CN1956779B - Affinity particle and affinity separation method - Google Patents
Affinity particle and affinity separation method Download PDFInfo
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- CN1956779B CN1956779B CN2005800165535A CN200580016553A CN1956779B CN 1956779 B CN1956779 B CN 1956779B CN 2005800165535 A CN2005800165535 A CN 2005800165535A CN 200580016553 A CN200580016553 A CN 200580016553A CN 1956779 B CN1956779 B CN 1956779B
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Abstract
The present invention is affinity particles that are characterized by having phosphorylcholine groups represented by the following formula (1) covalently bonded onto the surface of inorganic powder and also by having ligands having specific affinity with a certain target substance covalently bonded or adsorbed onto the surface of inorganic powder. The object of the present invention is to provide an affinity separation method that uses affinity particles utilizing inexpensive inorganic particles and is capable of separating the target substance easily and with high accuracy.
Description
Technical field
The present invention relates to affine particle and affine separation method.In particular, but relate to the affine particle that utilized inorganic particle and high accuracy and the affine separation method of isolation of target substances easily.But for high accuracy and the immuno-precipitation, latex agglutination method etc. that easily detect target substance also extremely useful for the various separation of representative, purifying, detection method.
Background technology
In the past, the separation and purification of organism material was to adopt column chromatography.But post has the fatal problem shown in following (1)-(3) in separating.
(1) in order to obtain target substance, must use multiple post, purification efficiency is poor.
(2) must confirm experiment, confirming whether to contain target substance in the separated component, so purifying needs the more time.
(3) loss during purifying is big, needs a large amount of samples.
For addressing the above problem, the affinity column or the affine particle (patent documentation 1, patent documentation 2) that support part in the separation and purification of target substance, have been adopted.
But carrying out separation and purification by affinity column has following problem.
(1) required target substance can't Selective Separation.That is, except that by the part captured object material, undesirable target substance also is adsorbed on the post.
(2) capturing efficiency is low, needs a large amount of liquor samples.
In addition, in that affine particle is dispersed in the affine separation method that separates in the liquor sample, use (non-patent literatures 1) such as agaroses, this has following problem.
(1) required target substance can't Selective Separation.That is, except that by the part captured object material, undesirable target substance also is adsorbed on the affine particle.
(2) light specific gravity is difficult to separate affine particle.
(3) disintegration of carrier takes place easily, durability is low.
On the other hand, because inorganic particle adsorbs more material than organic granular, so those skilled in the art do not expect utilizing inorganic particle as affine particle.
Patent documentation 1: the special fair 8-26076 communique of Japan
Patent documentation 2: Japanese Unexamined Patent Application Publication 2002-511141 communique
Non-patent literature 1:Bioconjugate Chem.; 2002; 13 (2); 163-166
Summary of the invention
Invent problem to be solved
The invention provides the affine separation method that is with historically new significance, this method has been by having utilized the affine particle of cheap inorganic particle, can be easy and isolation of target substances accurately.In addition, but for high accuracy and immunosedimentation, latex agglutination method of easily detecting target substance etc. be that various separation, purifying, the inspection method of representative is extremely useful.
Solve the method for problem
That is, the invention provides affine particle, it is characterized in that: the surface of inorganic particle has Phosphorylcholine base shown in the following formula (1) with the form of covalent bond.
[changing 4]
(1)
The present invention also provides affine particle, it is characterized in that: the surface of inorganic particle has the Phosphorylcholine base shown in the following formula (1) with the form of covalent bond, and the surface of inorganic particle has the reactive group or the adsorption group that can combine with the part that certain target substance is had pathoklisis with the form of covalent bond or absorption.
[changing 5]
(1)
The present invention provides affine particle again, it is characterized in that: the surface of inorganic particle has the Phosphorylcholine base shown in the following formula (1) with the form of covalent bond, and the surface of inorganic particle has the part that has pathoklisis with certain target substance with the form of covalent bond or absorption.
[changing 6]
(1)
The present invention also provides above-mentioned affine particle, it is characterized in that: above-mentioned inorganic particle is one or more inorganic particles that are selected from silica, titanium oxide, zinc white, aluminium oxide, iron oxide, talcum powder, mica, sericite, collaurum, its average grain diameter is 20nm-500 μ m, and proportion is 1.0g/cm
3More than.
The present invention provides above-mentioned affine particle again, it is characterized in that: above-mentioned part is one or more parts that are selected from various antibody, antigen, enzyme, substrate, acceptor, agglutinin, peptide, DNA, RNA, nucleic acid ligands, albumin A, Protein G, avidin, biotin, chelate compound, each metal ion species.
The present invention also provides the affine separation method of the target substance that is undertaken by inorganic particle, it is characterized in that comprising following steps: the 1st step that (1) makes the affine particle of claim 1 or 2 combine with part arbitrarily, (2) make the affine particle of making in the 1st step be scattered in the 2nd step in the liquor sample, wherein liquor sample contains by part selectivity captured object material arbitrarily, and (3) reclaim the 3rd step by the target substance of affine particle capture.
The present invention provides the affine separation method of the target substance that is undertaken by inorganic particle again, it is characterized in that comprising following steps: (1) makes the affine particle of claim 3 be scattered in the 1st step in the liquor sample, wherein said liquor sample contains by part selectivity captured object material arbitrarily, and (2) reclaim the 2nd step by the target substance of affine particle capture.When affine particle of the present invention being applied to the detection of antibody such as immuno-precipitation or latex agglutination method or albumen, do not need the recycling step of (2), can easily confirm by the variation of its dispersity by visual.
The invention effect
Affine particle of the present invention is only caught certain target substance (wish separate target substance) by part, and suppresses other material and be adsorbed on the particle, so separation selectivity is high.Because the dispersiveness of its excellence is separated from liquor sample with as easy as rolling off a log, by having utilized the affine particle of cheap inorganic powder grains, can be easy and isolation of target substances accurately.
That is to say that the separation method of target substance of the present invention can be in the short time and separated effectively, easily to be separated into the target substance of purpose.Usually, material has the character of absorption foreign matter, and affine particle in the past is difficult to an isolation of target substances effectively, but by modifying particle surface with the Phosphorylcholine base, but the utmost point prevents the non-specific adsorption of other material and affine particle effectively, can improve purification efficiency.
In addition, the Phosphorylcholine base has high hydrophily, in containing the liquor sample of water, also has the function of the dispersiveness raising that makes affine particle.
And common particle has the tendency because of the salt aggegation, for example will be from serum during the separate targets thing, owing to aggegation takes place the various salt in the serum, purification efficiency reduces, but affine particle of the present invention also seldom aggegation in the presence of salt can be reclaimed target substance effectively.
The affine particle that the present invention uses is made of inorganic particle, so the proportion height, by leaving standstill or mild centrifugation can easily reclaim.In addition this particle is adorned post as carrier, also can be used for the recovery of target substance with the form of affinity column.
