CN102145255A - Method for separately preparing water-soluble nano particles with same isoelectric points - Google Patents
Method for separately preparing water-soluble nano particles with same isoelectric points Download PDFInfo
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Abstract
The invention relates to a method for separately preparing water-soluble nano particles with the same isoelectric points, which comprises the following steps of: 1) preparing a sample: preparing the sample for isoelectric focusing electrophoresis by using a mixture of water-soluble nano particles and amphoteric electrolyte; 2) adding the sample prepared in the step 1) into an isoelectric focusing electrophoresis system, performing isoelectric focusing electrophoresis on the sample to separate the water-soluble nano particles with different isoelectric points, and obtaining multiple groups of samples subjected to isoelectric focusing electrophoresis separation, wherein the samples contain the water-soluble nano particles with the same isoelectric points; and 3) removing the amphoteric electrolyte: removing the amphoteric electrolyte in the sample of each group obtained in the step 2) to purify the water-soluble nano particles in the sample of each group, and finally obtaining the water-soluble nano particles with the same isoelectric points. By the method, the nano particles with the same surface charge properties can be obtained, and the nano particles with the same size are further purified.
Description
Technical field
The present invention relates to a kind of method of separating the identical water-soluble nanoparticles of preparation isoelectric point.
Background technology
Along with developing rapidly of nanosecond science and technology, it and more and more deep the intersecting of other subjects have produced many new theories and method, and from brand-new angle, existing scientific system is had an immense impact on, to (for example: treatment of cancer) provide new solution solving many global great difficult problems.Be used for medical nano particle at present and comprise quantum dot (QuantumDot), lipid nano-medicament carrier, nm of gold (Gold nanoparticle), Nano Silver and carbon nanomaterial etc.Studies show that nano particle (nmol/L level) under extremely low concentration just can suppress to influence subcellular structures such as cell membrane, cytoskeleton and nucleus.A plurality of research groups confirm that nano material has stronger antibiotic and antiviral activity, its mechanism of action comprises pair cell wall (film) structural damage, and to the inhibition of energetic supersession and respiratory chain, therefore the research of nano particle has in recent years caused the extensive concern of biomedical and environmental science circle.And the generation of nano effect is based on character such as the size of nano material, charged, surface texture, at present, the size of nano particle is the more clearly character of research, mainly be that size relatively is easy to observe and control, in fact charged character also is the critical nature that influences nano effect, the surperficial charge confrontation biological safety that studies show that nano material especially in recent years, reasonably designs and optimizes the nano material assembly very important directive significance is arranged.
Along with the growing interest of researcher to the nano material biological effect, the functional modification of nano molecular, the biocompatibility that strengthens nano molecular becomes the focus in nano materials research field.With carbon nanomaterial fullerene (Fullerene, C
60) be example, because its unique cage structure, regular nano-scale and stable biological effect have been widely studied and have been applied to fields such as oncotherapy and targeted drug carrying.The carbon basket structure of fullerene has strong lipophilicity, and in order to solve fullerene poorly soluble problem in water, the researcher introduces polar group on the fullerene carbon skeleton, improves that it is water-soluble.Water miscible modification makes fat-soluble nano particle have the character of amphiphilic.Typical case's representative of water-soluble fullerenes derivates is that richness is reined in pure C
60(OH) n, it is a kind of polyhydric fullerene derivate, has fine solubility in water.
But fullerene C
60Molecule is 32 bodies, is the summit with 60 carbon atoms, has formed the sealing sphere by 12 pentagons and 20 hexagons, the pentagon ring is a singly-bound on the sphere, and ring does not link to each other each other, and the public seamed edge of hexagonal rings then is two keys, and the ring ring interconnects, and water-soluble modification is easy to form isomer.The charged character of product after the water-soluble modification has very big-difference, and this phenomenon is prevalent in the fullerene family molecule, comprises C
60, C
70, C
82, Gd@C
82, CNT, quantum dot, nano-metal particle etc.Because charged character also is the critical nature that influences nano effect, medicinal application and nanometer property research there is very important meaning so obtain the identical water-soluble nanoparticles of charged character.
At CDerwent, EDerwent, MDerwent. retrieve in the database, 396 patents about isoelectric focusing (isoelectricfocusing) are arranged, 46.5% protein/polypeptide (B04-N04), 76.0% that is distributed in (unordered) is distributed in microorganism detection and 83.6% and is distributed in electrophoresis (S03-E03E) field respectively, and the patent that adopts isoelectric focusing to be used for the identical water-soluble nanoparticles separation and purification of isoelectric point is not seen open report.
Summary of the invention
At existing research, the purpose of this invention is to provide a kind of method of separating the identical water-soluble nanoparticles of preparation isoelectric point.The water-soluble nano molecule that this method obtains has identical charged character, can improve the stability of water-soluble nanoparticles, so that be applied to medicinal application and nanometer property research.
