CN1284795C - Magnetic nano particle nucleic acid separator, and its preparing method and use - Google Patents
Magnetic nano particle nucleic acid separator, and its preparing method and use Download PDFInfo
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- CN1284795C CN1284795C CN 03142274 CN03142274A CN1284795C CN 1284795 C CN1284795 C CN 1284795C CN 03142274 CN03142274 CN 03142274 CN 03142274 A CN03142274 A CN 03142274A CN 1284795 C CN1284795 C CN 1284795C
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Abstract
The present invention relates to a nucleic acid separator for magnetic nanometer particles, which comprises the following three layers of structures: (1) an inner nuclear layer which is composed of nuclear shell type magnetic nanometer particles; (2) a 3-triethoxysilyl trimethoxysilane intermediate layer coated on the inner nuclear layer; (3) a single-stranded DNA outer shell layer coated on the intermediate layer. The present invention also relates to a method for prparing the nucleic acid separator for magnetic nanometer particles. The nucleic acid separator for magnetic nanometer particles in the present invention can simply and effectively separate DNA or RNA of an object.
Description
Technical field
The present invention relates to a kind of nano biological separator, more particularly, the present invention relates to a kind of magnetic nano-particle separate nucleic acid device.The invention still further relates to the Preparation method and use of this separator.
Background technology
As everyone knows, field of nanometer technology is the most popular current scientific and technical research field, nanotechnology is applied to formed emerge science technology-nanometer biotechnology in the bio-science field, be the science of utilizing nanotechnology research and solving the significant problem in the life science, it is just becoming one of current important advanced scientific research field.
Now, the application prospect of magnetic nano-particle in biological technical field is subjected to people's attention day by day.Magnetic nano-particle is through being often used as the separating medium of biological sample magnetic field separation.Separation method with magnetic nano-particle is easy and simple to handle, required equipment is cheap, and velocity of separation is fast simultaneously, helps keeping the biological activity of sample.At present, utilization more and more widely.
The magnetic nano-particle general requirement that is used for biotechnology has following characteristic: (1) particle has superparamagnetism, is easy to absorption and wash-out; (2) particle needs the bigger specific magnetising moment, to guarantee the sensitivity of separation efficiency; (3) particle surface will be easy to carry out chemically modified, to be connected with drug molecule with different biologies; Require to have better biocompatibility when (4) being used in the body.Nanoparticle separator based on this purpose design is generally sandwich structure, inner nuclear layer is generally magnetic Nano material, the centre is a coating layer, the existing products great majority adopt macromolecular material as coating layer at present, outermost layer is then modified the functional group that specific effect is arranged with biological tissue, to satisfy the requirement of bioseparation.
But the polymer microsphere that separator of the prior art adopts letex polymerization to obtain more has wherein coated the magnetic-particle of nano-scale.Yet the structure of polymer microsphere is more loose usually, and the magnetic particle that wherein comprises breaks away from microballoon easily and can produce agglomeration in long-time preservation process, thereby influences the performance of product; On magnetic nano-particle, fixedly during active substance, come off easily with absorption or affinity interaction.
The invention provides a kind of magnetic nano-particle separate nucleic acid device, overcome the deficiencies in the prior art.
Summary of the invention
The purpose of this invention is to provide a kind of magnetic nano-particle separate nucleic acid device.
Another object of the present invention provides the method for this magnetic nano-particle separate nucleic acid device of preparation.
A further object of the present invention is the separation that this magnetic nano-particle separate nucleic acid device is applied to separate targets DNA or RNA.
In a first aspect of the present invention, a kind of magnetic nano-particle separate nucleic acid device is provided, it contains following three-decker:
(1) inner nuclear layer that constitutes by the core-shell type magnetic nano particle;
(2) MPTS (the 3-sulfydryl propyl trimethoxy silicane) middle layer of coating inner nuclear layer;
(3) the single stranded DNA outer shell in coating middle layer.
In another preference, described core-shell type magnetic nano particle inner nuclear layer comprises kernel and shell.The interior nuclear composition of described inner nuclear layer is selected from: the oxide compound of iron, nickel, nickel-ferro alloy or its combination.Preferably, the interior nuclear composition of described inner nuclear layer is the oxide compound of iron.The outer shell component of described inner nuclear layer is selected from silicon-dioxide, agarose, olefin polymer, polyacrylonitrile, epoxy compounds or its combination.Preferably, the outer shell component of described inner nuclear layer is a silicon-dioxide.