The accompanying drawing summary
Fig. 1 is the ideograph of expression affine particle of the present invention and the optionally difference of affine particle when capture protein in the past.
Fig. 2 is the structural formula and the NMR wave spectrum of the compound of synthesis example 1 manufacturing.
Fig. 3 is the structural formula and the NMR wave spectrum of the compound of synthesis example 2 preparations.
Fig. 4 is to use Af particle (A), (B), (C) of PC particle (A), (B), (C) and synthesis example 6 preparations of synthesis example 5 preparations to carry out the P quantitative results.
Fig. 5 is the 31P-CPMAS wave spectrum of the PC particle (A) of reference example 1 preparation.
Fig. 6 is the FT-IR wave spectrum of the PC particle (A) of reference example 1 preparation.
Fig. 7 is the 13C-CPMAS wave spectrum of the PC particle (C) of reference example 3 preparations.
Fig. 8 is the evaluation to the PC particle (A) of reference example 1,2,3 preparations, (B), the non-special absorption of (C) CKIs matter.
Fig. 9 is the antagonism BA antibody of the use Af particle (A) that carries out among the embodiment 1, the optionally evaluation of antihuman hemoglobin.
Figure 10 is the antagonism BA antibody of the use Af particle (B) that carries out among the embodiment 2, the optionally evaluation of antihuman hemoglobin.
Figure 11 is the antagonism BA antibody of the use Af particle (C) that carries out among the embodiment 3, the optionally evaluation of antihuman hemoglobin.
Figure 12 is optionally estimating goat is sero-fast of the use Af particle (A) that carries out among the embodiment 1,3, (C).
Figure 13 is the use Af particle (A) that carries out among the embodiment 1,3, the selective evaluation to antihuman hemoglobin in the lowlenthal serum of (C).
Figure 14 is the antagonism BA antibody of the amino particle of the use carried out in the comparative example 1, the optionally evaluation of antihuman hemoglobin.
The best mode that carries out an invention
Below describe the present invention in detail.
" inorganic particle "
Among the present invention, be not particularly limited for the inorganic particle that constitutes affine particle.It is inorganic object about 20nm-500 μ m that inorganic particle typically refers to average grain diameter.Concrete particle for example has: talcum powder, kaolin, mica, sericite, muscovite, phlogopite, synthetic mica, rubellan, biotite, vermiculite, magnesium carbonate, calcium carbonate, alumina silicate, barium silicate, calcium silicates, magnesium silicate, strontium silicate, the wolframic acid slaine, magnesium, silica, zeolite, barium sulfate, sintering calcium sulfate (bassanite), calcium phosphate, fluoridated apatite, hydroxyapatite, ceramic powders, metallic soap (Zinc tetradecanoate for example, calcium palmitate, aluminum stearate), boron nitride, cerium oxide, inorganic particles such as collaurum.
Particularly preferred particle is silica, titanium oxide, zinc white, aluminium oxide, iron oxide, talcum powder, mica, sericite, collaurum etc.Compare the inorganic particle of preferred atresia matter with the inorganic particle of porous matter.
Phosphorylcholine base of above-mentioned (1) formula and the combinative reactive group of part or adsorption group import particle surface by covalent bond, and therefore preferred its surface has amino particle.
Also the average grain diameter of preferred inorganic particle is 20nm-500 μ m, and proportion is 1.0g/cm
3More than.
Silica, titanium oxide, zinc white, aluminium oxide, iron oxide, talcum powder, mica, sericite, collaurum etc. are for example arranged.
" combinative reactive group of part or adsorption group "
As long as part can in conjunction with, be not particularly limited.For example in the covalent bond form, preferred amide, ester, polyurethane, ether, secondary amine, urea key, disulfide bond etc.Therefore, preferred part can form the reactive group of these covalent bond forms, preferred amino, hydroxyl, carboxyl, mercapto, aldehyde radical etc.Preferred avidin-the biotin of absorption shape, metal-chelate compound etc.Therefore preferred part can form the adsorption group of these absorption shapes, preferred avidin, biotin, chelate compound etc.
" part "
Among the present invention, part is the material that combines with certain target substance specificity, is various antibody, antigen, enzyme, substrate, acceptor, peptide, DNA, RNA, nucleic acid ligands, albumin A, Protein G, avidin, biotin, chelate compound, each metal ion species etc.For example, various antibody have IgG, IgM, IgA, IgD, IgE, IgY, and antigen has protein, polysaccharide, enzyme has glutathione-S-transferase, substrate has glutathione, and acceptor has hormone receptor, cytokine receptor, and chelate compound has nitrilotriacetic acid, each metal ion species that Ni is arranged
2+, Co
2+, Cu
2+, Zn
2+, Fe
3+
" manufacture method of affine particle of the present invention "
The surface of inorganic particle has the Phosphorylcholine base shown in the formula (1) with the form of covalent bond, directly there be the reactive group or the adsorption group that can combine with the part that certain target substance is had pathoklisis in the surface of inorganic particle with the form of covalent bond or absorption, this is an essence of the present invention, therefore its preparation method is unqualified, combination by any method.
But, the scheme that as mentioned above, use the polymer have Phosphorylcholine base and combinative reactive group of part or adsorption group in advance, do not have chemical bond, is just overlayed on particle surface does not comprise in the present invention.This is because the polymer of lining may be peeled off, and occurs because the influence that the lining polymer produces.
Affine particle of the present invention can be by preparations such as following methods.
Step 1: import the Phosphorylcholine base shown in the following formula (1) and combinative reactive group of part or adsorption group to particle.Unqualified to reactive group or adsorption group, can be amino or hydroxyl, carboxyl, aldehyde radical etc.
Step 2: for Phosphorylcholine base shown in the formula (1) and part are combined with the reactive group or the adsorption group of importing particle.The chemical constitution that exists between Phosphorylcholine base or part and reactive group or adsorption group (group at interval) is arbitrarily.For example the interval group can also be the alkylidene chain that contains one or more amino except that methene chain, oxyethylene group chain etc. arbitrarily.
When amino " be present in the reactive group of particle surface or adsorption group for "
Step 1: can amino be imported arbitrarily in the particle by the method for known method or exploitation from now on.Amino can directly import particle surface.Amino is primary amine or secondary amine.
Step 2: the oxidisability cleacvage reaction by glyceryl phosphoryl choline obtains aldehyde body or hydrate, and itself and the particle with amino are carried out the reductive amination reaction, and the Phosphorylcholine base directly is additional to particle surface.
Do not make the Phosphorylcholine base combine (conditioned reaction amount) with all amino, residual amino becomes the combinative substituting group of part.
Perhaps, obtain carboxysome by the oxidation cleacvage reaction of glyceryl phosphoryl choline, with its with have amino particle and carry out amidation process, the Phosphorylcholine base directly is additional to particle surface.
