CN101723389B - Method for preparing magnetic silica microspheres with surfaces modified by cations - Google Patents
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- CN101723389B CN101723389B CN2009102190118A CN200910219011A CN101723389B CN 101723389 B CN101723389 B CN 101723389B CN 2009102190118 A CN2009102190118 A CN 2009102190118A CN 200910219011 A CN200910219011 A CN 200910219011A CN 101723389 B CN101723389 B CN 101723389B
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Abstract
The invention relates to a method for preparing magnetic silica microspheres with surfaces modified by cations, and the number of electric charges of the modified cations varies with pH of solution. Ethyl orthosilicate is hydrolyzed to generate the magnetic silica microspheres by a sol-gel method in the presence of Fe3O4 nanoparticles. Epoxy silane reagent of 3-Glycidoxypropyltrimethoxy silane (GPTMS) is adopted to activate magnetic silica microparticles, and the cationic modifying agent with the pKa value within 4 to 8 is used as functional group, therefore, the magnetic silica microspheres with surfaces modified by cations are prepared by covalent interaction. The method comprises the preparation of the magnetic silica microspheres, the epoxidation of the magnetic silica microspheres and the cationic modification on the surfaces of the magnetic silica microspheres. The magnetic microspheres prepared by the method are used for separating nucleic acid from biological samples according to the variation of the electric charges on the surfaces.
Description
Technical field
The invention belongs to the synthetic field of material, relate to a kind of preparation methods that is used for the biological sample isolating nucleic acid, be specifically related to a kind of preparation method of magnetic silica microspheres with surfaces modified by cations.
Background technology
Biological magnetic separation technique is to grow up on the basis of traditional magnetic separation technique, is the high efficient separation technology of application with organisms such as cell, bacterium, nucleic acid and protein.It utilizes under magnetic or the effect of magnetic mark organism outside magnetic field and does this characteristic of orientation movement, to target organism extract, enrichment, separation and purifying.Early stage work mainly concentrates on the technology of preparing that develops the magnetic mark thing, comprises by flooding in magnetic fluid magnetic nanoparticle adsorption in polymer microballoons such as agarose, polyacrylamide, poly-glutaraldehyde and polyvinyladehydes.This microballoon is a feature evenly with all even magnetic particle content of particle size dispersion, has guaranteed the reproducibility and the controllability of different bioseparation processes.Biological magnetic separation technique has been compared certain advantage with the standard separating step.At first the magnetic sepn process is very simple usually, only relates to several treatment steps, and all purification steps all can take place in single test tube or container, does not need expensive liquid chromatographic system, whizzer, strainer or miscellaneous equipment.Sepn process can directly be carried out containing on the rough sample of suspended solid material.In some cases, for example extraction of intracellular protein, even can integrate cracking and separating step, thus shorten whole disengaging time.Organisms such as magnetic separation technique pair cell, protein and nucleic acid normally as mild as a dove.Compare with affinity chromatography with high speed centrifugation, magnetic separation technique has lower shearing force, can keep the vigor of separated cell and the biological activity of biomolecules preferably.
At present, biological magnetic separation technique is becoming an important means of life science.In biology field, nucleic acid is the important topic in modern biology, the medical research.As the carrier of genetic information, DNA is the basic substance of genetic expression, and except that the effect in vital movements such as organism normal growth, growth and breeding, it and life are also closely related as the generation of tumour, genetic diseases etc. unusually.Therefore, to the separate nucleic acid purifying be the important prerequisite and the guarantee of many research fields.
Common separate nucleic acid method has phenol extraction/precipitator method, chromatography, density gradient centrifugation etc. at present.These traditional methods are often more time-consuming, and complex steps needs to use organic reagent, diminishes operator's health.So be necessary to study a kind of new method of extracting genomic dna fast, easily.
Levison use a kind of with the magnetic agarose microbeads as matrix, the magnetic microsphere of modified with diethylaminoethyl (DEAE) is used for nucleic acid extraction.
Sun Minli synthesizes a kind of magnetic glucan-DEAE microballoon by the method for magnetic nano-particle and dextran-DEAE parcel, and is applied to extract plasmid in the big bacillus.