Wherein, described single stranded DNA outer shell is by crossing the single stranded DNA that sulfide linkage is connected with the middle layer.
In a second aspect of the present invention, a kind of method for preparing magnetic nano-particle separate nucleic acid device is provided, it may further comprise the steps:
(1) in the microemulsion system that contains magnetic nano-particle and inner nuclear layer shell formation agent (as tetraethoxy), by the water-in-oil-type reverse microemulsion process, form the core-shell type magnetic nano particle, described core-shell type magnetic nano particle comprises kernel and shell,
Described magnetic nano-particle is selected from: the oxide compound of iron, nickel, nickel-ferro alloy or its combination;
Described inner nuclear layer shell forms agent and is formed into the shell that branch is selected from down group: silicon-dioxide, agarose, olefin polymer, polyacrylonitrile, epoxy compounds or its combination;
(2) use 3-sulfydryl propyl trimethoxy silicane that the core-shell type magnetic nano particle surface of step (1) is modified, obtained modifying the magnetic nano-particle of sulfydryl;
(3) use modified the single stranded DNA of crossing sulfide linkage to the modification of step (2) surface of magnetic nano-particle of sulfydryl modify, form magnetic nano-particle separate nucleic acid device.
Wherein, in the described microemulsion system TritonX-100, n-hexyl alcohol, hexanaphthene in 1: (1～3): the ratio uniform mixing of (4～6).
In a third aspect of the present invention, the purposes of magnetic nano-particle separate nucleic acid device of the present invention is provided, it is used to DNA isolation or RNA.
In a fourth aspect of the present invention, a kind of method of isolating nucleic acid is provided, comprise step: this magnetic nano-particle separate nucleic acid device is joined in the solution that contains target single stranded DNA or RNA, make it at Tris-HCl and MgCl
2Mixing solutions in, under the certain temperature fully after the hybridization, under the guiding of outside magnetic field target single stranded DNA or RNA are separated.
Description of drawings
Fig. 1 is the surface-enhanced Raman effects figure of the magnetic nano-particle (FSM) of having modified sulfydryl, shows that sulfydryl is modified at the surface of magnetic nano-particle, and the sulfydryl that is modified at the magnetic nano particle sub-surface has good Raman reinforcing effect in the substrate of silver.
Fig. 2 is the surface-enhanced Raman effects figure of the magnetic nano-particle (FSMD) that connected single stranded DNA, shows that DNA is connected to the surface of magnetic nano-particle, and Raman reinforcing effect is preferably arranged.
Embodiment
The present inventor is through extensive and deep research, invented in the finishing of magnetic nano-particle and can discern and in conjunction with the material of DNA or RNA, remove to catch target dna or RNA with this magnetic nano-particle then, carry out isolating technology under the effect of outside magnetic field again.
The method for preparing magnetic nano-particle separate nucleic acid device of the present invention may further comprise the steps:
(1) preparation magnetic nano-particle
Prepare FeSO respectively with redistilled water
47H
2O and FeCl
36H
2The mixing solutions of O and NaOH solution.Fe in the mixing solutions of molysite
2+Ionic concentration is 0.1～0.2mol/l, Fe
3+Ionic concentration is 0.1～0.3mol/l, and the concentration of NaOH solution is 2～3mol/l.Under vigorous stirring be that half NaOH solution of mixing salt solution volume is added drop-wise in the mixing salt solution lentamente with volume.At 40 ℃～60 ℃ following ageing 12h, with redistilled water with sediment undergoes washing for several times, dry 24h under 40 ℃～80 ℃ condition again after the filtration promptly gets product after grinding in agate mortar with resulting solid precipitation.