Do not make the Phosphorylcholine base combine (conditioned reaction amount) with all amino, residual amino becomes the combinative substituting group of part.
" particle surface imports amino method "
Amino is imported the known method of particle, and (step 1) is as described below.
1. the surface reaction by plasma treatment imports amino
Under nitrogen atmosphere, import amino to particle surface by low temperature plasma.Specifically, particle is packed in the reaction vessel for plasma, will vacuumize in the reaction vessel, import nitrogen then with vavuum pump.Then can import amino to particle surface by glow discharge.Also the inorganic material grain through plasma treatment can be carried out machinery granulate.Document about plasma treatment is as follows.
1.M.Muller，C.oehr
Plasma?aminofunctionalisation?of?PVDF?microfiltration?membranes：comparison?of?the?in?plasma?modifications?with?a?grafting?method?usingESCA?and?an?amino-selective?fluorescent?probe
Surface?and?Coatings?Technology?116-119(1999)802-807
2.Lidija?Tusek，Mirko?Nitschke，Carsten?Werner，Karin?Stana-Kleinschek，Volker?Ribitsch
Surface?characterization?of?NH3?plasma?treated?polyamide?6?foils
Colloids?and?Surfaces?A：Physicochem.Eng.Aspects?195(2001)81-95
3.Fabienne?Poncin-Epaillard，Jean-Claude?Brosse，Thierry?Falher
Reactivity?of?surface?groups?formed?onto?a?plasma?treated?poly(propylene)film
Macromol.Chem.Phys.200.989-996(1999)
2. import amino by surface modifier
Use has surface modifiers such as amino alkoxy silane, chlorosilane, silazane, and the inorganic particle surfaces such as particle of containing silanol are handled.
For example,, silica dioxide granule is handled, imported amino by having the 3-TSL 8330 of primary amino radical.Specifically, silica is immersed in water-2-propyl alcohol mixed liquor, add the 3-TSL 8330, be heated to 100 ℃ then, reacted 6 hours.After being cooled to room temperature, with methyl alcohol silica is washed, drying can obtain the amino particle that directly imports to silica surface.Preferred handled particle also has particles such as glass, aluminium oxide, talcum powder, clay, mica, asbestos, titanium oxide, zinc white, iron oxide except that silica.
3. import amino (with reference to the special fair 1-54379 communique of Japan, the special fair 1-54380 communique of Japan, the special fair 1-54381 communique of Japan) by the organosilicon gas phase treatment
At first by 1,3,5, the 7-tetramethyl-ring tetrasiloxane is handled particle surface, makes Si-H base that imports to the surface and the monomer reaction with amino, obtains amidized surface.For example, with mica and 1,3,5, the 7-tetramethyl-ring tetrasiloxane is packed in the drier, outgases with lift pump.Reacted 16 hours down at 80 ℃, take out mica then, dry down at 120 ℃.The gained mica is scattered in the ethanol, adds allylamine, then add the ethanolic solution of chloroplatinic acid, stirred 2 hours down at 60 ℃, filter behind the reaction terminating, with the ethanol washing, drying under reduced pressure obtains the amination mica.This is fit to various inorganic particles (mica, talcum powder, kaolin, aluminium oxide, titanium oxide, zinc oxide, iron oxide, various inorganic pigment) are handled.
It is monomer that the monomer that uses in this method can use amine.Amine is that monomer is not limited to allylamine, as long as have amino and polymerisable vinyl, acrylic isoreactivity position.Amino can be by protections such as butoxy carbonyl, benzyloxycarbonyl.
In addition, even be not that amine is monomer, as epoxy radicals, also can be to have the monomer that for example can import amino functional group simply by reaction with diamines.
" to having the method that amino particle imports the Phosphorylcholine base "
Below, following expression imports the method (step 2) of Phosphorylcholine base to the amination particle surface.
Particle is soaked in the methyl alcohol, adds phosphatidyl glycerol aldehyde, at room temperature placed 6 hours.Add the cyano group Boratex down at 0 ℃ then, adding thermal agitation spends the night, to amino addition Phosphorylcholine base.With particle with methanol cleaning after, drying can obtain the surface and directly have the particle of Phosphorylcholine base.Reaction dissolvent is except that methyl alcohol, so long as protonic solvents such as ethanol, 2-propyl alcohol all can use, the importing rate when using methyl alcohol is higher.
Use the 3-TSL 8330 as surface modifier, with amino import silica, then to import the scheme of method of Phosphorylcholine base (being called for short PC) as follows.
[changing 7]
[changing 8]
As mentioned above, preparation has amino particle, oxidisability cleacvage reaction by glyceryl phosphoryl choline obtains aldehyde body or hydrate, by itself and the reductive amination reaction with amino particle, can prepare the particle that the Phosphorylcholine base directly is attached to particle surface.
This method has the importing rate height of Phosphorylcholine base, and the advantage that can modify the surface of various inorganic particles.
The above-mentioned compound that contains aldehyde carries out the oxidisability cracking by known method with known glyceryl phosphoryl choline and obtains, and is open-and-shut step.For example use oxidants such as periodic acid, periodate or bismuth trioxide, to 1, the 2-glycol carries out oxidation, makes the key cracking, obtains the aldehyde body.Reaction usually in water or contain in the organic solvent of water and carry out, reaction temperature be 0 ℃ to room temperature.The aldehyde body can become hydrate through balanced reaction in water, this is for not influence of reaction ensuing and amine.Below expression prepares an example of the scheme of the monofunctional aldehyde body that contains the Phosphorylcholine base.
[changing 9]
The reductive amination reaction that the aldehyde body (or hydrate) that oxidisability cleacvage reaction by glyceryl phosphoryl choline is obtained combines with the amino of particle can be by stirring both and easily carrying out in solvent.This reaction is both to be dissolved or dispersed in water or the alcohol (can mix ternary organic solvent), forms imines, by reducing agent it is reduced then, obtains secondary amine.The reducing agent that the preferred cyano group Boratex of reducing agent etc. are gentle as long as Phosphorylcholine is stable, also can use other reducing agent.Reaction is carried out to room temperature at 0 ℃ usually, also can according to circumstances heat.
Can make the compound shown in the formula (2) to measure arbitrarily, to react according to conventional method and above-mentioned amino, also can be with residual amino as combinative reactive group of part or adsorption group.
[changing 10]
(2)
The integer of n=1-12
Concrete grammar is as follows: for example can be with the compound of formula (2) and thionyl chloride at N, react in N '-dimethyl formamide, make the chloride thing, at N, react with particle in N '-dimethyl formamide, by the Phosphorylcholine base shown in the amido link introducing-type (1) with amino.
The compound of formula (2) can be synthetic by following proposal.