The present invention adopts isoelectric focusing electrophoresis to separate the identical water-soluble nanoparticles of preparation isoelectric point (pI).Described isoelectric focusing electrophoresis utilizes special buffer solution (being ampholytes solution) to make a pH gradient in gel, and the making of pH gradient is to utilize ampholytes to have close but different pKa and pI value, under External Electrical Field, forms the pH gradient naturally.Every kind of amphiprotic substance just will be moved to the pH place that equals its isoelectric point during electrophoresis, and this moment, amphiprotic substance no longer had clean plus or minus electric charge, formed very narrow district's band, can collect purifying.
The objective of the invention is to be achieved through the following technical solutions:
A kind of method of separating the identical water-soluble nanoparticles of preparation isoelectric point may further comprise the steps:
1) preparation sample:, prepare the sample that is used for isoelectric focusing electrophoresis with mixing of water-soluble nanoparticles and ampholytes (ampholyte);
2) the point focusing electrophoresis such as carry out: the described sample pipetting volume that step 1) is made is to the isoelectric focusing electrophoresis system, described sample such as is carried out at the point focusing electrophoresis to be separated with the described water-soluble nanoparticles that will have different isoelectric points, obtain the described sample after many groups are separated through isoelectric focusing electrophoresis, contain the identical described water-soluble nanoparticles of isoelectric point in this sample;
3) remove ampholytes: the described ampholytes removal step 2) in every group of sample of acquisition finally obtains the water-soluble nanoparticles with identical isoelectric point so that the described water-soluble nanoparticles in described every group of sample is carried out purifying.
Preferably, the concentration of described water-soluble nanoparticles is 0.1-10mg/mL, and the concentration of described ampholytes is 0.1-50% (m/v).
Preferably, described water-soluble nanoparticles is the nano particle that has carried out the surface water insoluble chemical modification of hydroxylating, carboxylated, amination, phosphonic acidsization etc.
Preferably, carry out step 2) before the point focusing electrophoresis such as described, carry out prerunning, the step of described prerunning comprises that the gel that will have fixing protonated concentration gradient is put on the coldplate, on described gel with fixing protonated concentration gradient, be coated with liquid mineral oil to avoid the generation of bubble, under constant-pressure conditions, described isoelectric focusing system carried out prerunning, point focusing electrophoresis times such as the described prerunning time is 30-120 minute, and is described are 30-180 minute.Described gel with fixing protonated concentration gradient comprises the gel of poly aromatic acid amides, polyacrylamide, agarose or starch, and the mean molecule quantity of described polyacrylamide is at 500-90, between 000 dalton.
Preferably, step 2) point focusing electrophoresis such as described comprises equilibrium ion exchange membrane, assembling isoelectric focusing electrophoresis instrument, loads described sample, focuses on operation, collects step.
Preferably, described water-soluble nanoparticles comprises water-soluble carbon nano material, water-soluble quantum dot nano particle, water-soluble nano metallic particles, and described water-soluble carbon nano material comprises surface water dissolubility modification fullerene nano particle, CNT etc.; Described water-soluble quantum dot nano particle comprises the nano particle of hydroxylating quantum dot, the nano particle of carboxylated quantum dot, the nano particle of amination quantum dot etc.; Described water-soluble nano metallic particles also comprises nanogold particle, colloid gold particle, nano-Ag particles etc.
Preferably, described surface water dissolubility modification fullerene nano particle comprises: gold filled belongs to the fullerene nano particle in hydroxylating fullerene nano particle, carboxylated fullerene nano particle, amination fullerene nano particle, the hydroxylating, the interior gold filled of carboxylated belongs to fullerene nano particle, the interior bag of amination metal fullerene nano particle etc.
Preferably, described CNT comprises: the CNT that hydroxylating SWCN, carboxylated SWCN, amination SWCN, surperficial polyethylene glycol (PEG) are modified etc.
Preferably, described ampholytes is the polymer of polyethylene polyamine, and the polymer of described polyethylene polyamine comprises: the co-product of one or more of ethylenediamine, diethylenetriamine, triethylene tetramine and TEPA.
Preferably, the described ampholytes of the described removal of step 3) be adopt that centrifugal, centrifugal filtration, target substance precipitation are centrifugal, dialysis, electrophoresis, column chromatography, precipitation, crystallization or organic solvent deposit washing centrifugation method again.
Preferably, described organic solvent deposit washing centrifugation method is used for the described surface water dissolubility modification of purifying fullerene nano particle.Described solvent is the mixed emulsion of isopropyl alcohol, ether and n-hexane.
Comparing the method that separation of the present invention prepares the identical water-soluble nanoparticles of isoelectric point with prior art has the following advantages:
1, the present invention is used for the method for isoelectric focusing electrophoresis the separation of water-soluble nanoparticles first, promptly utilize nanoparticle surface charging different in kind, method separating water-soluble nano particle with isoelectric focusing electrophoresis, to obtain the identical nano particle of surface charge property, be being further purified to measure-alike nano particle.