The magnetic microsphere of above-mentioned two kinds of finishing DEAE combines with nucleic acid, mainly is because the DEAE of magnetic microsphere finishing has positive charge in the aqueous solution, can combine with electronegative nucleic acid, thereby reaches the purpose of separation and purification nucleic acid.But DEAE and nucleic acid binding ability are strong, need the salts solution wash-out with high density in elution process.This makes troubles for the application of downstream nucleic acid, because nucleic acid just can be used for downstream application in the salts solution of lower concentration.So behind Levison and Sun Minli the magnetic microsphere extraction nucleic acid, also need adopt the method for ethanol sedimentation to remove the salt of high density, and then the nucleic acid precipitation is dissolved in the buffered soln again with finishing DEAE.Therefore, this nucleic acid purification process wastes time and energy, and causes the loss of nucleic acid in the process of ethanol sedimentation.
Summary of the invention
Purpose of the present invention:
In order to solve the above-mentioned technical problem that exists in the background technology, the invention provides a kind of preparation method of magnetic silica microspheres with surfaces modified by cations, the magnetic silica microspheres with surfaces modified by cations that this method of complying with is prepared does not need the salts solution wash-out of high density when extracting nucleic acid, time saving and energy saving, effectively reduced the loss of nucleic acid.
Solve technical scheme of the present invention:
A kind of preparation method of magnetic silica microspheres with surfaces modified by cations, this method with magnetic silica microballoon as template, as activator, be prepared into magnetic silica microspheres with surfaces modified by cations as modifier with oxirane coupling agent 3-glycidyl ether oxygen base propyl trimethoxy silicane (GPTMS) with positively charged ion; Described magnetic silica microballoon is to be nuclear with the ferroferric oxide nano granules, make the magnetic silica microballoon that the silicon source is a shell with tetraethoxy, it is characterized in that: described cation modified dose is a class positively charged compound in the aqueous solution, and this cation modified dose of pKa value is between 4～8.
Above-mentioned preparation method may further comprise the steps
(1) preparation magnetic silica microballoon;
(2) epoxy groupization of magnetic silica microballoon;
(3) cationic surface is modified:
(a) magnetic silica microballoon of the surface bond epoxy group(ing) that step (2) is made mixes with dilute hydrochloric acid, while stirring reacting by heating;
(b) after reaction finished, magnetic resolution obtained throw out, and throw out is washed with secondary water washing;
(c) cation modified dose is dissolved in the ultrapure water, regulating the pH value is 7～10, adds the throw out after step (3.2) is washed, and stirs reacting by heating;
(d) after reaction finished, magnetic resolution obtained throw out, and throw out is washed with secondary water washing, with after the preprocessing solution washing, put into preprocessing solution again, was prepared into magnetic silica microspheres with surfaces modified by cations.
Above-mentioned cation modified dose is Histidine, poly Histidine, imidazoles or derivatives thereof, 2-(N-morpholino) ethyl sulfonic acid or N, two (2-the hydroxyethyl)-2-aminoethyl sulfonic acid of N-.
The concentration of above-mentioned steps (a) dilute hydrochloric acid is 0.01～0.05mol/L, and effect was best when concentration was 0.01mol/L; The magnetic silica microballoon of surface bond epoxy group(ing) and the mass ratio of dilute hydrochloric acid are 1: 100～500, and this ratio is that 1: 100～300 o'clock effects are better, and this ratio is that 1: 150 o'clock effect is best; Stirring velocity is 600～800 rev/mins, is heated to 80～100 ℃, and the reaction times is 2～4 hours;
Cation modified dose of above-mentioned steps (c) is a class pKa value between 4～8 compound, and stirring velocity is 600～800 rev/mins, is heated to 55～100 ℃, and the reaction times is 4～6 hours;
The composition of above-mentioned steps (d) preprocessing solution is: 10mM MES, 1mM EDTA and 10mMNaCl.