" about combinative reactive group of part or adsorption group "
In the above-mentioned reaction, do not make the Phosphorylcholine base combine (conditioned reaction amount) with all amino, residual amino becomes combinative reactive group of part or adsorption group.This particle is the affine particle of claim 2, is the particle that Phosphorylcholine base shown in the formula (1) and the combinative reactive group of part or adsorption group directly are present in the surface of inorganic particle.The particle that part combines with this residual amino is the affine particle of claim 3, is the particle that Phosphorylcholine base shown in the formula (1) and part directly are present in the surface of inorganic particle.
The affine particle person of being to use of claim 2 is according to the material that will catch (target substance), the goods form that it is combined with part arbitrarily.The affine particle of claim 3 be in advance with the goods form that itself and part are combined.The affine particle of claim 1 is the affine particle that the Phosphorylcholine base of formula (1) at least is present in particle surface, the reactive group or the adsorption group that no matter part are not arranged or can combine with it, the user is according to the material that will catch (target substance), the goods form that it is combined with part arbitrarily.As long as the Phosphorylcholine base shown in the formula (1) is present in particle surface at least, then comprise any type of affine particle, for example also comprise the form of claim 2 and claim 3.
In the above-mentioned reaction, make amino residually with as combinative reactive group of part or adsorption group, the method for this reaction that can be at war with by the 3-TSL 8330 that makes the 3-TSL 8330 and imported the Phosphorylcholine base or conditioned reaction amount wait carries out.
Also can make the compound and the reaction of this amino that have functional group arbitrarily, make this functional group become combinative reactive group of part or adsorption group.For example can be glutaraldehyde, two imidic acid Arrcostabs, acid azide base class, isocyanates etc.
In the scheme when using the 3-TSL 8330 as above-mentioned surface modifier, regulate the reacting dose of surface modifier, make the hydroxyl (OH) that is present in particle surface residual, can utilize residual OH base as combinative reactive group of part or adsorption group.
" part and associated methods " with amino particle
When part is protein, can make the aldehyde radical and the reaction of the amino on the inorganic particle of glutaraldehyde, the amino in the protein is reacted with another aldehyde radical, thus conjugated protein.When being hydroxyl " be present in the reactive group of particle surface or adsorption group "
There is hydroxyl in the surface of most of inorganic particles, therefore need not new combinative reactive group of part or the adsorption group that resembles the above-mentioned amino that import, the hydroxyl (OH) that is present in particle surface be can directly utilize, Phosphorylcholine base and part or combinative reactive group of part or adsorption group imported.Affine particle of the present invention is preferably by this method preparation.
" import the method for Phosphorylcholine base to particle " with hydroxyl
From the Si-OMe dehydration of the compound of following formula (3) or (4), form chemical bond with the hydroxyl of particle surface.This chemical reaction is undertaken by add hot reflux in nearly all organic solvent, can quantitatively carry out easy as can.By this dehydration, can import the extremely stable Phosphorylcholine base of chemistry, physical property, thereby preferred.The compound that contains the Phosphorylcholine base shown in following formula (3) or the formula (4) is a new compound.
[changing 11]
(3)
[changing 12]
(4)
In the formula, m is 2-6, and n is 1-4.OMe can be OEt, Cl.In addition among OMe that combines with Si or OEt or the Cl maximum two can be methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group.
" preparation method who contains the Phosphorylcholine based compound of formula (3) "
Phosphorylcholine derivative shown in the following formula (5) is dissolved in distilled water.The Phosphorylcholine derivative of following formula (5) is known compound, can be available from market.
[changing 13]
(5)
The aqueous solution of the compound of formula (5) is cooled off in ice-water bath, add sodium metaperiodate, stirred 5 hours.The decompression concentration of reaction solution, drying under reduced pressure is by the Phosphorylcholine derivative with aldehyde radical shown in the following formula of methanol extraction (6).
[changing 14]
(6)
Then, in the methanol solution of formula (6), add the 3-TSL 8330 of 0.5 equivalent.This mixed solution is at room temperature stirred the stipulated time, ice-cooled then, add sodium cyanoborohydride in right amount, return back to room temperature, stirred 16 hours.Continue in reaction vessel, to feed drying nitrogen therebetween.Behind the filtering-depositing, obtain the methanol solution of formula (3) and/or (4).
Even m, n shown in formula (3) or (4) in the compound change, also fully similarly carry out according to said sequence.The order here is the situation of m=3, n=2.Reaction dissolvent is not particularly limited, except that above-mentioned methyl alcohol, can makes alcohol such as water, ethanol, propyl alcohol, butanols, non-protonic solvents such as DMF or DMSO.But, be the polymerization in preventing to react, preferred dehydrated solvent, methyl alcohol wherein preferably dewaters.
(3) or the methoxyl group in (4) (OMe) when being ethyoxyl (OEt), can change methyl alcohol into ethanol and react, during for Cl, can only change to dimethyl formamide or dimethyl sulfoxide (DMSO).
And, among OMe that combines with Si or OEt or the Cl, when 2 or 1 are replaced by one of them of methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, also can fully similarly prepare with said method.
" preparation method who contains the Phosphorylcholine based compound of formula (4) "
Phosphorylcholine derivative shown in the following formula (5) is dissolved in the mixed liquor of distilled water and acetonitrile.The Phosphorylcholine derivative of following formula (5) is known compound, can buy from market.
[changing 15]
(5)
The aqueous solution of the compound of formula (5) is cooled off in ice-water bath, add sodium metaperiodate and ruthenium trichloride, stirred 3 hours.The decompression concentration of reaction solution, drying under reduced pressure is by the Phosphorylcholine derivative with carboxyl shown in the following formula of methanol extraction (7).
[changing 16]
(7)
Then,, in formula (7), add thionyl chloride in N '-dimethyl formamide, make the chloride thing, then add 0.5 equivalent 3-TSL 8330 and 2 equivalent triethylamines at N.This mixed solution is at room temperature stirred the stipulated time, obtain the N of formula (4), N '-dimethyl formamide solution.
Even m, n in the compound shown in the formula (4) change, also similarly carry out according to said sequence fully.The order here is the situation of m=3, n=2.Reaction dissolvent is not particularly limited, removes above-mentioned N, outside N '-dimethyl formamide, can also use non-protonic solvents such as acetonitrile, oxolane, dimethyl sulfoxide (DMSO).Be the polymerization in preventing to react, preferred dehydrated solvent.
In addition, 2 or 1 also can fully similarly prepare with said method when one of them replaces by methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group among OMe that combines with Si or OEt or the Cl.
" about combinative reactive group of part or adsorption group "
In the above-mentioned reaction, do not make Phosphorylcholine base and all hydroxyl reactions (conditioned reaction amount), then residual hydroxyl becomes combinative reactive group of part or adsorption group.This particle is the affine particle of claim 2, is the particle that Phosphorylcholine base shown in the formula (1) and the combinative reactive group of part or adsorption group directly are present in the inorganic particle surface.Making this residual hydroxyl combine the affine particle that the particle that obtains then is a claim 3 with part, is the particle that Phosphorylcholine base shown in the formula (1) and part directly are present in the surface of inorganic particle.
