CN104209087B - A kind of dispersed nano magnetic bead for sample quick pretreatment and preparation method thereof - Google Patents
A kind of dispersed nano magnetic bead for sample quick pretreatment and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of dispersed nano magnetic bead for sample quick pretreatment and preparation method thereof, the chemical constitution of described nano magnetic material is expressed as Fe
3o
4siO
2-AEAPS, described nano magnetic material is the core-shell type multifunctional magnetic Nano material having target transport, single dispersing and specific bond ability concurrently, can enrichment pathogenic microorganism fast and efficiently when carrying out the diagnosis of on-the-spot epidemic, further, can be used for carrying out nucleic acid extraction to obtained cause of disease, material of the present invention is the high-tech product that nanometer technology, Protocols in Molecular Biology and biomedical technology combine.
Description
Technical field
The invention belongs to nano biological medical material field, relate to a kind of dispersed nano magnetic bead for sample quick pretreatment and preparation method thereof.
Background technology
The medium of usual infectious disease pathogens is the mixed systems such as soil, blood, urine, need the sample of collection just can be made to detect for subsequent biological through miscellaneous pre-treatment steps such as constantly centrifugal, extraction, dilutions, the time of this process consumes, constrain the development of pathogen Fast Detection Technique widely, make in numerous biology inspection means soon, be treated as the key determining detection efficiency and qualification result the early stage of sample.In recent years the immune magnetic separation technique risen, the magnetic material identification object utilizing antibody or gene probe to modify, out, substantially increases the efficiency of pre-treatment by target cause of disease concentration and separation from the sample medium of complexity under additional magnetic fields.
Usually, after the separated enrichment out of target cause of disease, through simple process, cause of disease to be measured is released, what carry out at first be also most basic Protocols in Molecular Biology is cause of disease DNA extractive technique, and the DNA extracted, the integrality of its primary structure, directly determines the success or failure of the series of genes engineering research next carried out.Traditional DNA extractive technique is as guanidinium isothiocyanate-phenol-chloroform extraction process, alkali density method, cetab extraction process etc., operating procedure complexity, length consuming time, easily cross pollution, the organic substance remained in DNA solution has inhibitory action to archaeal dna polymerase, in addition, the organic reagent such as phenol, chloroform easily causes environmental pollution, damage operator's health, based on above deficiency, traditional DNA extractive technique is greatly limited in application.The paramagnetic particle method nucleic acid extraction technology of rising in recent years, without any need for toxic solvent, does not need repeatedly centrifugal, is only by nucleic acid in conjunction with based on magnetic bead, just can reach the object extracting genomic DNA.
But paramagnetic particle method nucleic acid extraction technology employing conventional at present is magnetic bead, it is mostly polydispersion magnetic bead, owing to there is inhomogenous size and dimension, therefore in liquid environment, the dispersibility of polydispersion magnetic bead is poor, this inferior position makes their sedimentation time extend, insufficient with solution effects, thus cause the reduction of magnetic bead service efficiency.
Require that limited, the commercially available magnetic bead of higher, traditional polydispersion magnetic bead method for extracting nucleic acid application is mostly the problems such as polydispersion magnetic bead for the Sample pretreatment existed in current infectious disease pathogens Site Detection, the present invention, for the high-tech product combined by nanometer technology, Protocols in Molecular Biology and biomedical technology, provides the dispersed nano magnetic bead that can be applied to immune Magneto separate and nucleic acid extraction.
Summary of the invention
The object of this invention is to provide a kind of dispersed nano magnetic bead for sample quick pretreatment and preparation method thereof.
For achieving the above object, technical scheme of the present invention is as follows: a kind of dispersed nano magnetic bead for sample quick pretreatment, chemical constitution is expressed as Fe
3o
4siO
2-AEAPS.