2, of the present invention applied widely, it is applicable to the nano molecular aggregation that the surface is charged, comprise quantum dot (Quantum Dot), lipid nano-medicament carrier, nm of gold (Gold nanoparticle), Nano Silver and carbon nanomaterial, the especially soluble derivative of fullerene family.In the prior art, in the water-soluble fullerenes derivates system, because the isomer that the isomery effect produces is a lot, then the charged effect of each molecule is very inequality in the fullerene aggregation, causes the instability of fullerene aggregation, causes the precipitation of particle to be separated out.But the fullerene nano particle that the present invention utilizes the isoelectric point character of water-soluble nanoparticles will have identical charged character separates, and the nano particle that obtains has identical charged character, so the nanometer aqueous suspensions has better stability.Therefore adopt the water-soluble nanoparticles of method preparation of the present invention to have more excellent performances such as heat endurance.
Description of drawings
Fig. 1 is the structural representation of sample focus cell of the present invention;
Fig. 2 is the schematic diagram that the prerunning of isoelectric focusing electrophoresis of the present invention finishes;
Fig. 3 is the schematic diagram that isoelectric focusing electrophoresis of the present invention finishes;
Fig. 4 is the schematic diagram after isoelectric focusing electrophoresis product of the present invention is collected;
Fig. 5 is the pI of isoelectric focusing electrophoresis product of the present invention and the graph of a relation of cell quantity;
(a is that sample focuses on the product that cell 1 separates to the product of the different isoelectric points that Fig. 6 separates for isoelectric focusing electrophoresis of the present invention, b is that sample focuses on the product that cell 2 separates, c is that sample focuses on the product that cell 3 separates, and d is that sample focuses on the product that cell 4 separates) capillary electrophoresis analysis figure.
(a is that sample focuses on the product that cell 1 separates to the product of the different isoelectric points that Fig. 7 separates for isoelectric focusing electrophoresis of the present invention, b is that sample focuses on the product that cell 2 separates, c is that sample focuses on the product that cell 3 separates, and d is that sample focuses on the product that cell 4 separates) infrared analysis figure
(a is that sample focuses on the product that cell 1 separates to the product of the different isoelectric points that Fig. 8 separates for isoelectric focusing electrophoresis of the present invention, b is that sample focuses on the product that cell 2 separates, c is that sample focuses on the product that cell 3 separates, and d is that sample focuses on the product that cell 4 separates) XPS photoelectron spectroscopy analysis chart
Wherein: 1, electrode film (negative electrode); 2, sample focuses on cell; 3, electrode film (anode).
The specific embodiment
Separate the identical richness of preparation isoelectric point and rein in alcohol (C
60(OH)
24) method of nano particle, this method may further comprise the steps:
1) preparation sample: richness is reined in alcohol (C
60(OH)
24) nano particle and ampholytes mix, the mixed aqueous solution that obtains above-mentioned two kinds of materials is used for the sample of isoelectric focusing electrophoresis with preparation, and described richness is reined in alcohol (C
60(OH)
24) nano particle is by being used in commercially available fullerene nano particle C
60Toluene solution in, add NaOH, TBAH (TBAH) and H
2O
2Reaction obtains, and described richness is reined in alcohol (C
60(OH)
24) concentration of nano particle is 0.1-10mg/mL, described ampholytes is triethylene tetramine and the TEPA mixture of pH 3-10, its concentration is 0.1-50% (m/v).
2) prerunning, concrete steps are as follows:
Polyacrylamide gel is put on the coldplate, on described polyacrylamide gel, be coated with liquid mineral oil to avoid the generation of bubble, with anodal liquid and negative pole liquid wetting positive pole of difference and negative electrode bar, then described positive pole and negative electrode bar are put in respectively on the anode and negative electrode of isoelectric focusing electrophoresis system, with electrode alignment electrode strip center, add a cover, under starting voltage 200V, the isoelectric focusing system is carried out prerunning, move 30-120 minute.
Point focusing electrophoresis such as 3): adopt the Mini-MicroRotofor isoelectric focusing electrophoresis system of Bio-RAD company that sample is carried out isoelectric focusing electrophoresis and separate, concrete steps are as follows:
The equilibrium ion exchange membrane: dry amberplex is in the electrolyte infiltration of spending the night;
Assembling isoelectric focusing electrophoresis instrument:, insert an anode film and cathodic coating, assembling anode, two electrode assemblies of negative electrode at the two ends of sample focus cell with deionized water rinsing equilibrium ion exchange membrane;
Load sample: on described sample focus cell, be provided with two rounds, a round rubber belt sealing wherein, adjust on another round and the described electrode assemblie ventilating opening point-blank, with filled syringe 3 ml samples are added described sample focus cell, described sample can slowly be diffused into and fill up described sample and focus on cell, guarantees to drive away all bubbles, in order to avoid the bubble destruction electric field, dry the outer surface of described sample focus cell, with the described sample focus cell of rubber belt sealing; The sample focus cell focuses on cells by 10 samples to be formed, communicates between each cell but barrier film is arranged, and described barrier film has than macroscopic void, and purpose is the sample (seeing accompanying drawing 1) of isolating each cell collection under the condition that does not influence the sample electrophoresis migration.
