CN100593545C - Preparation method for immune nano magnetic glucan micro-sphere - Google Patents
Preparation method for immune nano magnetic glucan micro-sphere Download PDFInfo
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Abstract
The present invention relates to a preparation method of immune nano-magnetic dextran microspheres, which belongs to the technical field of biological separation. In the process steps, nano-size magnetic iron oxide particles are prepared by the method of coprecipitation; a solution is prepared with ferrous ion, iron ion and nickel ion according to the molar ratio of 1:(1.0 to 4.0):(0.2 to 0.8); glucan enwraps the magnetic iron oxide particles; the proteins of the nano-magnetic dextran microspheres are linked. The present invention has the advantages of realization of the narrow diameter of particles, good reproducibility, low price and natural activity of the immune nano-magnetic dextran microspheres.
Description
Technical field
The invention belongs to the bioseparation technology field, particularly provide a kind of biomagnetism to separate short-cut method, be applicable to the separation of various biology aspect.
Background technology
The present invention relates to the preparation method of dextran nano-magnetic microsphere, the magnetic microsphere particle diameter is mainly used in nucleic acid about 50-100nm, protein, cell, the application of immunology test aspect.
Dextran (dextran), have another name called dextran, it is a class polysaccharide with wire main chain, mainly again 1, the 6-a-D-pyranoside links together, itself and degraded product in vivo (CO2, H2O) thereof are nontoxic to human body, use the history of existing five more than ten years clinically as the blood expander, in addition.Dextran molecule can both stable existence (JANES KA under weak acid or weakly alkaline environment, CALVO P, ALONSO MJ, Polusaccharide colloidal particles as delivery systems for macromolecules[J] .Adv Drug Deliv Rev, 2001,47 (1): 83-97.), contain a large amount of " OH " in the structure, can be connected closely with some macromole formation.Between recent two decades, it is found that dextran and some macromole are coupled at together after, shown some beyond thought effects.As being connected with some drugs or protein, enzyme etc., can strengthen medicine or protein biologically stable in vivo, prolong half-life, and reducing its immunogenicity. dextran is the natural polysaecharides chromatography media, just occurred as far back as mid-term in 20th century, it has the numerous characteristics of perfect medium: high-hydrophilic, particle diameter be little, contain more activated hydroxyl, with biomacromolecule non-specific adsorption does not take place, and is one of medium of most widely used up to now a kind of chromatography, hydrophobic and ion-exchange chromatography.In recent years, nucleic acid, cell, albumen, the immunology test plays an important role with purifying in the biology real work.Along with the development of recombinant DNA technology and cell-fusion techniques, the mass production of various biologically active substances such as cell, enzyme, antibody and nucleic acid etc. becomes possibility.The value added of these biologically active substances is high, but content is lower in stock liquid.Therefore, low-cost, high-level efficiency purifying purpose product reaches effectively removes objectionable impurities, and reaching a highly purified separated product is the target that these biologic separation and purification processes are pursued.Separating from sample liquid, purifying in detecting various nucleic acid DNAs, RNA, protein and cell etc. is important link in life science and the clinical medicine.Separation in the actual sample liquid, purifying all account for quite great proportion in time with on the funds, even account for more than 80%.Separation method commonly used at present has the precipitator method, centrifuging, and ion exchange method, various chromatographys etc., this is that some method needs relatively more expensive plant and instrument, although the simple operation of some method, inferior separating effect; Some method complicated operation is time-consuming, and they are greatly limited on using.Modern is to realize automatization, miniaturization to demands of sample device; Do not use or use less as far as possible toxic reagent; Speed is fast, efficient; Characteristics such as product is suitable for follow-up biological operation, and cost is low.Beginning the eighties in 20th century has the scholar magnetically stabilized fluidized bed to be used for the separation of biologic.Magnetically stabilized fluidized bed is that a kind of advantage of integrated part of sepn process, velocity of separation bed, fixed bed and magnetic separation technique rolls into one, both can reduce unit operation steps such as centrifugal or filtration, velocity of separation is fast, operational condition is gentle, process efficiency is high, its lytic activity yield height, reduce the cost of separation and purification, has tempting DEVELOPMENT PROSPECT in the bioseparation.The core of magnetically stabilized fluidized bed is the magnetic resolution medium, is applicable to that therefore the synthetic of magnetic resolution medium of magnetically stabilized fluidized bed is the key that the magnetically stabilized fluidized bed operation realizes.