The method of the epoxy groupization of above-mentioned steps (2) magnetic silica microballoon is:
(a) getting magnetic silica microballoon that step (1) makes and toluene is 1: 1000～1100 to be made into mixing solutions by mass ratio, and this ratio is that 1: 1020～1080 o'clock effects are best; With the frequency supersound process of 50～70Hz 10～30 minutes;
(b) be under the nitrogen protection of 2～3mL/min at gas flow rate, add 3-glycidyl ether oxygen base propyl trimethoxy silicane, while dripping with 600～800 rev/mins of stirrings, refluxed 12～24 hours with 90～110 ℃ again, obtain throw out, the mass ratio of the mixing solutions of described 3-glycidyl ether oxygen base propyl trimethoxy silicane and step (a) is 1: 22～26;
(c) with external magnet the throw out that step (b) obtains is separated, after this throw out used dimethyl formamide, acetone, dehydrated alcohol, water washing successively, put into vacuum drying oven, 50～70 ℃ of dryings 12～24 hours are prepared into the magnetic silica microballoon of surface bond epoxy group(ing).
The preparation method of above-mentioned steps (1) magnetic silica microballoon is:
(a) be 1: 12～20 to mix cetyl trimethylammonium bromide and toluene by mass ratio, this ratio is that 1: 12～18 o'clock effects are best; With 800～1000 rev/mins this mixture is stirred;
(b) with FeCl
24H
2O and FeCl
36H
2O is to be dissolved in secondary water at 1: 3 by mass ratio, is mixed with the aqueous solution that concentration is 130～150g/L; This concentration is that 140～150g/L effect is best;
(c) be to be added drop-wise in the mixture of step (a) with the speed of 4～5mL/h the aqueous solution under 2～3mL/min nitrogen protection at gas flow rate, continue to stir 4～6 hours with 600～800 rev/mins while dripping with step (b);
(d) the adding mass concentration is 25% ammoniacal liquor, continue to stir 2～4 hours with 600～800 rev/mins, have ferroferric oxide nano granules to form, the mass ratio of described ammoniacal liquor and cetyl trimethylammonium bromide is 0.1～0.2: 1, and this ratio is that 0.15～0.2: 1 o'clock effect is best;
(e) drip tetraethoxy with the speed of 2～3mL/h again, while dripping with 600～800 rev/mins of stirring reactions, regulate pH value to 8～9, after dropwising 1～2 hour, stop logical nitrogen, stirred 4～5 days with 600～800 rev/mins under the normal temperature and pressure, obtain product, the mass ratio of described tetraethoxy and cetyl trimethylammonium bromide is 1～1.5: 1, and this ratio is that 1.2～1.5: 1 o'clock effect is best;
(f) in product, add ethanol, with 1500～2000 rev/mins centrifugal 30～50 minutes, obtain upper strata liquid and throw out, the mass ratio of described ethanol and cetyl trimethylammonium bromide is 2～2.5: 1, this ratio is that 2.1～2.4: 1 o'clock effect is best;
(g) abandon upper strata liquid, throw out was refluxed 12～24 hours with 70～80 ℃ in ethanol, obtain upper strata bright yellow solution and black precipitate, the mass ratio of described ethanol and cetyl trimethylammonium bromide is 20～25: 1, and this ratio is that 20～24: 1 o'clock effect is best;
(h) abandon the upper strata bright yellow solution, with black precipitate ethanol, water, acetone alternately after the washing, are put into vacuum drying oven, and 50～70 ℃ of dryings 12～24 hours are prepared into magnetic silica microballoon.
Used cation modified dose is a class positively charged compound in the aqueous solution in the aforesaid method, and this cation modified dose of pKa value is between 4～8, and along with the rising of pH value of solution value, cation group can change not electriferous state into by electriferous state.With this type of cationic magnetic silica microballoon of finishing that aforesaid method is prepared, can utilize the electrically charged variation of magnetic microsphere surface institute to extract nucleic acid.The electronegative nucleic acid combination in desirable surface when the magnetic microsphere surface is positively charged, thus separate nucleic acid is come out, change pH value in the solution again, make magnetic microsphere surface neutral, nucleic acid is eluted.
Advantage of the present invention is:
1, adopts the magnetic silica microspheres with surfaces modified by cations of this method preparation, because the positively charged ion of modifying is a class pKa value between 4～8 compound, do not need the salts solution wash-out of high density when extracting nucleic acid with it, thereby just do not need to adopt the method for ethanol sedimentation to remove the salt of high density, time saving and energy saving, can be because of the loss that in the process of ethanol sedimentation, causes nucleic acid yet.