The affine particle person of being to use of claim 2 is according to the material that will catch (target substance), the goods form that it is combined with part arbitrarily.The affine particle of claim 3 is the goods forms that combine with part in advance.The affine particle of claim 1 is the affine particle that the Phosphorylcholine base of formula (1) at least is present in particle surface, be no matter not part or combinative reactive group of part or adsorption group are arranged, the user is according to the protein that will catch (certain target substance), the goods form that it is combined with part arbitrarily.In addition, so long as the Phosphorylcholine base of formula (1) is present in particle surface at least, then the present invention comprises any type of affine particle, for example also comprises the form of claim 2 and claim 3.
" part and associated methods " with particle of hydroxyl
When part is protein, use the hydroxyl on the cyanogen bromide-activated particle.Make amino and its reaction in the protein, with this conjugated protein.
Make any compound and this hydroxyl reaction, can make this functional group become combinable reactive group of part or adsorption group with functional group.
When being carboxyl " import to reactive group in the particle or adsorption group "
Step 1: the method by known method or exploitation from now on imports carboxyl in any particle.Carboxyl can directly import particle surface.
Step 2: make compound that contains Phosphorylcholine shown in the following formula (2) and particle reaction by conventional method, make the Phosphorylcholine base form amido link, make residual carboxyl as combinative reactive group of part or adsorption group with carboxyl.
Do not make the Phosphorylcholine base combine (conditioned reaction amount) with all carboxyls, then residual carboxyl becomes combinative reactive group of part or adsorption group.
[changing 17]
(8)
" import the method for carboxyl to particle surface "
(step 1) is as described below for the known method of importing carboxyl in particle.
1. import carboxyl by surface modifier
Use has surface modifiers such as the alkoxy silane, chlorosilane, silazane of carboxyl, and the inorganic particle surfaces such as particle of containing silicon ammonia alcohol are handled.
For example silica dioxide granule is handled, imported carboxyl by the triethoxysilylpropyltetrasulfide succinyl oxide.Specifically, the triethoxysilylpropyltetrasulfide succinyl oxide is dissolved in N, dinethylformamide adds distilled water and 4-dimethylaminopyridine, at room temperature stirs 16 hours, obtains the silane coupler with carboxylic acid shown in the following formula (3).This reaction is the hydrolysis of the succinyl oxide that undertaken by 4-dimethylaminopyridine.
By having the silane coupler of carboxylic acid, silica dioxide granule is handled, import carboxyl.Specifically, silica is immersed in water-2-propyl alcohol mixed liquor, adds silane coupler, be heated to 100 ℃ then, reacted 6 hours with carboxylic acid.Be cooled to room temperature, use methanol wash silica then, drying can obtain the particle that carboxyl directly imports to silica surface.The particle of preferably taking this processing also has particles such as glass, aluminium oxide, talcum powder, clay, mica, asbestos, titanium oxide, zinc white, iron oxide except that silica.
2. import carboxyl (with reference to the special fair 1-54379 communique of Japan, the special fair 1-54380 communique of Japan, the special fair 1-54381 communique of Japan) by the organosilicon gas phase treatment
At first by 1,3,5, the 7-tetramethyl-ring tetrasiloxane is handled particle surface, makes Si-H base that imports to the surface and the monomer reaction with carboxyl, obtains the carboxylated surface.Preferably various inorganic particles (mica, talcum powder, kaolin, aluminium oxide, titanium oxide, zinc oxide, iron oxide, various inorganic pigment) are handled.
It is monomer that the monomer that uses in this method can use carboxyl.As long as being monomer, carboxyl has carboxyl and polymerisable vinyl, acrylic isoreactivity position.
" in particle, import the method for Phosphorylcholine base " with carboxyl
Then, below represent to import the method (step 2) of Phosphorylcholine base to the carboxylated particle surface.
The particle that the surface is had carboxyl is immersed in the solution of N-hydroxy-succinamide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the surface of particle is covered by active ester groups.To wherein adding the amino Phosphorylcholine derivative solution that has shown in the formula (7), import the Phosphorylcholine base.
" about combinative reactive group of part or adsorption group "
In the above-mentioned reaction, do not make the Phosphorylcholine base combine (conditioned reaction amount) with whole carboxyl, then residual carboxyl becomes combinative reactive group of part or adsorption group.This particle is the affine particle of claim 2, is the particle that Phosphorylcholine base shown in the formula (1) and the combinative reactive group of part or adsorption group directly are present in the inorganic particle surface.This part combines with combinative reactive group of this part or adsorption group, then becomes the affine particle of claim 3, is the particle that Phosphorylcholine base shown in the formula (1) and part directly are present in the surface of inorganic particle.
The affine particle person of being to use of claim 2 is according to the material that will catch (target substance), the goods form that it is combined with part arbitrarily.The affine particle of claim 3 is the goods forms that combine with part in advance.The affine particle of claim 1 is the affine particle that there is the Phosphorylcholine base of formula (1) at least in particle surface, be no matter not part or combinative reactive group of part or adsorption group are arranged, the user is according to the material that will catch (target substance), the goods form that it is combined with any part.So long as there is the Phosphorylcholine base of formula (1) at least in particle surface, then the present invention comprises any type of affine particle, for example also comprises the form of claim 2 and claim 3.
In the above-mentioned reaction, make carboxyl residual as combinative reactive group of part or adsorption group, this can be undertaken by the reacting dose that adjusting imports to the silane coupler with carboxylic acid in the Phosphorylcholine base.
Make the compound and this carboxyl reaction that have functional group arbitrarily, this functional group can be used as combinative reactive group of part or adsorption group.
" part and associated methods " with particle of carboxyl
When part was protein, the carboxyl on the inorganic particle was immersed in the solution of N-hydroxy-succinamide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, made particle surface form active ester.Make amino and its reaction in the protein, with this conjugated protein.Make the compound and this hydroxyl reaction that have functional group arbitrarily, then this functional group can be used as combinative reactive group of protein or adsorption group.
" the affine separation method of target substance "
Use above-mentioned gained affine particle of the present invention, can carry out the affine separation method of target substance of the present invention.
The inventive method is utilized inorganic particle, can be easy carry out high-precision separation, put from this, be the isolation and purification method of the target substance that is with historically new significance.
Method of the present invention comprises following three steps.When being the affine particle of part combination in advance (claim 2), the 1st step is carried out, and therefore omits.
1. make part and chemically combined the 1st step of affine particle arbitrarily; wherein; affine particle is characterised in that: the surface of inorganic particle has the affine particle of the Phosphorylcholine base shown in the formula (1) with the form of covalent bond; perhaps the surface that has Phosphorylcholine base shown in the formula (1) and inorganic particle with the form of covalent bond, the surface of inorganic particle has part with the form of covalent bond or absorption, and this part and certain target substance have pathoklisis.