For a preparation method for the dispersed nano magnetic bead of sample quick pretreatment, comprise the following steps:
(1) solvent-thermal method prepares monodispersed ferriferrous oxide nano sphere;
(2) adopt the St ber method improved, at ferriferrous oxide nano sphere outer cladding silica, obtain monodispersed Fe
3o
4siO
2nanosphere, be specially: take the ferriferrous oxide nano sphere that 0.1g step (1) prepares, adding 20ml deionized water, 80ml absolute ethyl alcohol and 1ml mass fraction wherein is successively the ammoniacal liquor of 28%, ultrasonic mixing in backward mixed liquor adds 1ml ethyl orthosilicate, mechanical agitation 6 hours under room temperature (20 ° of C); After completion of the reaction, by gained solution centrifugal, and use deionized water and ethanol purge 3 times successively, obtained monodispersed coated with silica ferriferrous oxide nano sphere (Fe
3o
4siO
2nanosphere), by products therefrom dried for standby in 60 ° of C baking ovens;
(3) Fe3O that silane coupler aminoethylaminopropyl dimethyl silicone polymer obtains step (2) is used
4siO
2carry out silanization treatment, obtain Fe
3o
4siO
2-AEAPS; Be specially: take the Fe that 0.1g step (2) obtains
3o
4siO
2nanosphere, joins in 20ml dimethyl formamide, obtains solution A after ultrasonic disperse; After the aminoethylaminopropyl dimethyl silicone polymer of 10ml and the dimethyl formamide of 20ml being mixed, add a certain amount of succinic anhydride and make mixed solution pH value maintain 3.9 ~ 4.1, under 60 ° of C, mechanical agitation 3 hours, obtains solution B; Add 20ml deionized water by after solution A, B mixing, under 60 ° of C, continue mechanical agitation after 5 hours, by gained solution centrifugal, and use deionized water and ethanol purge 3 times successively, the coated with silica ferriferrous oxide nano sphere (Fe of obtained monodispersed silanization
3o
4siO
2-AEAPS).
For an application for the dispersed nano magnetic bead of sample quick pretreatment, this is applied as and described dispersed nano magnetic bead is used for pathogenic microorganism immunity Magneto separate and on-the-spot Diagnosis of Infectious Diseases carries out nucleic acid extraction.
Compared with prior art, the invention has the beneficial effects as follows: the dispersed nano magnetic material for immune Magneto separate and nucleic acid extraction disclosed by the invention, there is monodispersity, superparamagnetism and pathoklisis, specifically, magnetic particle of the present invention there is more homogeneous size and shape, specific area is large, be convenient to efficiently with target product coupling; Single dispersing magnetic bead sinking speed in homemade buffer solution is slow, is convenient to magnetic bead and fully contacts with nucleic acid, increases extraction efficiency; Nano ferriferrous oxide has superparamagnetism, is separated very simple under the effect of externally-applied magnetic field, makes it be convenient to very much be separated; Outer field nano silicon has nontoxicity, good water-soluble, good biocompatibility, after silylation modification, its rich surface is containing a large amount of functional group, be convenient to conjugated biological molecules, especially to nucleic acid, there is special affinity under certain condition, when external condition changes, nanometer magnetic bead can discharge again the nucleic acid being adsorbed on its surface, the nucleic acid fragment extracted is large, purity is high, steady quality is reliable, nanometer magnetic bead preparation process of the present invention is simple, method environmental protection, cost price are cheap, is applicable to large-scale production.
Accompanying drawing explanation
Fig. 1 is used for the dispersed nano magnetic bead structural characterization figure of immune Magneto separate and nucleic acid extraction;
Fig. 2 is used for the real-time fluorescence quantitative PCR result figure of the dispersed nano Beads enrichment cause of disease sample of immune Magneto separate;
Fig. 3 is used for the real-time fluorescence quantitative PCR result figure of the dispersed nano magnetic bead extraction cause of disease sample DNA of nucleic acid extraction.