2, to extract the nucleic acid amount big for the magnetic silica microspheres with surfaces modified by cations prepared of this method.
3, this method is reasonable in design, technological feasibility good, and is simple to operate.
Description of drawings
Fig. 1 is the VSM magnetic hysteresis loop of the magnetic silica microballoon of embodiment 1 finishing Histidine.
Fig. 2-a is the electron scanning micrograph of the magnetic silica microballoon of embodiment 1 preparation.
Fig. 2-b is the transmission electron microscope photo of the magnetic silica microballoon of embodiment 1 preparation.
Fig. 2-c is the electron scanning micrograph of magnetic silica microballoon of the finishing Histidine of embodiment 1 preparation.
Fig. 3 is the magnetic silica microballoon XPS photoelectron spectrum figure of the finishing Histidine of embodiment 1 preparation.
Fig. 4 is the chemical shift spectrogram of magnetic silica microballoon XPS photoelectron spectrum N element of the finishing Histidine of embodiment 1 preparation.
Fig. 5 is the full-automatic X-ray diffracting spectrum of magnetic silica microballoon of the finishing Histidine of embodiment 1 preparation.
Embodiment
Below in conjunction with drawings and Examples the present invention is described in further detail, but protection scope of the present invention is not limited to these embodiment.
The magnetic silica microballoon of embodiment 1 preparation finishing Histidine
1. preparation magnetic silica microballoon
Get the 7.2892g cetyl trimethylammonium bromide and 109.3g toluene joins in the four-hole boiling flask, stir with 1000 rev/mins with stirrer, 0.329g FeCl
24H
2O and 0.987g FeCl
36H
2O is dissolved in and is mixed with the aqueous solution that concentration is 140g/L in the 9.4397g secondary water; under 2～3mL/min nitrogen protection, be added drop-wise in the said mixture with 4～5mL/h; 800 rev/mins continue to stir 4～6 hours; adding mass concentration again is 25% ammoniacal liquor 2mL; 800 rev/mins continue to stir 2 hours; in mixture; there is ferroferric oxide nano granules to form; slowly add tetraethoxy 9.79mL, 800 rev/mins of stirring reactions are regulated pH to 8; dripping tetraethoxy with 2～3mL/h finished back 1 hour; stop nitrogen protection, 800 rev/mins of stirrings, normal temperature and pressure aging 5 days down; in product, add ethanol 16.4g; with 2000 rev/mins centrifugal 40 minutes, remove upper strata liquid, 80 ℃ of throw outs refluxed 12 hours with ethanol 164g; remove the upper strata bright yellow solution; black precipitate ethanol; water; acetone is washing alternately, puts into 60 ℃ of dryings of vacuum drying oven 12～24 hours, is prepared into magnetic silica microballoon.
2. the epoxy groupization of magnetic silica microballoon
Get magnetic silica microballoon 0.1g, toluene 105g adds there-necked flask, is the supersound process 10 minutes of 60Hz with the ultrasonic cleaning unit frequency, at 2～3mL/min N
2Under the gas shiled; add 3-glycidyl ether oxygen base propyl trimethoxy silicane 4.2g again; 800 rev/mins of stirrings; 110 ℃ were refluxed 12 hours; with external magnet throw out is separated; after alternately washing 3 times with dimethyl formamide, acetone, dehydrated alcohol, water successively, put into 60 ℃ of dryings of vacuum drying oven 12～24 hours, be prepared into the magnetic silica microballoon of surface bond epoxy group(ing).
3. Histidine finishing
Get the 4.13g Histidine and be dissolved in the 90mL ultrapure water, regulate pH value to 8.0 with the NaOH solid.The magnetic silica microballoon 0.3g that adds the surface bond epoxy group(ing) is heated to 80 ℃ of reactions 4 hours, is prepared into the magnetic silica microballoon of finishing Histidine.