(2) silicon-dioxide is at γ-Fe
2O
3The modification on surface
With TritonX-100, n-hexyl alcohol, hexanaphthene in 1: (1～3): the ratio uniform mixing of (4～6) forms the microemulsion system of transparent and stable.Place ultrasonic wave to handle 30-60 minute above-mentioned microemulsion system, again to the γ-Fe that wherein adds 0.01～1g
2O
3(magnetic nano-particle) takes out upper strata liquid with ultrasonication and pours in the three-necked flask after 3 minutes, stir and made it even in 30～60 minutes.Get the certain density strong aqua of 1ml with the dilution of 2ml redistilled water, it is slowly joined in the microemulsion of continuous stirring, continue stirring and ammoniacal liquor was dispersed in the microemulsion in 10～60 minutes.After 1 hour, in microemulsion, drip the tetraethoxy (the inner nuclear layer shell forms agent) of 1～5ml, constantly stirred 10 hours simultaneously, and the temperature of system is remained between 15～40 ℃.In system, add acetone and make particle precipitation, perhaps the system standing over night is made the particle natural sedimentation, use the ethanol wash particle.Particle after cleaning placed under 300～700 ℃ the condition calcination 1～4 hour, and collected the core-shell type magnetic nano particle.
(3) with 3-sulfydryl propyl trimethoxy silicane (MPTS) modified magnetic nano particles surface
Get in the acetate and alcoholic acid mixed solution of 15mg magnetic nano-particle adding 2～10ml, use ultrasonication 20～60 minutes; Take by weighing 0.01～1g MPTS, use ultrasonication 10～60 minutes; These two kinds of solution are mixed, and reaction is 1 hour under 10～40 ℃ condition, takes out particle then and also cleans 3 times with acetate and alcoholic acid mixed solution, then 100-300 ℃ of vacuum-drying 2 hours, collects particle (FSM).
(4) single stranded DNA is in the modification of magnetic nano particle sub-surface
Get a certain amount of single stranded DNA of crossing sulfide linkage of having modified, join the NaHCO of 500 μ l
3And Na
2CO
3Mixed solution in, after mixing, in the solution of this single stranded DNA, add a small amount of FSM, make it to be uniformly dispersed, and in a humid environment in reacting 12～36 hours (sulfydryl of crossing on sulfide linkage and the middle layer on the DNA reacts, and makes dna molecular be directly connected in the middle layer by crossing sulfide linkage) under 10～50 ℃ the condition.Separate wash particle and vacuum-drying 10 hours under 20～80 ℃ condition, use redistilled water dispersed particle (FSMD) then, obtain magnetic nano-particle separate nucleic acid device.
The length that is applicable to single stranded DNA of the present invention has no particular limits.Usually mean length is about 5～200bp, preferably is about 10～50bp.
The interior nuclear composition of core-shell type magnetic nano particle inner nuclear layer also has the alloy of nickel (Ni) or nickel (Ni) and iron (Fe) etc. except the oxide compound of iron.
The outer shell component of core-shell type magnetic nano particle inner nuclear layer also has organic integuments such as agarose, olefin polymer, polyacrylonitrile, epoxy compounds except inorganic integuments such as silicon-dioxide.For selected outer shell component, can select for use suitable inner nuclear layer shell to form agent according to prior art.For example when outer shell component is silicon-dioxide, can select for use tetraethoxy or other suitable inner nuclear layer shell to form agent.
Major advantage of the present invention is:
(1) magnetic nano-particle of Shi Yonging is a monodispersity, does not produce agglomeration, and the shape of magnetic nano-particle and diameter are easy to control;
(2) single stranded DNA of energy recognition objective DNA or RNA is connected with magnetic nano-particle by chemical bond, and stability is good, difficult drop-off;
(3) compare with traditional electrophoretic method, the inventive method is easy and efficient.
Below in conjunction with specific embodiment, the invention will be further elaborated.Should be understood that and the invention is not restricted to these specific embodiments.
Embodiment 1
The preparation method of the inner nuclear layer of magnetic nano-particle
Adopt improved chemical coprecipitation to prepare the inner nuclear layer of magnetic particle, concrete grammar is as follows: prepare FeSO respectively with redistilled water
47H
2O and FeCl
36H
2The mixing solutions of O and NaOH solution.Fe in the mixing solutions of molysite
2+Ionic concentration is 0.1～0.2mol/l, Fe
3+Ionic concentration is 0.1～0.3mol/l, and the concentration of NaOH solution is 2～3mol/l.Under vigorous stirring be that half NaOH solution of mixing salt solution volume is added drop-wise in the mixing salt solution lentamente with volume.At 40 ℃～60 ℃ ageing 12h, with redistilled water with sediment undergoes washing for several times, dry 24h under 40 ℃～80 ℃ condition again after the filtration promptly gets product after grinding in agate mortar with resulting solid precipitation.