Focus on operation: will assemble the described sample focus cell of described electrode assemblie, and use the 10ml syringe, the negative electrode in described electrode assemblie adds 0.1MNaOH respectively, and the anode in described electrode assemblie adds 0.1M H
3PO
4, cover coldplate, open rotary switch, the placement machine case lid applies electric field to described negative electrode and anode, and power is 1W, when working voltage no longer increases, etc. point focusing electrophoresis operation 30-180 minute;
Collect: the isoelectric focusing operation is finished, as early as possible the sample focus cell is inserted on the vacuum sample collection module, sample vacuum in the sample focus cell is sucked collecting chamber (seeing accompanying drawing 4), obtain the different described sample of many group isoelectric points, contain the identical described richness of isoelectric point in every group of sample and rein in alcohol (C
60(OH)
24) nano particle, reduce described sample focus cell vibrations, to avoid diffusion as far as possible;
PH measures: use the interior pH of each isoelectric focusing electrophoresis cell of pH determination of electrode of minor diameter, promptly measure the isoelectric point (pI) of nano particle.
4) remove ampholytes: the described water-soluble nanoparticles that every group of sample that step 3) is obtained adopts the emulsion of isopropyl alcohol, ether and n-hexane (ratio of the mixed emulsion of described isopropyl alcohol, ether and n-hexane is 1: 1: 1 or 1: 2: 3 or 3: 2: 1) to wash in the centrifugal next described sample carries out purifying, to remove ampholytes, the nano particle that final acquisition has identical isoelectric point.The molecular weight ranges 300-1 of common ampholytes, 200 dalton combine with weak static with target nanoparticle, and this may disturb the character and the activity of nanoparticle.Many methods (as centrifugal filtration, dialysis, electrophoresis etc.) can be used to from target nanoparticle separate ampholytes, and other method the application is not repeated.
The richness of the different isoelectric points that separation and purification obtains through isoelectric focusing electrophoresis is reined in alcohol (C
60(OH)
24) nano particle has the difference (see figure 6) through the retention time that records of CE (Capillary Electrophoresis), show richness rein in alcohol isoelectric point have difference, the richness of the different isoelectric points of further infrared spectrum graph discovery is reined in pure surface group and be there are differences, especially different (see figure 7)s of the state of hydroxyl, the analysis of XPS photoelectron spectroscopy also as can be seen, there is more complicated variation (see figure 8) in the hydroxyl that richness is reined in alcohol surface.
Separate the identical water-soluble carboxyl fullerene (C of preparation isoelectric point
60(C (COOH)
2)
2) method of nano particle, said method comprising the steps of:
1) preparation sample: with water-soluble carboxyl fullerene (C
60(C (COOH)
2)
2) nano particle and ampholytes mixing, the mixed aqueous solution that obtains above-mentioned two kinds of materials is used for the sample of isoelectric focusing electrophoresis with preparation, this water-soluble carboxyl fullerene obtains different adduct number purpose C60 malonate derivatives with commercially available fullerene nano particle by classical Bingle-Hirsch reaction, the ester cracking reaction obtains under alkali condition then, described water-soluble carboxyl fullerene (C
60(C (COOH)
2)
2) concentration of nano particle is 1mg/mL, ampholytes is triethylene tetramine and the TEPA mixture of pH 3-10, its concentration is 40% (m/v).
2) concrete steps of prerunning are with embodiment 1, wherein the 50 minutes time of prerunning operation;
3) etc. the concrete steps of point focusing electrophoresis are with embodiment 1, its medium point focusing electrophoresis operation 100 minutes;
4) remove ampholytes: every group of sample that step 3) is obtained precipitates centrifugal filtration by absolute ethyl alcohol, again with deionized water redissolution carboxylated fullerene (C
60(C (COOH)
2)
2) nano particle carries out purifying, the final carboxylated fullerene (C with identical isoelectric point of obtaining
60(C (COOH)
2)
2) nano particle.
Carboxylated fullerene (the C of the different isoelectric points that separation and purification obtains through isoelectric focusing electrophoresis
60(C (COOH)
2)
2) nano particle can pass through means such as CE (Capillary Electrophoresis), infrared spectrum, XPS photoelectron spectroscopy, TEM transmission electron microscope and detect difference, the application is not repeated.
Separate bag fullerene Gd@C in the identical water soluble hydroxy metal of preparation isoelectric point
82(OH)
22The method of nano particle said method comprising the steps of:
1) preparation sample: with bag fullerene Gd@C in the water soluble hydroxy metal
82(OH)
22Nano particle and ampholytes mix, and the mixed aqueous solution that obtains above-mentioned two kinds of materials is used for the sample of isoelectric focusing electrophoresis, this Gd@C with preparation
82(OH)
22Nano particle is with commercially available Metal Gd and C
82By the synthetic embedded metal fullerene Gd@C of arc discharge method
82Particle, this embedded metal fullerene Gd@C
82Particle is by adding NaOH, TBAH (TBAH) reaction acquisition, bag fullerene Gd@C in the described water-soluble metal
82(OH)
22The concentration of nano particle is 2mg/mL, and described ampholytes is triethylene tetramine and the TEPA mixture of pH 3-10, and its concentration is 30% (m/v).