The magnetic silica microballoon of embodiment 2 preparation finishing poly Histidines
1. preparation magnetic silica microballoon
Get the 7.2892g cetyl trimethylammonium bromide and 109.3g toluene joins in the four-hole boiling flask, stir with 1000 rev/mins with stirrer, 0.329g FeCl
24H
2O and 0.987g FeCl
36H
2O is dissolved in and is mixed with the aqueous solution that concentration is 140g/L in the 9.4397g secondary water; under 2～3mL/min nitrogen protection, be added drop-wise in the said mixture with 4～5mL/h; 800 rev/mins continue to stir 4～6 hours; adding mass concentration again is 25% ammoniacal liquor 2mL; 800 rev/mins continue to stir 2 hours; in mixture; there is ferroferric oxide nano granules to form; slowly add tetraethoxy 9.79mL, 800 rev/mins of stirring reactions are regulated pH to 8; dripping tetraethoxy with 2～3mL/h finished back 1 hour; stop nitrogen protection, 800 rev/mins of stirrings, normal temperature and pressure aging 5 days down; in product, add ethanol 16.4g; with 2000 rev/mins centrifugal 40 minutes, remove upper strata liquid, 80 ℃ of throw outs refluxed 12 hours with ethanol 164g; remove the upper strata bright yellow solution; black precipitate ethanol; water; acetone is washing alternately, puts into 60 ℃ of dryings of vacuum drying oven 12～24 hours, is prepared into magnetic silica microballoon.
2. the epoxy groupization of magnetic silica microballoon
Get magnetic silica microballoon 0.1g, toluene 105g adds there-necked flask, is the supersound process 10 minutes of 60Hz with the ultrasonic cleaning unit frequency, at 2～3mL/min N
2Under the gas shiled; add 3-glycidyl ether oxygen base propyl trimethoxy silicane 4.2g again; 800 rev/mins of stirrings; 110 ℃ were refluxed 12 hours; with external magnet throw out is separated; after alternately washing 3 times with dimethyl formamide, acetone, dehydrated alcohol, water successively, put into 60 ℃ of dryings of vacuum drying oven 12～24 hours, be prepared into the magnetic silica microballoon of surface bond epoxy group(ing).
3. poly Histidine finishing
Get 5.6g poly Histidine and be dissolved in the 90mL ultrapure water, regulate pH value to 8.0 with the NaOH solid.Add the magnetic silica microballoon 0.3g of surface bond epoxy group(ing), be heated to 80 ℃ of reactions and spend the night.Be prepared into the magnetic silica microballoon of finishing poly Histidine.
The magnetic silica microballoon of embodiment 3 preparation finishing imidazoles
1. preparation magnetic silica microballoon
Get the 7.2892g cetyl trimethylammonium bromide and 109.3g toluene joins in the four-hole boiling flask, stir with 1000 rev/mins with stirrer, 0.329g FeCl
24H
2O and 0.987g FeCl
36H
2O is dissolved in and is mixed with the aqueous solution that concentration is 140g/L in the 9.4397g secondary water; under 2～3mL/min nitrogen protection, be added drop-wise in the said mixture with 4～5mL/h; 800 rev/mins continue to stir 4～6 hours; adding mass concentration again is 25% ammoniacal liquor 2mL; 800 rev/mins continue to stir 2 hours; in mixture; there is ferroferric oxide nano granules to form; slowly add tetraethoxy 9.79mL, 800 rev/mins of stirring reactions are regulated pH to 8; dripping tetraethoxy with 2～3mL/h finished back 1 hour; stop nitrogen protection, 800 rev/mins of stirrings, normal temperature and pressure aging 5 days down; in product, add ethanol 16.4g; with 2000 rev/mins centrifugal 40 minutes, remove upper strata liquid, 80 ℃ of throw outs refluxed 12 hours with ethanol 164g; remove the upper strata bright yellow solution; black precipitate ethanol; water; acetone is washing alternately, puts into 60 ℃ of dryings of vacuum drying oven 12～24 hours, is prepared into magnetic silica microballoon.
