNA enzyme 5 μ L of 40 μ g/ μ L behind the mixing, and fully 30min is hatched in 37 ℃ of water-baths behind the mixing;
Said cell pyrolysis liquid consists of: 3M NaI, 5M urea, 40g/L Triton-X-100,10mM EDTA Disodium and 25mM Tris-HCl;
(4) add the pomegranate-shaped magnetic nanometer particle congery dispersion liquid 10 μ L that claim 1 method makes, fully mixing leaves standstill 5min, adds 40 μ L Virahols, leaves standstill 5min;
(5) magnetic separates, and abandons supernatant, adds mass concentration 70% ethanol 1mL washing, and magnetic separates, and abandons supernatant;
(6) add 50 μ L TE damping fluids, 5min is hatched in 37 ℃ of water-baths, and magnetic separates behind the mixing, gets supernatant, is K562 cell DNA elutriant;
Said TE damping fluid consists of: 10mM Tris-HCl, 1mM EDTA Disodium, pH8.0.
4. the application of the prepared pomegranate-shaped magnetic nanometer particle congery of the described preparation method of claim 1 is characterized in that being used for the separation and purification of transgenic soybean DNA, and step is following:
(1) 3 genetically engineered soybean seed is cleaned, and soaks imbibition 24h in the pure water, and manual peeling adds 0.2g silica sand, grinds to form paste under the ice bath, changes in the 10mL centrifuge tube;
(2) extracting solution that adds 65 ℃ of preheatings fast fluctuates to the 9mL place, 65 ℃ of water-bath 30min, and every therebetween 5min puts upside down three times, and the centrifugal 10min of 10000rpm gets supernatant;
Said extracting solution consists of: pH8.0,0.1M Tris-HCl; PH8.0,0.02M EDTA Disodium; 1.5M NaCl, mass concentration 2% Vinylpyrrolidone polymer 40, mass concentration 2% cetyl trimethylammonium bromide and mass concentration 3% beta-mercaptoethanol;
(3) supernatant adds equal-volume chloroform-primary isoamyl alcohol mixing solutions, and the chloroform in the mixing solutions: the primary isoamyl alcohol volume ratio is 24: 1, slowly turns upside down 30 times, and the centrifugal 10min of 10000rpm gets supernatant 1mL and is added in the 1.5mL centrifuge tube;
(4) add the pomegranate-shaped magnetic nanometer particle congery dispersion liquid 10 μ L that claim 1 method makes, fully mixing leaves standstill 5min, magnetic resolution, and deposition is washed 3 times with absolute ethyl alcohol, naturally volatilization 30min;
(5) add 100 μ LTE damping fluids, 5min is hatched in 37 ℃ of water-baths, and magnetic separates behind the mixing, gets supernatant, is the transgenic soybean DNA elutriant;
Said TE damping fluid consists of: 10mM Tris-HCl, 1mM EDTA Disodium, pH8.0.
5. the application of the prepared pomegranate-shaped magnetic nanometer particle congery of the described preparation method of claim 1 is characterized in that being used for the separation and purification of plant-animal DNA.
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| CN101845435A (en) * | 2010-06-11 | 2010-09-29 | 江南大学 | Method for extracting transgenic soybean DNA by using magnetic nanoparticles |
| CN105039314A (en) * | 2015-08-27 | 2015-11-11 | 开平牵牛生化制药有限公司 | Method for extracting polynucleotide by utilizing superparamagnetic nano microsphere |
| CN113584017A (en) * | 2021-08-24 | 2021-11-02 | 北京化工大学 | Magnetic response DNA separation medium and preparation and use method thereof |
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| CN1736881A (en) * | 2005-07-07 | 2006-02-22 | 华中科技大学 | Super paramagnetic ferric oxide composite nanometre particle preparation method |
| WO2008074804A2 (en) * | 2006-12-18 | 2008-06-26 | Colorobbia Italia S.P.A. | Magnetic nanoparticles for the application in hyperthermia, preparation thereof and use in constructs having a pharmacological application |
| CN101417822A (en) * | 2008-11-24 | 2009-04-29 | 中国科学院长春应用化学研究所 | Method for preparing super paramagnetic mesoporous ferriferrous oxide nano particle |
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| CN1736881A (en) * | 2005-07-07 | 2006-02-22 | 华中科技大学 | Super paramagnetic ferric oxide composite nanometre particle preparation method |
| WO2008074804A2 (en) * | 2006-12-18 | 2008-06-26 | Colorobbia Italia S.P.A. | Magnetic nanoparticles for the application in hyperthermia, preparation thereof and use in constructs having a pharmacological application |
| CN101417822A (en) * | 2008-11-24 | 2009-04-29 | 中国科学院长春应用化学研究所 | Method for preparing super paramagnetic mesoporous ferriferrous oxide nano particle |
| CN101612541A (en) * | 2009-07-17 | 2009-12-30 | 江南大学 | The preparation of polyacrylic acid coated ferroferric oxide magnetic nano-particles and application thereof |
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