2) etc. the concrete steps of point focusing electrophoresis are with embodiment 1, its medium point focusing electrophoresis operation 80 minutes;
3) removal ampholytes: with step 2) the every group of sample that obtains obtains bag fullerene Gd@C in the identical water-soluble metal of isoelectric point by the centrifugal filtration of absolute ethyl alcohol precipitation
82(OH)
22Nano particle is again with bag fullerene Gd@C in the deionized water redissolution water-soluble metal
82(OH)
22Nano particle carries out purifying.
Bag fullerene Gd@C in the water-soluble metal of the different isoelectric points that separation and purification obtains through isoelectric focusing electrophoresis
82(OH)
22Nano particle can pass through means such as CE (Capillary Electrophoresis), infrared spectrum, XPS photoelectron spectroscopy, TEM transmission electron microscope and detect difference, and the application is not repeated.
Separate the method for identical water-soluble fullerenes derivates list addition methylene fullerene [60] di 2 ethylhexyl phosphonic acid tetraacethyl (MMPF) nano particle of preparation isoelectric point, said method comprising the steps of:
1) preparation sample: water-soluble fullerenes derivates list addition methylene fullerene [60] di 2 ethylhexyl phosphonic acid tetraacethyl (MMPF) nano particle and ampholytes are mixed, the mixed aqueous solution that obtains above-mentioned two kinds of materials is used for the sample of isoelectric focusing electrophoresis with preparation, single addition methylene fullerene [60] di 2 ethylhexyl phosphonic acid tetraacethyl (MMPF) adopts commercial obtainable fullerene nano particle by under the effect of alkaline environment and iodine, obtain Cyclopropanated product with methylene biphosphonic acid esters tetra-ethyl ester that contains the activity methylene and Iod R, ester cracking reaction through alkali condition obtains again, the concentration of described single addition methylene fullerene [60] di 2 ethylhexyl phosphonic acid tetraacethyl is 0.5mg/mL, described ampholytes is triethylene tetramine and the TEPA mixture of pH 3-7, and its concentration is 10% (m/v).
2) concrete steps of prerunning are with embodiment 1, and the wherein said prerunning time is 30 minutes;
3) etc. the concrete steps of point focusing electrophoresis are with embodiment 1, its medium point focusing electrophoresis operation 90 minutes;
4) remove ampholytes: the emulsion that every group of sample employing ratio that step 3) is obtained is 1: 1: 1 isopropyl alcohol, ether and n-hexane is washed the centrifugal purifying that carries out, to remove ampholytes, obtain identical water-soluble fullerenes derivates list addition methylene fullerene [60] the di 2 ethylhexyl phosphonic acid tetraacethyl nano particle of isoelectric point.
Single addition methylene fullerene [60] di 2 ethylhexyl phosphonic acid tetraacethyl nano particle of the different isoelectric points that separation and purification obtains through isoelectric focusing electrophoresis can pass through means such as CE (Capillary Electrophoresis), infrared spectrum, XPS photoelectron spectroscopy, TEM transmission electron microscope and detect difference, and the application is not repeated.
Embodiment 5
Separate the identical carboxylated SWCN SWCT (C (COOH) of preparation isoelectric point
2)
nMethod, said method comprising the steps of:
1) preparation sample: with carboxylated SWCN SWCT (C (COOH)
2)
nMix with ampholytes; the mixed aqueous solution that obtains above-mentioned two kinds of materials is used for the sample of isoelectric focusing electrophoresis with preparation; this carboxylated SWCN obtains described carboxylated SWCN SWCT (C (COOH) with commercially available single-walled nanotube by acidylate, esterification oxidation reaction
2)
nConcentration be 0.1-10mg/mL, ampholytes is diethylenetriamine and the TEPA mixture of pH 3-7, its concentration is 0.1-50% (m/v);
2) etc. the concrete steps of point focusing electrophoresis are with embodiment 1, wherein, and etc. point focusing electrophoresis operation 30-180 minute;
3) removal ampholytes: with step 2) the every group of sample that obtains adopts the method for ultracentrifugation filtration to carry out purifying, and centrifugal rotational speed 10000-80000g finally obtains the identical carboxylated SWCN SWCT (C (COOH) of isoelectric point
2)
n
The water miscible carboxylated SWCN SWCT (C (COOH) of the different isoelectric points that separation and purification obtains through isoelectric focusing electrophoresis
2)
nThe means such as infrared spectrum, XPS photoelectron spectroscopy, TEM transmission electron microscope of can passing through detect difference, and the application is not repeated.