2. the epoxy groupization of magnetic silica microballoon
Get magnetic silica microballoon 0.1g, toluene 105g adds there-necked flask, is the supersound process 10 minutes of 60Hz with the ultrasonic cleaning unit frequency, at 2～3mL/min N
2Under the gas shiled; add 3-glycidyl ether oxygen base propyl trimethoxy silicane 4.2g again; 800 rev/mins of stirrings; 110 ℃ were refluxed 12 hours; with external magnet throw out is separated; after alternately washing 3 times with dimethyl formamide, acetone, dehydrated alcohol, water successively, put into 60 ℃ of dryings of vacuum drying oven 12～24 hours, be prepared into the magnetic silica microballoon of surface bond epoxy group(ing).
3. imidazoles finishing
Get the 3.6g imidazoles and be dissolved in the 90mL ultrapure water, regulate pH value to 8.0 with the NaOH solid.Add the magnetic silica microballoon 0.3g of surface bond epoxy group(ing), be heated to 80 ℃ of reactions and spend the night.Be prepared into the magnetic silica microballoon of finishing imidazoles.
In order to verify beneficial effect of the present invention, with the magnetic silica microballoon of the finishing Histidine of embodiment 1 preparation with VSM hysteresiscope, XPS photoelectron spectrograph, automatically x-ray diffractometer, scanning electronic microscope, transmission electron microscope, elemental analyser, Atomic Absorption Spectroscopy AAS characterize it; Utilize agarose gel electrophoresis to test the performance of the magnetic silica microballoon purified genomic dna of finishing Histidine, various test situation are as follows:
Observe article: the magnetic silica microballoon of finishing Histidine
Laboratory apparatus: the vibrating sample magnetometer model is vsm-9500; The high resolution transmission electron microscopy model is JEM-3010; XPS photoelectron spectrograph model is KRATOS AXIS ULTRA; Full-automatic x-ray diffractometer model is D/Max-3c; The environmental scanning electron microscope model is Quanta 200; Elemental analyser; Atomic absorption spectrophotometry degree meter model is TAS 986 (G).
1, observes
Magnetic silica microballoon is modified front and back observe by transmission electron microscope, scanning electronic microscope using method, observed result is seen Fig. 2-a, Fig. 2-b and Fig. 2-c.By Fig. 2-a, Fig. 2-b as seen, transmission electron microscope observing and sem observation magnetic silica microballoon are class sphere, structural integrity; By Fig. 2-c as seen, the sem observation magnetic silica microballoon still is the class sphere, structural integrity after modifying.
2, test
Press VSM hysteresiscope, XPS photoelectron spectrograph, full-automatic x-ray diffractometer, elemental analyser, Atomic Absorption Spectroscopy AAS, the testing method of electrophoretic analysis is tested surperficial huge legendary turtle metal ionic magnetic silica microballoon.
3, observed result
Magnetic silica microballoon with VSM hysteresiscope test surfaces modification Histidine the results are shown in Figure 1.As seen from Figure 1, the magnetic silica microballoon paramagnetism of finishing Histidine is good, no coercive force, and saturation magnetization is 30.081emu/g.Photoelectron spectrum figure with XPS photoelectron spectrograph test sees Fig. 3, as seen from Figure 3, being combined on the magnetic silica microballoon of Histidine success, the content of each element sees Table 1.
The magnetic silica microballoon XPS photoelectron spectrum constituent content of table 1 finishing Histidine
| Element | Peak area (cts-eV/s) | Sensitive constant | Concentration (%) |
| C1s | 2649.0 | 0.278 | 31.52 |
| Si2p | 2311.1 | 0.328 | 23.57 |
| O1s | 11093.1 | 0.78 | 43.31 |
| N1s | 238.5 | 0.477 | 1.6 |
By table 1 as seen, thus obtained microsphere contains elements such as Si, C, O, N, shows that the magnetic silica microballoon of finishing Histidine contains the corresponding composition of this microballoon of preparation.
The chemical shift spectrogram of the magnetic silica microballoon XPS photoelectron spectrum N element of finishing Histidine is seen Fig. 4.As seen from Figure 4, N element peak illustrates that at 399.4ev the N element exists with the tertiary amine form.
The X-ray diffracting spectrum of measuring the magnetic silica Nano microsphere of finishing Histidine with full-automatic x-ray diffractometer is shown in Fig. 5.As seen from Figure 5, X-ray diffracting spectrum 2 θ locate to have occurred Fe at 18.2 °, 30.613 °, 35.690 °, 43.420 °, 53.949 ° and 57.214 ° etc.