But up to the present commercial product mainly concentrates on external product, though performance is relatively good, but cost an arm and a leg, allow some research institutions and medium-sized and small enterprises hang back, and the magnetic microsphere particle diameter mainly concentrates on micron order, particle diameter is big, the organic layer of the quilt that wraps of most of magnetic microsphere of while, some bag is still formed owing to physical adsorption or by simple chemical bond connection by the material of some carbohydrates, easily come off, and grain is wide through distributing, inhomogeneous (Stefan Miltenyi.:High Gradient Magnetic Cell Separation With MACS.JCytometry 11:231-238 (1990), be restricted for some separation like this, simultaneously the albumen bag by process in the process, usually use cyanogen bromide, yet cyanogen bromide has severe toxicity, aglucon on the matrix after the cyanogen bromide-activated usually has leakage phenomenon (Clonis YD in addition, Small DAP.High-performance dye-ligandChromatograph.In Reactive in Protein and EnzymeTechnology (M) .London:Macmillan Press, 1987.87-100).
Therefore an object of the present invention is to have prepared and a kind ofly make in the blocky natural active matter dextran of ferric oxide coated outside one deck and be connected with immune magnetic microsphere by adding a kind of linking agent, particle diameter is little, narrow diameter distribution, has natural biological activity, highly versatile, cheap, coupling agent is nontoxic chemical coupling agent, and the aglucon of reactivity mechanism is perfect.
Summary of the invention
The invention reside in the preparation method that a kind of immune nano magnetic glucan micro-sphere is provided, realized that particle diameter is narrow, good reproducibility, cheap, make immune nano dextran magnetic microsphere have natural radioactivity.
Processing step of the present invention is as follows:
1, coprecipitation method prepares nano level magnetic iron oxide particles 0.05-0.6mol/L, and affiliated ferric oxide is Fe
3O
4Or
YFe
2O
3
2, Fe
2+Ion: Fe
3+Ion: Ni
2+Ion is 1: (1.0-4.0): (0.2-0.8) the mol ratio wiring solution-forming 1; put into the confined reaction bottle; be evacuated; feeding nitrogen protects; temperature is warming up to 30-60 ℃; by above-mentioned solion 1: sodium hydroxide (0.5-5mol/L) solution is 1: (0.2-1) volume ratio is added drop-wise to sodium hydroxide solution in the above-mentioned solion 1 with 4-20 ml/min speed; under 500-3000 rev/min of stirring, react; after sodium hydroxide solution drips; temperature is elevated to 60-100 ℃, reacts after 30-120 minute, uses washed with de-ionized water 3-6 time; be kept in the middle of the 10-50mM phosphate buffered saline buffer 2-8 ℃ of preservation.
3, dextran coated magnetic iron oxide particles
(1) activation of dextran: the acetate solution 2 of 0.5-3g/ml dextran is under 100-500 rev/min of stirring, by dextran acetum 2: sodium periodate (0.05-0.3mol/L) solution is with 1: (0.08-0.5) volume ratio is added sodium periodate solution in dextran solution 2, mix, 20-25 ℃ of following lucifuge reacted 30-70 minute, and reaction finishes the back and dialysed 20-24 hour in 2-8 ℃ of deionized water; The dextran of oxidation is kept in the deionized water 2-8 ℃ of preservation.
(2) nano magnetic glucan micro-sphere: activatory 50% dextran of 1 volume and 3-8 volume 50% non-activated dextran are mixed wiring solution-forming 3, press dextran solution 3: ((30-80mg/ml) phosphate solution is with 1: 1 mixed of volume ratio for ferric oxide, 100-500 rev/min of stirring, 2-8 ℃ was reacted 24-50 hour, press dextran solution 3: ferric oxide (30-80mg/ml) phosphate solution is with volume ratio 1: mixed (0.04-0.25), mixing, 2-8 ℃ was reacted 60-150 minute; With above-mentioned solution in phosphate buffered saline buffer 2-8 ℃ the dialysis 20-24 hour; Be kept in the phosphate buffer 4 ℃ of storages.