Separate the identical amination SWCN SWCT (NH of preparation isoelectric point
2)
nMethod, said method comprising the steps of:
1) preparation sample: with amination SWCN SWCT (NH
2)
nMix with ampholytes; the mixed aqueous solution that obtains above-mentioned two kinds of materials is used for the sample of isoelectric focusing electrophoresis with preparation; this amination SWCN obtains described amination SWCN SWCT (NH with commercially available single-walled nanotube by the synthetic in advance functional amido reaction of acylation reaction
2)
nConcentration be 5mg/mL, described ampholytes is triethylene tetramine and the TEPA mixture of pH 3-10, its concentration is 20% (m/v);
2) concrete steps of prerunning are with embodiment 1, and wherein the prerunning time is 90 minutes;
3) etc. the concrete steps of point focusing electrophoresis are with embodiment 1, its medium point focusing electrophoresis operation 90 minutes;
4) remove ampholytes: the method that every group of sample that step 3) is obtained adopts ultracentrifugation to filter is carried out purifying, and centrifugal rotational speed 10000-80000g finally obtains the identical amination SWCN SWCT (NH of described isoelectric point
2)
n
The water miscible amination SWCN SWCT (NH of the different isoelectric points that separation and purification obtains through isoelectric focusing electrophoresis
2)
nNano particle can pass through means such as infrared spectrum, XPS photoelectron spectroscopy, TEM transmission electron microscope and detect difference, and the application is not repeated.
Embodiment 7
Separate the identical hydroxylating SWCN SWCT (OH) of preparation isoelectric point
nMethod, said method comprising the steps of:
1) preparation sample: with hydroxylating SWCN SWCT (OH)
nMix with ampholytes, the mixed aqueous solution that obtains above-mentioned two kinds of materials is used for the sample of isoelectric focusing electrophoresis with preparation, and this hydroxylating SWCN passes through NaOH or H with commercially available single-walled nanotube
2O
2Hydroxylating obtain described hydroxylating SWCN SWCT (OH)
nConcentration be 10mg/mL, described ampholytes is ethylenediamine and the TEPA mixture of pH 3-10, its concentration is 0.1% (m/v);
2) concrete steps of prerunning are with embodiment 1, and wherein the prerunning time is 40 minutes;
3) etc. the concrete steps of point focusing electrophoresis are with embodiment 1, its medium point focusing electrophoresis operation 180 minutes;
4) remove ampholytes: the method that every group of sample that step 3) is obtained adopts ultracentrifugation to filter is carried out purifying, and centrifugal rotational speed 10000-80000g finally obtains the identical hydroxylating SWCN SWCT (OH) of described isoelectric point
n
The water miscible hydroxylating SWCN SWCT (OH) of the different isoelectric points that separation and purification obtains through isoelectric focusing electrophoresis
nNano particle can pass through means such as infrared spectrum, XPS photoelectron spectroscopy, TEM transmission electron microscope and detect difference, and the application is not repeated.
Separate the method for the nano particle of the identical amination quantum dot of preparation isoelectric point, may further comprise the steps:
1) preparation sample: the nano particle (buying from Sigma company) and the ampholytes of amination quantum dot are mixed, the mixed aqueous solution that obtains above-mentioned two kinds of materials is used for the sample of isoelectric focusing electrophoresis with preparation, the concentration of the nano particle of described amination quantum dot is 7mg/mL, described ampholytes is triethylene tetramine and the TEPA mixture of pH 3-10, and its concentration is 5% (m/v);
2) concrete steps of prerunning are with embodiment 1, and the wherein said prerunning time is 120 minutes;
3) etc. the concrete steps of point focusing electrophoresis are with embodiment 1, its medium point focusing electrophoresis operation 120 minutes;
4) remove ampholytes: the method that every group of sample that step 3) is obtained adopts ultracentrifugation to filter is carried out purifying, and centrifugal rotational speed 40000g finally obtains the nano particle of the identical amination quantum dot of described isoelectric point.
The nano particle of the water soluble amino quantum dot of the different isoelectric points that separation and purification obtains through isoelectric focusing electrophoresis can pass through means such as infrared spectrum, XPS photoelectron spectroscopy, TEM transmission electron microscope and detect difference, and the application is not repeated.
Embodiment 9
Separate the method for the nano particle of the identical hydroxylating quantum dot of preparation isoelectric point, may further comprise the steps:
1) preparation sample: the nano particle (buying from Sigma company) and the ampholytes of hydroxylating quantum dot are mixed, the mixed aqueous solution that obtains above-mentioned two kinds of materials is used for the sample of isoelectric focusing electrophoresis with preparation, the concentration of the nano particle of described hydroxylating quantum dot is 8mg/mL, described ampholytes is triethylene tetramine and the TEPA mixture of pH 3-10, and its concentration is 10% (m/v);
2) concrete steps of prerunning are with embodiment 1, and the wherein said prerunning time is 90 minutes;
3) etc. the concrete steps of point focusing electrophoresis are with embodiment 1, its medium point focusing electrophoresis operation 100 minutes;
4) remove ampholytes: the method that every group of sample that step 3) is obtained adopts ultracentrifugation to filter is carried out purifying, and centrifugal rotational speed 80000g finally obtains the nano particle of the identical hydroxylating quantum dot of described isoelectric point.
The nano particle of the water soluble hydroxy quantum dot of the different isoelectric points that separation and purification obtains through isoelectric focusing electrophoresis can pass through means such as infrared spectrum, XPS photoelectron spectroscopy, TEM transmission electron microscope and detect difference, and the application is not repeated.