3O
4The strong diffraction peak of feature, SiO has appearred at places such as 22.700 °
2The strong diffraction peak of feature, these diffracted signals and isometric system Fe
3O
4The diffraction peak [JCPDS, 65-3107] of lattice plane such as (111), (220), (311), (400), (422), (511) consistent, SiO has appearred near 22.700 °
2Unformed characteristic diffraction peak.
Test the constituent content of the magnetic silica Nano microsphere of magnetic silica microballoon that obtains and finishing Histidine with elemental analyser.Test result sees Table 2.
The constituent content of the magnetic silica microballoon of table 2 magnetic silica microballoon and finishing Histidine
By table 2 as seen, the magnetic silica microballoon of finishing Histidine is compared with magnetic silica microballoon, and the content of C, H, N significantly raises, and illustrates that magnetic silica microballoon modified by corresponding organic composition.
Claims (9)
1. the preparation method of a magnetic silica microspheres with surfaces modified by cations, this method with magnetic silica microballoon as template, earlier carry out the activation on magnetic silica microballoon surface as activator, be prepared into magnetic silica microspheres with surfaces modified by cations with positively charged ion as modifier again with oxirane coupling agent 3-glycidyl ether oxygen base propyl trimethoxy silicane; Described magnetic silica microballoon is to be nuclear with the ferroferric oxide nano granules, make the magnetic silica microballoon that the silicon source is a shell with tetraethoxy, it is characterized in that: described cation modified dose is a class positively charged compound in the aqueous solution, and this cation modified dose of pKa value is between 4～8; Described cation modified dose is Histidine, poly Histidine, imidazoles, 2-(N-morpholino) ethyl sulfonic acid or N, two (2-the hydroxyethyl)-2-aminoethyl sulfonic acid of N-.
2. the preparation method of magnetic silica microspheres with surfaces modified by cations according to claim 1, it is characterized in that: this method may further comprise the steps
(1) preparation magnetic silica microballoon;
(2) activation on magnetic silica microballoon surface;
(3) cationic surface is modified:
(a) magnetic silica microballoon of the surface bond epoxy group(ing) that step (2) is made mixes with dilute hydrochloric acid, while stirring reacting by heating;
(b) after reaction finished, magnetic resolution obtained throw out, and throw out is washed with secondary water washing;
(c) cation modified dose is dissolved in the ultrapure water, regulating the pH value is 7～10, adds the throw out after step (b) is washed, and stirs reacting by heating;
(d) after reaction finished, magnetic resolution obtained throw out, and throw out is washed with secondary water washing, with after the preprocessing solution washing, put into preprocessing solution again, was prepared into magnetic silica microspheres with surfaces modified by cations; The composition of step (d) preprocessing solution is: 10mM MES, 1mM EDTA and 10mMNaCl.
3. the preparation method of magnetic silica microspheres with surfaces modified by cations according to claim 2 is characterized in that:
The concentration of described step (a) dilute hydrochloric acid is 0.01～0.05mol/L, the magnetic silica microballoon of surface bond epoxy group(ing) and the mass ratio of dilute hydrochloric acid are 1: 100～500, stirring velocity is 600～800 rev/mins, is heated to 80～100 ℃, and the reaction times is 2～4 hours;
Step (c) stirring velocity is 600～2000 rev/mins, is heated to 55～100 ℃, and the reaction times is 4～6 hours;
The composition of step (d) preprocessing solution is: 10mM MES, 1mM EDTA and 10mM NaCl.
4. the preparation method of magnetic silica microspheres with surfaces modified by cations according to claim 2 is characterized in that:
The activatory method on step (2) magnetic silica microballoon surface is:
(a) getting magnetic silica microballoon that step (1) makes and toluene was 1: 1000～1100 to be made into mixing solutions by mass ratio, with the frequency supersound process of 50～70Hz 10～30 minutes;
(b) be under the nitrogen protection of 2～3mL/min at gas flow rate, add 3-glycidyl ether oxygen base propyl trimethoxy silicane, while dripping with 600～800 rev/mins of stirrings, refluxed 12～24 hours with 90～110 ℃ again, obtain throw out, the mass ratio of the mixing solutions of described 3-glycidyl ether oxygen base propyl trimethoxy silicane and step (a) is 1: 22～26;
(c) with external magnet the throw out that step (b) obtains is separated, after this throw out used dimethyl formamide, acetone, dehydrated alcohol, water washing successively, put into vacuum drying oven, 50～70 ℃ of dryings 12～24 hours are prepared into the magnetic silica microballoon of surface bond epoxy group(ing).