Embodiment 2
The preparation method of hud typed nucleic acid magnetic nano-particle
With TritonX-100, n-hexyl alcohol, hexanaphthene ratio uniform mixing, form the microemulsion system of transparent and stable in 1: 2: 5.Place ultrasonic wave to handle 30～60 minutes above-mentioned microemulsion system, again to the γ-Fe that wherein adds 0.5g
2O
3, take out upper strata liquid after 6 minutes with ultrasonication and pour in the three-necked flask, stir and made it even in 30 minutes.Get the certain density strong aqua of 1ml with the dilution of 2ml redistilled water, after 30 minutes it is slowly joined in the microemulsion of continuous stirring, continue stirring and ammoniacal liquor was dispersed in the microemulsion in 30 minutes.After 1 hour, in microemulsion, drip the tetraethoxy of 1～3ml, constantly stirred 10 hours simultaneously, and the temperature of system is remained between 15～30 ℃.In system, add acetone and make particle precipitation, perhaps the system standing over night is made the particle natural sedimentation, use the ethanol wash particle.Particle after cleaning placed under 400～700 ℃ the condition, particle is collected in calcination 1～4 hour.
Embodiment 3
The preparation of magnetic nano-particle separate nucleic acid device
Get the magnetic nano-particle that makes among the 15mg embodiment 2 and add in the acetate and alcoholic acid mixed solution of 2ml, use ultrasonication 20～60 minutes; Take by weighing 0.01g MPTS, use ultrasonication 10～60 minutes; These two kinds of solution are mixed, and reaction is 1 hour under 10～30 ℃ condition, takes out particle then and also cleans 3 times with acetate and alcoholic acid mixed solution, then 100～300 ℃ of vacuum-dryings 2 hours, collects particle (FSM).As can be seen from Figure 1, sulfydryl is modified the surface of magnetic nano-particle, and good Raman reinforcing effect is arranged.
Get a certain amount of single stranded DNA (mean length 7bp) of crossing sulfide linkage of having modified, join the NaHCO of 500 μ l
3And Na
2CO
3Mixed solution in, after mixing, in the solution of this single stranded DNA, add a small amount of FSM, make it to be uniformly dispersed, and reaction 24 hours under 20～40 ℃ condition in a humid environment.Separate wash particle and vacuum-drying 10 hours under 20～80 ℃ condition, use redistilled water dispersed particle (FSMD) then, obtain magnetic nano-particle separate nucleic acid device.As can be seen from Figure 2, dna probe has been connected to the magnetic nano particle sub-surface, and tangible Raman reinforcing effect is arranged.
Embodiment 4
The preparation of magnetic nano-particle separate nucleic acid device
Get the magnetic nano-particle that 15mg embodiment 2 makes and add in the acetate and alcoholic acid mixed solution of 6ml, with ultrasonication 20-60 minute; Take by weighing 0.5g MPTS, use ultrasonication 10～60 minutes; These two kinds of solution are mixed, and reaction is 1 hour under 10～30 ℃ condition, takes out particle then and also cleans 3 times with acetate and alcoholic acid mixed solution, then 100～300 ℃ of vacuum-dryings 2 hours, collects particle (FSM).
Get a certain amount of single stranded DNA (mean length 50bp) of crossing sulfide linkage of having modified, join the NaHCO of 500 μ l
3And Na
2CO
3Mixed solution in, after mixing, in the solution of this single stranded DNA, add a small amount of FSM, make it to be uniformly dispersed, and reaction 24 hours under 20～40 ℃ condition in a humid environment.Separate wash particle and vacuum-drying 10 hours under 20～80 ℃ condition, use redistilled water dispersed particle (FSMD) then, obtain magnetic nano-particle separate nucleic acid device.
Embodiment 5
The preparation of magnetic nano-particle separate nucleic acid device
Get the magnetic nano-particle that 15mg embodiment 2 makes and add in the acetate and alcoholic acid mixed solution of 10ml, with ultrasonication 20～60 minutes; Take by weighing 1g MPTS, use ultrasonication 10～60 minutes; These two kinds of solution are mixed, and reaction is 1 hour under 10～30 ℃ condition, takes out particle then and also cleans 3 times with acetate and alcoholic acid mixed solution, then 100～300 ℃ of vacuum-dryings 2 hours, collects particle (FSM).