For example, the surface of inorganic particle has the Phosphorylcholine base shown in the formula (1) and combinative reactive group of part or adsorption group, wherein the former is with covalent bonds, the latter is with covalent bond or adsorption form combination, with so affine particle and 1ml arbitrarily part PBS solution pack in the 2ml microcentrifugal tube, 4 ℃ of slowly vibrations 30 minutes down.With 5000rpm centrifugal 5 minutes, supernatant inclined.In order to clean, add 1ml PBS solution, slowly vibration, with 5000rpm centrifugal 5 minutes, supernatant inclined.With this cleaning operation triplicate.
2. the affine particle for preparing in the first step is scattered in the 2nd step in the liquor sample, wherein said liquor sample contains can pass through part selectivity captured object material arbitrarily.
For example, the affine particle for preparing in the 1st step is scattered in the liquor sample, wherein contain can be by the captured object material optionally of part arbitrarily for liquor sample, 4 ℃ of slowly vibrations 30 minutes down.With 5000rpm centrifugal 5 minutes, supernatant inclined.In order to clean, add 1ml PBS solution, slowly vibration, with 5000rpm centrifugal 5 minutes, supernatant inclined.With this cleaning operation triplicate.
3. from isolated affine particle, reclaim the 3rd step of captured object material.
For example, in order to reclaim the captured object material from affine particle, add the 1ml elution buffer, slowly vibrated 30 minutes down at 4 ℃, wash-out target substance from particle reclaims supernatant.Add the 1ml elution buffer, slowly vibration with 5000rpm centrifugal 5 minutes, is reclaimed supernatant.Should operate and repeat twice.
Fig. 1 is the ideograph of expression by the optionally difference of affine particle of the present invention and affine particle capture target substance in the past.
Embodiment
Further describe the present invention according to embodiment below.The present invention is not subjected to the qualification of these embodiment.The Phosphorylcholine base that imports to particle surface can confirm by FT-IR and elementary analysis, quantitatively.
" synthesis example 1 "
" aldehyde compound that contains the Phosphorylcholine base "
6.29g 1-α-glyceryl phosphoryl choline is dissolved in 210ml distilled water, in ice-water bath, cools off.Add the 10.23g sodium metaperiodate, stirred 5 hours.The decompression concentration of reaction solution, drying under reduced pressure is by the methanol extraction object.Following compound (6) is represented its structure.
The 1H NMR wave spectrum of the compound of formula (6) in heavy water as shown in Figure 2.The compound of formula (6) becomes poised state with formula (9) in water, therefore actual wave spectrum has reacted formula (6) and formula (9) both sides.
[changing 18]
(6)
[changing 19]
(9)
" synthesis example 2 "
" carboxylic acid compound that contains the Phosphorylcholine base "
In the 200ml flask, add 5g glyceryl phosphoryl choline, 17g sodium metaperiodate, 81mg ruthenium trichloride n hydrate and 70% ion exchange water, 30g acetonitrile.At room temperature stirred 2 hours, and filtered then, from filtrate, remove and desolvate.From the gained solid matter, extract target compound with methyl alcohol, then obtain target compound (7) by removing methyl alcohol.
The 1H NMR wave spectrum of the compound of formula (7) in heavy water as shown in Figure 3.
[changing 20]
(7)
" synthesis example three "
" compound of formula (10) "
The compound of 5.0g synthesis example 1 is dissolved in the 55ml dehydration methyl alcohol, with gas in the drying nitrogen displacement container.Then in the methanol solution of compound 1, add 2.84g 3-TSL 8330.This mixed solution at room temperature stirred spend the night, ice-cooled then, add the 1.39g sodium cyanoborohydride, return back to room temperature, stirred 5 hours.Continue to feed drying nitrogen therebetween in the reaction vessel.Filtering-depositing obtains containing the methanol solution as the following formula (10) of target substance then.
[changing 21]
(10)
" synthesis example 4 "
" compound of formula (11) "
Make the compound of 9.0g synthesis example 4 be scattered in 300ml N, in N '-dimethyl formamide, with gas in the drying nitrogen displacement container.Then add the 4.5g thionyl chloride, stirred 15 minutes, add 2.84g 3-TSL 8330,9.5g triethylamine then.This mixed solution at room temperature stirred spend the night, filtering-depositing obtains containing target substance then---the N of the compound of following formula (11), N '-dimethyl formamide solution.
[changing 22]
(11)
" reference example 1 "
" surface of inorganic particle has the Phosphorylcholine particle (PC particle (A)) of Phosphorylcholine base with the form of covalent bond "
Get the methanol solution that 97.7 μ l contain the compound of the formula (10) of preparation in the 50 μ mol synthesis examples 3, add 47.5ml methyl alcohol, 2.5ml distilled water, adding the 5g average grain diameter again is that 1.5 μ m, specific area are 6m
2The silica gel of/g.Make this particle dispersion soln flow through night next time, carry out coupling at 80 ℃.Use the methyl alcohol eccentric cleaning after refluxing, obtain the PC particle (hereinafter referred to as PC particle (A)) of claim 1.The P quantitative assay of the PC particle of handling according to above order, with the surface modifier of synthesis example 3 (A) as shown in Figure 4.The PC import volume of obtaining thus is the particle of 3.1 μ mol/g, can confirm that the PC group has imported particle surface.
" reference example 2 "
" surface of inorganic particle has the Phosphorylcholine particle (PC particle (B)) of Phosphorylcholine base with the form of covalent bond "
Get the dimethyl formamide solution that 27.8 μ l contain the compound of the formula (11) of preparation in the 50 μ mol synthesis examples 4, add the 50ml dimethyl formamide, adding the 5g average grain diameter again is that 1.5 μ m, specific area are 6m
2The silica gel of/g.Make this particle dispersion soln flow through night next time, carry out coupling at 160 ℃.Use the methyl alcohol eccentric cleaning after refluxing, obtain the PC particle (hereinafter referred to as PC particle (B)) of claim 1.According to above order, the P quantitative assay of the PC particle (B) that the surface modifier of usefulness synthesis example 4 is handled as shown in Figure 4.The PC import volume of obtaining thus is the particle of 3.4 μ mol/g, can confirm that the PC group has imported particle surface.
" reference example 3 "
" surface of inorganic particle has the Phosphorylcholine particle (PC particle (C)) of Phosphorylcholine base with the form of covalent bond "
Get the dimethyl formamide solution that 27.8 μ l contain the compound of the formula (11) of preparation in the 50 μ mol synthesis examples 4, add 47.5ml dimethyl formamide, 2.5ml distilled water, adding the 5g average grain diameter again is that 1.5 μ m, specific area are 6m
2The silica gel of/g.Make this particle dispersion soln flow through night next time, carry out coupling at 160 ℃.Use the methyl alcohol eccentric cleaning after refluxing, obtain the PC particle (hereinafter referred to as PC particle (C)) of claim 1.According to above order, the P quantitative assay of the PC particle (C) that the surface modifier of usefulness synthesis example 4 is handled as shown in Figure 4.The PC import volume of obtaining thus is the particle of 7.3 μ mol/g, can confirm that the PC group has imported particle surface.