4, the proteic connection of nano magnetic glucan micro-sphere
(1) activation of nano magnetic glucan micro-sphere: nanometer dextran magnetic microsphere is drained, be mixed with solution in the sodium hydroxide solution of 0.1-0.5g/ml nanometer dextran magnetic microsphere, mix, press nanometer dextran magnetic microsphere solution: epoxy chloropropane is with 1: the 0.08-0.4 volume ratio is mixed, 20-25 ℃ was reacted acetone, deionized water rinse 3-5 time 6-10 hour; Be kept in the middle of the phosphate buffered saline buffer 2-8 ℃ of preservation.
(2) proteinic connection: 30-100mg/ml nano magnetic glucan micro-sphere and 0.5-2mg/ml protein mass are than 40-60: 1 is connected, 20-25 ℃ was reacted 2-4 hour down, separated on magnetic sheet then, with phosphate buffered saline buffer washing 5 times, phosphate buffered saline buffer is preserved, 2-8 ℃ of storage.
The present invention has used following reagent to be:
1. dextran can be following reagent: dextran T20, Dextran T 40, dextran T100 (T represents the dextran of different model particle diameter);
2. ferric oxide can be Fe
3O
4Or
YFe
2O
3,
3. phosphate solution concentration is 10-50mM, and the pH value is 7.0-7.5
4. the acetate strength of solution is 0.01-0.3M, and the pH value is 5.0-6.0
The present invention has the following advantages:
1. the at first synthetic a kind of nano-scale magnetic ferric oxide of present method, and adding a kind of nickel ion, to improve ferric oxide spherical in shape, particle diameter is littler, and particle size range is narrow, and magnetic is stronger.
Present method adopt chemical process ferric oxide outside bread by one deck have the dextran of natural radioactivity, have natural radioactivity, solid, firm.
3. present method has adopted a kind of hypotoxicity, and long-armed epoxy chloropropane carries out the activated dextran magnetic microsphere, has reduced the pollution to environment, has improved operability.
4. present method provides the universal method that a kind of albumen connects, the convenience that has reduced the error rate in the reagent use like this and used.
5. the foundation of present method can provide a kind of for the separation of biology aspect, and particle diameter is little, and narrow diameter distribution has natural biological activity, highly versatile, cheap reagent.
It is as follows to the influence of immune nano magnetic glucan micro-sphere to change different technical parameters
The constant rate of iron ion and ferrous ion changes the concentration of iron ion and ferrous ion, to the influence of the magnetic responsiveness of magnetic glucan complex microsphere:
Constant other conditions are constant, the constant rate of iron ion and ferrous ion, the concentration of change iron ion and ferrous ion, preparation magnetic glucan complex microsphere, on the vibrations sample magnetometer (VSM) of TM-VSM2050HGC, measure, measure magnetic responsiveness, see table one for details
The constant rate of table one iron ion and ferrous ion changes the concentration of iron ion and ferrous ion, to the influence of the magnetic responsiveness of magnetic glucan complex microsphere
| Iron ion/ferrous ion ratio | 0.06 | 0.2 | 0.3 |
| Magnetic correspondence emu/g | 20 | 35 | 50 |
The ratio of activatory dextran and non-activated dextran is to the influence of magnetic glucan micro-sphere particle diameter:
Constant other conditions, the ratio of change activatory dextran and non-activated dextran, the nano magnetic glucan micro-sphere of preparation.Transmissioning electric mirror determining nano magnetic glucan micro-sphere particle diameter with HIACHI H-600STEM sees following table two for details
Table two changes the influence of the ratio of activatory dextran and non-activated dextran to the magnetic glucan micro-sphere particle diameter
| Activatory dextran/non-activated dextran | 1/3 | 1/4 | 1/5 | 1/6 |
| Particle diameter/nm | 30.5 | 55.2 | 80.8 | 99.9 |
Proteinic consumption is to the influence of 100mg nano magnetic glucan micro-sphere in conjunction with conjugated protein amount:
Constant other conditions change proteinic consumption, and the preparation magnetic glucan micro-sphere is measured the supernatant protein concentration then, sees table three for details.