5. the preparation method of magnetic silica microspheres with surfaces modified by cations according to claim 2 is characterized in that:
The preparation method of step (1) magnetic silica microballoon is:
(a) be 1: 12～20 to mix cetyl trimethylammonium bromide and toluene by mass ratio, this mixture stirred with 800～1000 rev/mins;
(b) with FeCl
24H
2O and FeCl
36H
2O is to be dissolved in secondary water at 1: 3 by mass ratio, is mixed with the aqueous solution that concentration is 130～150g/L;
(c) be to be added drop-wise in the mixture of step (a) with the speed of 4～5mL/h the aqueous solution under 2～3mL/min nitrogen protection at gas flow rate, continue to stir 4～6 hours with 600～800 rev/mins while dripping with step (b);
(d) the adding mass concentration is 25% ammoniacal liquor, continues to stir 2～4 hours with 600～800 rev/mins, has ferroferric oxide nano granules to form, and the mass ratio of described ammoniacal liquor and cetyl trimethylammonium bromide is 0.1～0.2: 1;
(e) drip tetraethoxy with the speed of 2～3mL/h again, while dripping with 600～800 rev/mins of stirring reactions, regulate pH value to 8～9, after dropwising 1～2 hour, stop logical nitrogen, stirred 4～5 days with 600～800 rev/mins under the normal temperature and pressure, obtain product, the mass ratio of described tetraethoxy and cetyl trimethylammonium bromide is 1～1.5: 1;
(f) in product, add ethanol, with 1500～2000 rev/mins centrifugal 30～50 minutes, obtain upper strata liquid and throw out, the mass ratio of described ethanol and cetyl trimethylammonium bromide is 2～2.5: 1;
(g) abandon upper strata liquid, throw out was refluxed 12～24 hours with 70～80 ℃ in ethanol, obtain upper strata bright yellow solution and black precipitate, the mass ratio of described ethanol and cetyl trimethylammonium bromide is 20～25: 1;
(h) abandon the upper strata bright yellow solution, with black precipitate ethanol, water, acetone alternately after the washing, are put into vacuum drying oven, and 50～70 ℃ of dryings 12～24 hours are prepared into magnetic silica microballoon.
6. the preparation method of magnetic silica microspheres with surfaces modified by cations according to claim 5, it is characterized in that: the mass ratio of step (a) cetyl trimethylammonium bromide and toluene is 1: 12～18; The concentration of aqueous solution that step (b) is mixed with is 140～150g/L; The mass ratio of step (d) ammoniacal liquor and cetyl trimethylammonium bromide is 0.15～0.2: 1; The mass ratio of step (e) tetraethoxy and cetyl trimethylammonium bromide is 1.2～1.5: 1; The mass ratio of step (f) ethanol and cetyl trimethylammonium bromide is 2.1～2.4: 1; The mass ratio of step (g) ethanol and cetyl trimethylammonium bromide is 20～24: 1.
7. the preparation method of magnetic silica microspheres with surfaces modified by cations according to claim 4, it is characterized in that: the mass ratio of described step (a) magnetic silica microballoon and toluene is 1: 1020～1080.
8. the preparation method of magnetic silica microspheres with surfaces modified by cations according to claim 3, it is characterized in that: the magnetic silica microballoon of described step (a) surface bond epoxy group(ing) and the mass ratio of dilute hydrochloric acid are 1: 100～300, and the concentration of this dilute hydrochloric acid is 0.01mol/L.
9. the preparation method of magnetic silica microspheres with surfaces modified by cations according to claim 8, it is characterized in that: the magnetic silica microballoon of described step (a) surface bond epoxy group(ing) and the mass ratio of dilute hydrochloric acid are 1: 150.
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