Separate the method for the nano particle of the identical carboxylated quantum dot of preparation isoelectric point, may further comprise the steps:
1) preparation sample: the nano particle (buying from Sigma company) and the ampholytes of carboxylated quantum dot are mixed, the mixed aqueous solution that obtains above-mentioned two kinds of materials is used for the sample of isoelectric focusing electrophoresis with preparation, the concentration of the nano particle of described carboxylated quantum dot is 0.1mg/mL, described ampholytes is ethylenediamine and the diethylenetriamine mixture of pH 3-10, and its concentration is 2% (m/v);
2) concrete steps of prerunning are with embodiment 1, and the wherein said prerunning time is 50 minutes;
3) etc. the concrete steps of point focusing electrophoresis are with embodiment 1, its medium point focusing electrophoresis operation 30 minutes;
4) remove ampholytes: the method that every group of sample that step 3) is obtained adopts ultracentrifugation to filter is carried out purifying, and centrifugal rotational speed 60000g finally obtains the nano particle of the identical carboxylated quantum dot of described isoelectric point.
The nano particle of the water-soluble carboxyl quantum dot of the different isoelectric points that separation and purification obtains through isoelectric focusing electrophoresis can pass through means such as infrared spectrum, XPS photoelectron spectroscopy, TEM transmission electron microscope and detect difference, and the application is not repeated.
Embodiment 11
Separate the method for the identical water-soluble nano gold grain of preparation isoelectric point, may further comprise the steps:
1) preparation sample: water-soluble nano gold grain (buying from BBI and NANOCS company) and ampholytes are mixed, the mixed aqueous solution that obtains above-mentioned two kinds of materials is 10mg/mL with the concentration that preparation is used for the described water-soluble nano gold grain of sample of isoelectric focusing electrophoresis, described ampholytes is ethylenediamine and the triethylene tetramine mixture of pH 3-10, and its concentration is 50% (m/v);
2) concrete steps of prerunning are with embodiment 1, and the wherein said prerunning time is 70 minutes;
3) etc. the concrete steps of point focusing electrophoresis are with embodiment 1, its medium point focusing electrophoresis operation 180 minutes;
4) remove ampholytes: every group of sample that step 3) is obtained adopts the method for deionized water dialysis to carry out purifying, finally obtains the identical water-soluble nano gold grain of described isoelectric point.
The water-soluble nano gold grain of the different isoelectric points that separation and purification obtains through isoelectric focusing electrophoresis can pass through means such as infrared spectrum, XPS photoelectron spectroscopy, TEM transmission electron microscope and detect difference, and the application is not repeated.
Separate the method for the identical water-soluble nano silver particle of preparation isoelectric point, may further comprise the steps:
1) preparation sample: water-soluble nano silver particle (buying from BBI and NANOCS company) and ampholytes are mixed, the mixed aqueous solution that obtains above-mentioned two kinds of materials is used for the sample of isoelectric focusing electrophoresis with preparation, the concentration of described water-soluble nano silver particle is 3.5mg/mL, described ampholytes is ethylenediamine and the triethylene tetramine mixture of pH 3-10, and its concentration is 30% (m/v);
2) concrete steps of prerunning are with embodiment 1, and the wherein said prerunning time is 50 minutes;
3) etc. the concrete steps of point focusing electrophoresis are with embodiment 1, its medium point focusing electrophoresis operation 60 minutes;
4) remove ampholytes: every group of sample that step 3) is obtained adopts the method for deionized water dialysis to carry out purifying, finally obtains the identical water-soluble nano silver particle of described isoelectric point.
The water-soluble nano silver particle of the different isoelectric points that separation and purification obtains through isoelectric focusing electrophoresis can pass through means such as infrared spectrum, XPS photoelectron spectroscopy, TEM transmission electron microscope and detect difference, and the application is not repeated.
Embodiment 13
Separate the method for the identical water-soluble colloid gold grain of preparation isoelectric point, may further comprise the steps:
1) preparation sample: water-soluble colloid gold grain (buying from BBI and NANOCS company) and ampholytes are mixed, the mixed aqueous solution that obtains above-mentioned two kinds of materials is used for the sample of isoelectric focusing electrophoresis with preparation, the concentration of described water-soluble colloid gold grain is 0.4mg/mL, described ampholytes is the triethylene tetramine of pH 3-10, and its mass concentration is 5% (m/v);
2) concrete steps of prerunning are with embodiment 1, and wherein the prerunning time is 40 minutes;
3) etc. the concrete steps of point focusing electrophoresis are with embodiment 1, its medium point focusing electrophoresis operation 120 minutes;
4) remove ampholytes: every group of sample that step 3) is obtained adopts the method for deionized water dialysis to carry out purifying, finally obtains the identical water-soluble colloid gold grain of described isoelectric point.
The water-soluble colloid gold grain of the different isoelectric points that separation and purification obtains through isoelectric focusing electrophoresis can pass through means such as infrared spectrum, XPS photoelectron spectroscopy, TEM transmission electron microscope and detect difference, and the application is not repeated.
Just in order better to explain the present invention, it should not be construed limitation of the present invention to the above embodiments.Those skilled in the art is according to equivalents of the present invention or be equal to the technical scheme that replacement forms, and all drops within the rights protection scope of the present invention.