The consumption of table protein iii is tested supernatant liquor to the influence of the protein-bonded amount of 100mg nano magnetic glucan micro-sphere on KLB ULTROSPEC II type ultraviolet-visible pectrophotometer, calculate the amount of magnetic microsphere conjugated protein thus, sees table three for details.
| Protein mg | 1 | 1.5 | 2 | 2.5 |
| The amount mg/100mg of magnetic microsphere conjugated protein | 0.97 | 1.43 | 1.90 | 2.1 |
Embodiment
30 milliliters of mixing liquids of the ferric sulfate of embodiment 1:2g and 0.5g iron protochloride, and add 0.1 the gram single nickel salt, be heated to 50 ℃ then, 2000 rev/mins of speed stir the sodium hydroxide solution that drips 10 milliliters of 0.6mol/L down with 4-20 ml/min speed, after sodium hydroxide dropwises, temperature is elevated to 80 ℃, reacts 1 hour; With washed with de-ionized water 4 times.Be kept in the middle of the 20mmol/L phosphate buffered saline buffer 4 ℃ of preservations.
Embodiment 2:30g dextran topic 40 joins dissolving and mixing in 30 milliliters of 0.1M acetate buffers; The 0.1mol/L sodium periodate solution of adding 9ml, 25 ℃ of lucifuges were reacted 1 hour, used in the deionized water under 4 ℃ of conditions after reaction finishes and dialysed 20 hours; The dextran of oxidation is kept in the 30ml deionized water, in 30 milliliters of 40mg/ml ferric oxide 20mmol/ml phosphate buffered saline buffers, slowly splash into the 50% activatory dextran of 1 volume of 30ml and 50% non-activated dextran mixture of 5 volumes, 500 change stirring, 4 ℃ were reacted 48 hours, the sodium borohydride solution that adds the 4mg/ml of 3ml, mixing, 4 ℃ were reacted 2 hours.With above-mentioned solution with 4 ℃ of dialysis in the 0.15M phosphate buffered saline buffer 20 hours.Be kept in the phosphate buffer 4 ℃ of storages.
Embodiment 3: 2g nanometer dextran magnetic microsphere is drained, the 0.8mol/L sodium hydroxide solution that adds 10ml, mixing, add the 1.5g epoxy chloropropane, mixing, 25 ℃ were reacted 8 hours, and acetone, deionized water rinse 5 times are diluted to 80mg/ml with the 25ml phosphate buffered saline buffer with nano magnetic glucan micro-sphere; The 0.5mg/m l protein soln that adds 0.04g, 40 ℃ were reacted 8 hours, separate on the magnetic sheet, phosphate buffered saline buffer washing 5 times, phosphate buffered saline buffer is preserved, 4 ℃ of storages.