Detailed description of the invention
Dispersed nano magnetic bead has more homogeneous size and shape, has good dispersibility in certain medium, has larger specific area, effectively can shorten the sedimentation time of magnetic material, above advantage uses paramagnetic particle method to carry out required for nucleic acid extraction work just.
Based on above principle, the invention provides a kind of dispersed nano magnetic bead for sample quick pretreatment, the chemical constitution of this dispersed nano magnetic bead is expressed as Fe
3o
4siO
2-AEAPS.
Embodiment 1, the preparation of dispersed nano magnetic bead.
(1) solvent-thermal method is adopted to prepare monodispersed ferriferrous oxide nano sphere, concrete preparation process is as follows: take 2.0g sodium acetate and 0.25g ferric chloride hexahydrate respectively, join in 50ml ethylene glycol solution, system was transferred in reactor after 1 hour by magnetic agitation, reacted 10 hours under 100 ° of C.After completion of the reaction, by centrifugal for gained dark solution, and use deionized water and ethanol purge 3 times successively, obtained monodispersed ferriferrous oxide nano sphere (Fe
3o
4), by products therefrom dried for standby in 60 ° of C baking ovens; Monodispersed ferriferrous oxide nano sphere ESEM characterization result as shown in Figure 1a, magnetic Nano material prepared is as can be seen from Figure nanosphere structure, rough surface (piled up by small-particle and form), there is nanometer monodispersity, be conducive to material dispersed in the solution, particle diameter, at about 300nm, does not find agglomeration, this magnetic nano-balls also demonstrating preparation has superparamagnetism, i.e. good Magneto separate ability;
(2) adopt the St ber legal system improved for monodispersed coated with silica ferriferrous oxide nano sphere, concrete preparation process is as follows: take the ferriferrous oxide nano sphere that 0.1g step (1) prepares, adding 20ml deionized water, 80ml absolute ethyl alcohol and 1ml mass fraction wherein is successively the ammoniacal liquor of 28%, ultrasonic mixing in backward mixed liquor adds 1ml ethyl orthosilicate, mechanical agitation 6 hours under room temperature.After completion of the reaction, by centrifugal for gained dark solution, and use deionized water and ethanol purge 3 times successively, obtained monodispersed coated with silica ferriferrous oxide nano sphere (Fe
3o
4siO
2), by products therefrom dried for standby in 60 ° of C baking ovens; Monodispersed coated with silica ferriferrous oxide nano sphere ESEM characterization result as shown in Figure 1 b, silicon matrix layer is coated on the surface of magnetic nano-balls equably as can be seen from Figure, form the composite of nucleocapsid structure, smooth surface, very well dispersed, particle diameter, at about 320nm, illustrates that coated silicon matrix layer shell is about about 20nm; After using silane coupler aminoethylaminopropyl dimethyl silicone polymer to carry out silanization treatment to coated with silica ferriferrous oxide nano sphere, material morphology does not change.
(3) Fe that 0.1g step (2) obtains is taken
3o
4siO
2nanosphere, joins in 20ml dimethyl formamide, obtains solution A after ultrasonic disperse; After the aminoethylaminopropyl dimethyl silicone polymer of 10ml and the dimethyl formamide of 20ml being mixed, add a certain amount of succinic anhydride and make mixed solution pH value maintain 3.9 ~ 4.1, under 60 ° of C, mechanical agitation 3 hours, obtains solution B; 20ml deionized water is added by after solution A, B mixing, mechanical agitation is continued after 5 hours under 60 ° of C, by centrifugal for gained dark solution, and use deionized water and ethanol purge 3 times successively, the coated with silica ferriferrous oxide nano sphere (Fe of obtained monodispersed silanization
3o
4siO
2-AEAPS), products therefrom is dry in 60 ° of C baking ovens.
Embodiment 2, dispersed nano magnetic bead embodiment 1 prepared is applied to pathogenic microorganism immunity Magneto separate, comprises the following steps:
(1) preparation simulation cause of disease sample solution, complex dielectrics dilution.