Claims (10)
1. one kind is separated the method for preparing the identical water-soluble nanoparticles of isoelectric point, may further comprise the steps:
1) preparation sample: water-soluble nanoparticles is mixed with ampholytes, and preparation is used for the sample of isoelectric focusing electrophoresis;
2) the point focusing electrophoresis such as carry out: the described sample pipetting volume that step 1) is made is to the isoelectric focusing electrophoresis system, described sample such as is carried out at the point focusing electrophoresis to be separated with the described water-soluble nanoparticles that will have different isoelectric points, obtain the described sample after many groups are separated through isoelectric focusing electrophoresis, contain the identical described water-soluble nanoparticles of isoelectric point in this sample;
3) remove ampholytes: the described ampholytes removal step 2) in every group of sample of acquisition finally obtains the water-soluble nanoparticles with identical isoelectric point so that the described water-soluble nanoparticles in described every group of sample is carried out purifying.
2. method according to claim 1 is characterized in that, the concentration of described water-soluble nanoparticles is 0.1-10mg/mL, and the concentration of described ampholytes is 0.1-50% (m/v).
3. method according to claim 1 is characterized in that, described water-soluble nanoparticles is to have carried out the nano particle that the surface water insoluble chemical of hydroxylating, carboxylated, amination, phosphonic acidsization is modified.
4. method according to claim 1, it is characterized in that, carry out step 2) before the point focusing electrophoresis such as described, carry out prerunning, the step of described prerunning comprises that the gel that will have fixing protonated concentration gradient is put on the coldplate, on described gel with fixing protonated concentration gradient, be coated with liquid mineral oil to avoid the generation of bubble, under constant-pressure conditions, described isoelectric focusing system is carried out prerunning, point focusing electrophoresis times such as the described prerunning time is 30-120 minute, and is described are 30-180 minute.
5. method according to claim 1 is characterized in that step 2) point focusing electrophoresis such as described comprises equilibrium ion exchange membrane, assembling isoelectric focusing electrophoresis instrument, loads described sample, focuses on operation, collects step.
6. according to each described method in the claim 1 to 5, it is characterized in that, described water-soluble nanoparticles comprises water-soluble carbon nano material, water-soluble quantum dot nano particle, water-soluble nano metallic particles, and described water-soluble carbon nano material comprises surface water dissolubility modification fullerene nano particle, CNT; Described water-soluble quantum dot nano particle comprises the nano particle of hydroxylating quantum dot, the nano particle of carboxylated quantum dot, the nano particle of amination quantum dot; Described water-soluble nano metallic particles also comprises nanogold particle, colloid gold particle, nano-Ag particles.
7. method according to claim 6, it is characterized in that described surface water dissolubility is modified the fullerene nano particle and comprised: fullerene nano particle, the interior bag fullerene nano particle in fullerene nano particle, the amination metal that belongs to covered with gold leaf of carboxylated of belonging to covered with gold leaf in hydroxylating fullerene nano particle, carboxylated fullerene nano particle, amination fullerene nano particle, the hydroxylating.
8. method according to claim 6 is characterized in that, described CNT comprises: hydroxylating SWCN, carboxylated SWCN, amination SWCN, the polyethyleneglycol modified CNT in surface.
9. according to each described method in the claim 1 to 5, it is characterized in that, described ampholytes is the polymer of polyethylene polyamine, and the polymer of described polyethylene polyamine comprises: the co-product of one or more of ethylenediamine, diethylenetriamine, triethylene tetramine and TEPA.
10. method according to claim 1, it is characterized in that, the described ampholytes of the described removal of step 3) be adopt that centrifugal, centrifugal filtration, target substance precipitation are centrifugal, dialysis, electrophoresis, column chromatography, precipitation, crystallization or organic solvent deposit washing centrifugation method again.
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| CN103396793A (en) * | 2013-07-24 | 2013-11-20 | 中国科学院长春光学精密机械与物理研究所 | Multicolor luminous carbon nanodot as well as preparation method and application thereof |
| CN105858985A (en) * | 2016-06-06 | 2016-08-17 | 北京师范大学 | Method for recovering metal oxide nano-particles from water |
| CN110760306A (en) * | 2018-07-27 | 2020-02-07 | Tcl集团股份有限公司 | Quantum dot purification method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396793A (en) * | 2013-07-24 | 2013-11-20 | 中国科学院长春光学精密机械与物理研究所 | Multicolor luminous carbon nanodot as well as preparation method and application thereof |
| CN105858985A (en) * | 2016-06-06 | 2016-08-17 | 北京师范大学 | Method for recovering metal oxide nano-particles from water |
| CN105858985B (en) * | 2016-06-06 | 2018-05-08 | 北京师范大学 | A kind of method of metal oxide nanoparticles thing in recycle-water |
| CN110760306A (en) * | 2018-07-27 | 2020-02-07 | Tcl集团股份有限公司 | Quantum dot purification method |
| CN110760306B (en) * | 2018-07-27 | 2021-11-23 | Tcl科技集团股份有限公司 | Quantum dot purification method |
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