Claims (4)
1, a kind of preparation method of immune nano magnetic glucan micro-sphere, processing step is:
(1) coprecipitation method prepares nano level magnetic iron oxide particles 0.05-0.6mol/L; Fe
2+: Fe
3+: Ni
2+Be 1: (1.0-4.0): (0.2-0.8) mol ratio is made into solion, put into the confined reaction bottle, be evacuated, feeding nitrogen protects, temperature is warming up to 30-60 ℃, by above-mentioned solion: sodium hydroxide solution is 1: the 0.2-1 volume ratio is added drop-wise to sodium hydroxide solution in the above-mentioned solion with 4-20 ml/min speed, under 500-3000 rev/min of stirring, react, after sodium hydroxide dropwised, temperature was elevated to 60-100 ℃, reacted 30-120 minute, with washed with de-ionized water 3-6 time, be kept in the middle of the 20-50mM phosphate buffered saline buffer, 2-8 ℃ of preservation obtains the ferric oxide phosphate solution;
(2) dextran coated magnetic iron oxide particles
The activation of a, dextran: the acetate solution of 0.5-3g/ml dextran is under 100-500 rev/min of stirring, by the dextran acetum: sodium periodate solution is with 1: the 0.08-0.5 volume ratio is added sodium periodate solution in dextran solution, mix, 20-25 ℃ of following lucifuge reacted 30-70 minute, and reaction finishes the back and dialysed 20-24 hour in 2-8 ℃ of deionized water; The dextran of oxidation is kept in the deionized water 2-8 ℃ of preservation;
B, nano magnetic glucan micro-sphere: activatory 50% dextran of 1 volume and 3-8 volume 50% non-activated dextran are mixed wiring solution-forming, by dextran solution: the ferric oxide phosphate solution that step (1) obtains is with 1: 1 mixed of volume ratio, 100-500 rev/min of stirring, 2-8 ℃ the reaction 24-50 hour, with above-mentioned solution in phosphate buffered saline buffer 2-8 ℃ the dialysis 20-24 hour; Be kept in the phosphate buffer 2-8 ℃ of storage;
(3) the proteic connection of nano magnetic glucan micro-sphere
A. the activation of nano magnetic glucan micro-sphere: nanometer dextran magnetic microsphere is drained, be mixed with the sodium hydroxide solution of 0.5g/ml nanometer dextran magnetic microsphere, press nanometer dextran magnetic microsphere solution: epoxy chloropropane is with 1: the 0.08-0.4 volume ratio is mixed, 20-25 ℃ was reacted acetone, deionized water rinse 3-5 time 6-10 hour; Be kept in the middle of the phosphate buffered saline buffer 2-8 ℃ of preservation;
B. proteinic connection: 30-100mg/ml nano magnetic glucan micro-sphere and 0.5-2mg/ml protein mass are than 40-60: 1 is connected, 20-25 ℃ was reacted 2-4 hour down, separated on magnetic sheet then, with phosphate buffered saline buffer washing 3-5 time, phosphate buffered saline buffer is preserved, 2-8 ℃ of storage.
2, in accordance with the method for claim 1, it is characterized in that described ferric oxide is Fe
3O
4Or Fe
2O
3
3, in accordance with the method for claim 1, it is characterized in that described dextran is dextran T20, Dextran T 40, dextran T100.
4, in accordance with the method for claim 1, it is characterized in that the concentration of described sodium hydroxide solution is 0.5-5mol/L; Sodium periodate solution concentration is 0.05-0.3mol/L; The concentration of ferric oxide phosphate solution is 30-80mg/ml.
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| CN101870500A (en) * | 2010-06-24 | 2010-10-27 | 浙江大学 | Preparation method of ferric oxide nanometer particle and magetic-control heating thereof |
| CN103601791A (en) * | 2013-11-25 | 2014-02-26 | 天津大学 | Method for promoting protein renaturation by increasing charge density of microspherical medium through ligand modification |
| CN104190383A (en) * | 2014-08-22 | 2014-12-10 | 南京农业大学 | Low-molecular-weight heparin affinity purification medium and method for purifying low-molecular-weight heparin |
| EP3578255B1 (en) * | 2017-01-04 | 2022-06-22 | Nanjing GenScript Biotech Co., Ltd. | High-loading and alkali-resistant protein a magnetic bead |
| CN109270064B (en) * | 2018-11-12 | 2020-12-01 | 新乡医学院 | Immunoprecipitation reagent based on dextran microspheres and preparation method and application thereof |
| CN116272702B (en) * | 2022-11-22 | 2023-09-26 | 广州蔚捷生物医药科技有限公司 | Biological nanometer microsphere and preparation method and application thereof |
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Application publication date: 20080319 Assignee: Jiangyin Zecheng Biotech Co.,Ltd. Assignor: BEIJING BIOMAI CENTURY BIOTECHNOLOGY Co.,Ltd. Contract record no.: 2013110000031 Denomination of invention: Preparation method for immune nano magnetic glucan micro-sphere Granted publication date: 20100310 License type: Exclusive License Record date: 20130624 |
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