Get a certain amount of single stranded DNA (mean length 95bp) of crossing sulfide linkage of having modified, join the NaHCO of 500 μ l
3And Na
2CO
3Mixed solution in, after mixing, in the solution of this single stranded DNA, add a small amount of FSM, make it to be uniformly dispersed, and reaction 24 hours under 20～40 ℃ condition in a humid environment.Separate wash particle and vacuum-drying 10 hours under 20～80 ℃ condition, use redistilled water dispersed particle (FSMD) then, obtain magnetic nano-particle separate nucleic acid device.
Embodiment 6
Get the magnetic nano-particle separate nucleic acid device that makes among the embodiment 3～5, add in the total RNA aqueous solution of 100 μ l, add sodium citrate buffer solution 20～50 μ l again, mixing is 10～50 ℃ of hybridization down.
Under the effect in external magnetic field, collect magnetic nano-particle, remove clear liquid.Clean magnetic nano-particle three times with sodium citrate buffer solution.Magnetic nano-particle is suspended in water, remove magnetic nano-particle under the outside magnetic field effect, collect water, and under-80 ℃ condition, preserve standby or be used for RT-PCR, cDNA storehouse structure etc.
The result: the hud typed nucleic acid magnetic nano-particle that embodiment 3-5 makes is a monodispersity, can not produce agglomeration, and its shape and diameter are easy to control.In addition, the single stranded DNA of energy recognition objective DNA or RNA is connected with this magnetic nano-particle by chemical bond, and stability is good, difficult drop-off, so separating effect is very good.
Under the prerequisite that does not depart from the spirit and scope of the invention, those skilled in the art can carry out various changes or modification to the present invention, and these forms of equal value drop in the application's appended claims institute restricted portion equally.
Claims (7)
1. magnetic nano-particle separate nucleic acid device is characterized in that it contains following three-decker:
(1) inner nuclear layer that constitutes by the core-shell type magnetic nano particle, wherein, described core-shell type magnetic nano particle inner nuclear layer comprises kernel and shell, the interior nuclear composition of described inner nuclear layer is selected from: the oxide compound of iron, nickel, nickel-ferro alloy or its combination; The outer shell component of described inner nuclear layer is selected from silicon-dioxide, agarose, olefin polymer, polyacrylonitrile, epoxy compounds or its combination;
(2) the 3-sulfydryl propyl trimethoxy silicane middle layer of coating inner nuclear layer;
(3) the single stranded DNA outer shell in coating middle layer.
2. magnetic nano-particle separate nucleic acid device according to claim 1 is characterized in that the interior nuclear composition of described inner nuclear layer is the oxide compound of iron.
3. magnetic nano-particle separate nucleic acid device according to claim 1 is characterized in that the outer shell component of described inner nuclear layer is a silicon-dioxide.
4. magnetic nano-particle separate nucleic acid device according to claim 1 is characterized in that, described single stranded DNA outer shell is by crossing the single stranded DNA that sulfide linkage is connected with the middle layer.
5. method for preparing magnetic nano-particle separate nucleic acid device is characterized in that it may further comprise the steps:
(1) in the microemulsion system that contains magnetic nano-particle and inner nuclear layer shell formation agent, by the water-in-oil-type reverse microemulsion process, form the core-shell type magnetic nano particle, described core-shell type magnetic nano particle comprises kernel and shell,
Described magnetic nano-particle is selected from: the oxide compound of iron, nickel, nickel-ferro alloy or its combination;
Described inner nuclear layer shell forms agent and is formed into the shell that branch is selected from down group: silicon-dioxide, agarose, olefin polymer, polyacrylonitrile, epoxy compounds or its combination;
(2) use 3-sulfydryl propyl trimethoxy silicane that the core-shell type magnetic nano particle surface of step (1) is modified, obtained modifying the magnetic nano-particle of sulfydryl;
(3) use modified the single stranded DNA of crossing sulfide linkage to the modification of step (2) surface of magnetic nano-particle of sulfydryl modify, form magnetic nano-particle separate nucleic acid device.
6. method according to claim 5 is characterized in that, TritonX-100 in the described microemulsion system, n-hexyl alcohol, hexanaphthene are in 1: 1～3: 4～6 ratio uniform mixing.
7. the purposes of magnetic nano-particle separate nucleic acid device as claimed in claim 1 is characterized in that, is used for DNA isolation or RNA.
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