Simulation cause of disease sample solution: 20ul enterovirus EV 71 (10MOI) is joined in 20mlPBS buffer solution, mixes;
Complex dielectrics dilution: get normal person's fecal sample (solid is as pea size) in Biohazard Safety Equipment, be dissolved in 20mlPBS buffer solution, make to mix with turbula shaker concuss;
(2) with the coated Fe of antibody
3o
4siO
2-AEAPS dispersed nano magnetic bead: monoclonal antibody and the 0.1g dispersed nano magnetic bead at room temperature night incubation of getting 40 μ l (100 μ g/ml) EV71 Structural protein VP1, in order to eliminate the non-specific adsorption of magnetic bead surfaces to EV71, by the magnetic bead of hatching and BSA(bovine serum albumin(BSA)) night incubation under 4 ° of environment; Fe
3o
4siO
2-AEAPS dispersed nano magnetic bead, can specific recognition target pathogenic microorganism after antibody is coated.
(3) 100ml small beaker is got, the coated Fe of antibody that two kinds of solution step (1) prepared mix afterwards and prepared by step (2)
3o
4siO
2-AEAPS dispersed nano magnetic bead mixes, at room temperature jointly hatch 0.5 hour, cause of disease in complex dielectrics dilution is fully combined with the disease-resistant former monoclonal antibody of magnetic bead surfaces, then under magnetic fields, the magnetic bead in beaker is all separated, so far complete the immune Magneto separate process of a separation and concentration pathogenic microorganism sample from complex dielectrics.The Fe of antigen will be combined with
3o
4siO
2-AEAPS dispersed nano magnetic bead is transferred in 100ml acidic buffer, release surface antigen, extracts cause of disease nucleic acid investigate immune Magneto separate effect finally by commercial nucleic acid extraction (DNA/RNA) kit.
Embodiment 3, dispersed nano magnetic bead is applied to the real-time fluorescence quantitative PCR result of pathogenic microorganism immunity Magneto separate as shown in Figure 2, nanometer magnetic bead through specific antibody modify after can with target antigen specific binding, the antigenantibody complex obtained after concentration and separation, using fluorescence quantitative PCR detection.In order to avoid error as much as possible, we have done three parallel sampleses and a negative control, as seen from the figure, curve 4 is negative control, its CT value is 26.62, and curve 1,2,3 is three parallel sampleses, and its CT value is respectively 14.21,14.64,15.02, illustrate that the antigen concentration captured by immune Magneto separate is higher, separating effect is desirable.
Embodiment 3, dispersed nano magnetic bead embodiment 1 prepared can be applicable to on-the-spot Diagnosis of Infectious Diseases and carries out nucleic acid extraction.
Concrete implementation step is as follows:
(1) get fresh shigella dysenteriae nutrient solution 1mL and be placed in 1.5mLEP pipe, 12000rpm1min is centrifugal, collects thalline, adds 200 μ L thalline re-suspension liquid, lashes mixing or vibration mixing, makes thalline resuspended;
(2) in step (1) EP pipe, add 220 μ L lysates, 56 ° of C water-baths, hatch 10min;
(3) add 200 μ L nucleic acid precipitated liquid in the EP pipe after hatching to step (2), lash mixing or vibration mixing, room temperature leaves standstill 3min;
(4) 30ul bead suspension (10mg dispersed nano magnetic bead ultrasonic disperse is in 1ml ultra-pure water) is added in the EP pipe after leaving standstill to step (3), lash mixing, EP pipe after step (3) being left standstill is placed on magnetic frame, leaves standstill 30s, when magnetic bead adsorbs completely, carefully removes liquid;
(5) step (4) EP pipe is taken off from magnetic frame, add 500 μ L cleaning solutions 1, after lashing mixing or vibration mixing, EP pipe is placed on magnetic frame, leaves standstill 30s, when magnetic bead adsorbs completely, carefully remove liquid, again EP pipe is taken off from magnetic frame, add 1000 μ L cleaning solutions 2, after lashing mixing or vibration mixing, EP pipe is placed on magnetic frame, leaves standstill 30s, when magnetic bead adsorbs completely, carefully remove liquid, room temperature leaves standstill 10min;
(6) step (5) EP pipe is taken off from magnetic frame, add 50 μ L eluents, after lashing mixing or vibration mixing, EP pipe is placed in 56 ° of C water-baths, hatch 5min, after taking-up, EP pipe is placed on magnetic frame and leaves standstill 30s, carefully DNA solution is transferred in collecting pipe when magnetic bead adsorbs completely, is placed in-20 ° of C and saves backup.