The 13C-CPMAS wave spectrum of the PC particle (A) of reference example 1 and 13C-PSTMAS wave spectrum are as shown in Figure 5.The PSTMAS wave spectrum is the method for wave spectrum of strand of motion of optionally gaining freedom, and is the method that is widely used in the modification link analysis of particle surface.Among Fig. 5, observe wave spectrum at the 54.2ppm place from the carbon of Phosphorylcholine base.
In the 31P-CPMAS wave spectrum of the PC particle (A) of reference example 1 shown in Figure 6, the NaH that is measuring as object
2PO
4Chemical displacement value place much at one detects peak value, can confirm the existence of phosphate thus.Above result can think can import the Phosphorylcholine base carrier silica gel surface.
Near 9ppm, 23ppm, observe wave spectrum among Fig. 5, near 60ppm, 69ppm, observe wave spectrum from the ethyl in the Phosphorylcholine from the carbon of interval group propyl group.As known from the above: the structure of formula (10) and (11) is not destroyed can to import in the silica gel.
Fig. 7 represents the FT-IR wave spectrum of the PC particle (C) of reference example 3.At 1650cm
-1Near can observe the distinctive absorption of amido link.
" the non-special absorption evaluation of Phosphorylcholine particle and protein "
Get in the 25mg reference example 1 PC particle (A), (B), (C) of preparation in the not importing Phosphorylcholine base that uses, untreated silica gel particle (abbreviation be untreated particle) and the reference example 1,2,3 respectively, add 1ml distilled water, carry out ultrasonic wave processing in 1 minute.The centrifugal distilled water of removing adds 1ml 100 μ g/ml albumin or 100 μ g/ml lysozymes, at room temperature reacts 1 hour, carries out 5 centrifugal purifications (5000g) with PBS, washing.Then add 1ml1%SDS, at room temperature reacted 1 hour, centrifugal (5000g) carries out quantitatively supernatant with MICRO BCA method, and its result as shown in Figure 8.Compare with the particle that is untreated, with PC particle (A) the strong inhibition albumin of Phosphorylcholine base processing, the absorption of lysozyme.PC particle (B), (C) and be untreated particle or PC particle (A) are compared, and albumin, lysozyme all further are suppressed absorption.
" embodiment 1 "
" surface of inorganic particle has Phosphorylcholine base and amino affine particle (Af particle (A)) with the form of covalent bond "
Get 87.9 μ l and contain the methanol solution of the compound of the formula (10) of preparation in the 45 μ mol synthesis examples 3 and the methanol solution that 50 μ l contain 5 μ mol 3-TSL 8330s, add 47.5ml methyl alcohol, 2.5ml distilled water, adding the 5g average grain diameter again is that 1.5 μ m, specific area are 6m
2The silica gel of/g.Make this particle dispersion soln flow through night next time, carry out coupling at 80 ℃.Use the methyl alcohol eccentric cleaning after refluxing, obtain the affine particle (hereinafter referred to as Af particle (A)) of claim 1.By above order, the P quantitative assay of the Af particle of handling with the surface modifier of synthesis example 3 (A) as shown in Figure 4.Obtain the particle that the PC import volume is 2.7 μ mol/g thus, can confirm that the PC group has imported particle surface.
" embodiment 2 "
" surface of inorganic particle has Phosphorylcholine base and amino affine particle (Af particle (B)) with the form of covalent bond "
Get 250 μ l and contain the dimethyl formamide solution of the compound of the formula (11) of preparation in the 45 μ mol synthesis examples 4 and the dimethyl formamide solution that 50 μ l contain 5 μ mol 3-TSL 8330s, adding the 5g average grain diameter again is that 1.5 μ m, specific area are 6m
2The silica gel of/g.Make this particle dispersion soln flow through night next time, carry out coupling at 160 ℃.Use the methyl alcohol eccentric cleaning after refluxing, obtain the affine particle (hereinafter referred to as Af particle (B)) of claim 1.By above order, the P quantitative assay of the Af particle (B) that the surface modifier of usefulness synthesis example 4 is handled as shown in Figure 4.The PC import volume of obtaining thus is the particle of 3.3 μ mol/g, can confirm that the PC group has imported particle surface.
" embodiment 3 "
" surface of inorganic particle has Phosphorylcholine base and amino affine particle (Af particle (C)) with the form of covalent bond "
Get 250 μ l and contain the dimethyl formamide solution of the compound of the formula (11) of preparation in the 45 μ mol synthesis examples 4 and the dimethyl formamide solution that 50 μ l contain 5 μ mol 3-TSL 8330s, add 47.5ml dimethyl formamide, 2.5ml distilled water, adding the 5g average grain diameter again is that 1.5 μ m, specific area are 6m
2The silica gel of/g.Make this particle dispersion soln flow through night next time, carry out coupling at 160 ℃.Use the methyl alcohol eccentric cleaning after refluxing, obtain the affine particle (hereinafter referred to as Af particle (C)) of claim 1.By above order, the P quantitative assay of the Af particle (C) that the surface modifier of usefulness synthesis example 4 is handled as shown in Figure 4.The PC import volume of obtaining thus is the particle of 6.3 μ mol/g, can confirm that the PC group has imported particle surface.
" selective evaluation 1 of affine particle "
Then provide the affine separation method shown in the claim 6.In 25mg embodiment 1,2,3, add 1ml distilled water among the Af particle (A) of preparation, (B), (C), carry out 1 minute ultrasonic wave and handle.By the centrifugal distilled water of removing, add the sodium cyanoborohydride (シ ア ノ ト リ ヒ De ロ ホ ウ acid Na ト リ ウ system) that 1ml 8% glutaraldehyde solution and 10mg are used for stablizing schiff bases, at room temperature reacted 5 hours, use MQ water centrifugal purification (5000g) 5 times.Obtaining glutaraldehyde is the affine particle of the claim 2 of combinative reactive group of part or adsorption group.Then add 1ml 1mg/ml BA or 1mg/ml human hemoglobin and 10mg sodium borohydride, at room temperature reacted 1 day, carry out 4 centrifugal purifications (5000g) with PBS.This BA or human hemoglobin are part.It below is the affine separation method shown in the claim 7.In order to make residual glutaraldehyde base inactivation, add 1ml ethanolamine hydrochloric salt (0.5M, pH 7.1) and 10mg sodium borohydride (ト リ ヒ De ロ ホ ウ acid Na ト リ ウ system), at room temperature reacted 1 hour, and carried out 4 centrifugal purifications (5000g), obtain the affine particle of claim 3 with PBS.Then add anti-BA antibody of 1ml HRP mark (10 μ g/ml) or HRP mark antihuman hemoglobin antibody (10 μ g/ml), at room temperature reacted 1 hour, use PBS centrifugal purification (5000g) 5 times.Add 1ml PBS again, stir, 10 μ l are transferred to 96 orifice plates,, measure with 450nm with the substrate TMBZ experiment that develops the color.Its result such as Fig. 9, Figure 10, shown in Figure 11.Af particle (A) has selectivity to human hemoglobin-HRP mark antihuman hemoglobin antibody.Af particle (B), Af particle (C) all have selectivity to the anti-BA antibody of BA-HRP mark, human hemoglobin-HRP mark antihuman hemoglobin antibody.