Dispersed nano magnetic bead can be applicable to on-the-spot Diagnosis of Infectious Diseases and carries out the real-time fluorescence quantitative PCR result of nucleic acid extraction as shown in Figure 3.In order to avoid error as much as possible, we have done three parallel sampleses and a negative control, as seen from the figure, curve 4 is negative control, its CT value is 26.28, and curve 1,2,3 is three parallel sampleses, and its CT value is respectively 6.22,6.72,6.92, sample CT value less explanation template DNA concentration is larger, and CT value little is like this that polydispersion magnetic bead is very inaccessible.RT-PCR result shows: first is very high for the dispersed nano magnetic bead extraction efficiency of nucleic acid extraction, and the second sample DNA purity extracted is high, there is not suppression to fluorescent quantitation.
Claims (2)
1., for a preparation method for the dispersed nano magnetic bead of sample quick pretreatment, the chemical constitution of described dispersed nano magnetic bead is expressed as Fe
3o
4siO
2-AEAPS, described sample quick pretreatment is that pathogenic microorganism immunity Magneto separate or Diagnosis of Infectious Diseases carry out nucleic acid extraction; It is characterized in that, the method comprises the following steps:
(1) solvent-thermal method prepares monodispersed ferriferrous oxide nano sphere;
(2) adopt the St ber method improved, at ferriferrous oxide nano sphere outer cladding silica, obtain monodispersed Fe
3o
4siO
2nanosphere, be specially: take the ferriferrous oxide nano sphere that 0.1g step (1) prepares, adding 20mL deionized water, 80mL absolute ethyl alcohol and 1mL mass fraction wherein is successively the ammoniacal liquor of 28%, ultrasonic mixing in backward mixed liquor adds 1mL ethyl orthosilicate, mechanical agitation 6 hours under room temperature; After completion of the reaction, by gained solution centrifugal, and use deionized water and ethanol purge 3 times successively, obtained monodispersed coated with silica ferriferrous oxide nano sphere, by products therefrom dried for standby in 60 ° of C baking ovens;
(3) Fe that silane coupler aminoethylaminopropyl dimethyl silicone polymer obtains step (2) is used
3o
4siO
2carry out silanization treatment, obtain Fe
3o
4siO
2-AEAPS; Be specially: take the Fe that 0.1g step (2) obtains
3o
4siO
2nanosphere, joins in 20mL dimethyl formamide, obtains solution A after ultrasonic disperse; After the aminoethylaminopropyl dimethyl silicone polymer of 10mL and the dimethyl formamide of 20mL being mixed, add succinic anhydride and regulate mixed solution pH value to 3.9 ~ 4.1, under 60 ° of C, mechanical agitation 3 hours, obtains solution B; Add 20mL deionized water by after solution A, B mixing, under 60 ° of C, continue mechanical agitation after 5 hours, by gained solution centrifugal, and use deionized water and ethanol purge 3 times successively, the coated with silica ferriferrous oxide nano sphere of obtained monodispersed silanization.
2. the application of dispersed nano magnetic bead prepared of method described in claim 1, is characterized in that, this is applied as and described dispersed nano magnetic bead is used for pathogenic microorganism immunity Magneto separate and on-the-spot Diagnosis of Infectious Diseases carries out nucleic acid extraction.
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