" selective evaluation 2 of affine particle "
Use the goat antiserum of antihuman hemoglobin to carry out the selectivity experiment.In 25mg embodiment 1,3, add 1ml distilled water among the Af particle (A) of preparation, (C), carry out 1 minute ultrasonic wave and handle.The centrifugal distilled water of removing adds the sodium cyanoborohydride that 1ml 8% glutaraldehyde solution and 10mg are used for stablizing schiff bases, at room temperature reacts 5 hours, with MQ water centrifugal purification (5000g), cleans 5 times.Obtaining glutaraldehyde is the affine particle of the claim 2 of combinative reactive group of part or adsorption group.Then add 1ml 1mg/ml human hemoglobin and 10mg sodium borohydride, at room temperature reacted 1 day, use PBS centrifugal purification (5000g) 4 times.This human hemoglobin is a part.It below is the affine separation method shown in the claim 7.In order to make residual glutaraldehyde base inactivation, add 1ml ethanolamine hydrochloric salt (0.5M, pH7.1) and 10mg sodium borohydride, at room temperature reacted 1 hour, carry out 4 centrifugal purifications (5000g) with PBS, obtain the affine particle of claim 3.Then add 1ml dilution and be 100 times goat antiserum, at room temperature reacted 1 hour.Then centrifugal (5000g) obtains supernatant (supernatant component).Carry out 5 centrifugal purifications (5000g) with PBS.Then add 1mlGly-HCl buffer solution (0.2M, pH 2.5), at room temperature reacted 1 hour, wash-out antihuman hemoglobin antibody, centrifugal (5000g) obtains supernatant (elution fraction).This supernatant component and elution fraction are crossed SDS-PAGE, carry out silver and dye, the result as shown in figure 12.For Af particle (A), (C), all the heavy chain band of antibody in the elution fraction of visible color depth is not seen other band, is the particle of highly selective capture antibody as can be known therefore.
" selective evaluation 3 of affine particle "
The goat antiserum that use has mixed antihuman hemoglobin carries out the selectivity experiment.In 25mg embodiment 1,3, add 1ml distilled water among the Af particle (A) of preparation, (C), carry out 1 minute ultrasonic wave and handle.The centrifugal distilled water of removing adds the sodium cyanoborohydride that 1ml 8% glutaraldehyde solution and 10mg are used for stablizing schiff bases, at room temperature reacts 5 hours, with MQ water centrifugal purification (5000g), cleans 5 times.Obtaining glutaraldehyde is the affine particle of the claim 2 of combinative reactive group of part or adsorption group.Then add 1ml 1mg/ml human hemoglobin and 10mg sodium borohydride, at room temperature reacted 1 day, use PBS centrifugal purification (5000g) 4 times.This human hemoglobin is a part.It below is the affine separation method shown in the claim 7.In order to make residual glutaraldehyde base inactivation, add 1ml ethanolamine hydrochloric salt (0.5M, pH7.1) and 10mg sodium borohydride, at room temperature reacted 1 hour, carry out 4 centrifugal purifications (5000g) with PBS, obtain the affine particle of claim 3.Then add dilution that 1ml mixed 50 μ g antihuman hemoglobins and be 100 times lowlenthal serum, at room temperature reacted 1 hour.Then centrifugal (5000g) obtains supernatant (supernatant component).Carry out 5 centrifugal purifications (5000g) with PBS.Then add 1ml Gly-HCl buffer solution (0.2M, pH 2.5), at room temperature reacted 1 hour, wash-out antihuman hemoglobin antibody, centrifugal (5000g) obtains supernatant (elution fraction).This supernatant component and elution fraction are crossed SDS-PAGE, carry out silver and dye, the result as shown in figure 13.For Af particle (A), (C), the heavy chain band of antibody in the elution fraction of all visible color depth, therefore the band that other visible color is shallow is the particle of highly selective capture antibody as can be known.From the concentration of band, the antibody capture amount is about 10-20 μ g.By the antibody activity of sandwich ELISA affirmation elution fraction, Af particle (A) is that 13.0 μ g, Af particle (C) are the activity about 10.1 μ g as can be known.
" comparative example 1 "
" surface of inorganic particle has amino affine particle (amino particle) with the form of covalent bond "
Get the methanol solution that 500 μ l contain 50 μ mol 3-TSL 8330s, add 47.5ml methyl alcohol, 2.5ml distilled water, adding the 5g average grain diameter again is that 1.5 μ m, specific area are 6m
2The silica gel of/g.Make this particle dispersion soln flow through night next time, carry out coupling at 80 ℃.Use the methyl alcohol eccentric cleaning after refluxing, obtain amino particle.Should add 1ml distilled water in the amino particle to 25mg, and carry out 1 minute ultrasonic wave and handle.By the centrifugal distilled water of removing, add the sodium cyanoborohydride that 1ml 8% glutaraldehyde solution and 10mg are used for stablizing schiff bases, at room temperature reacted 5 hours, with MQ water centrifugal purification (5000g), clean 5 times.Glutaraldehyde is combinative reactive group of part or adsorption group.Then add 1ml1mg/ml BA or 1mg/ml human hemoglobin and 10mg sodium borohydride, at room temperature reacted 1 day, carry out 4 centrifugal purifications (5000g) with PBS.This BA or human hemoglobin are part.For making residual glutaraldehyde base inactivation, add 1ml ethanolamine hydrochloric salt (0.5M, pH 7.1) and 10mg sodium borohydride, at room temperature reacted 1 hour, carry out 4 centrifugal purifications (5000g) with PBS, obtain the affine particle of claim 3.Then add the anti-BA antibody of HRP mark of 1ml10 μ g/ml or the HRP mark antihuman hemoglobin antibody of 10 μ g/ml, at room temperature reacted 1 hour, carry out 5 centrifugal purifications (5000g) with PBS.Add 1ml PBS again, stir, 10 μ l are transferred to 96 orifice plates, use the substrate TMBZ experiment that develops the color, measure with 450nm.Its result as shown in figure 14.The non-specific adsorption of protein is many, and selectivity is low.
Industrial applicability
Affine particle of the present invention is only caught the target protein that will separate, and is therefore selectively high. Other excellent dispersion is as easy as rolling off a logly separated from liquor sample. By having utilized the affine particle of cheap inorganic particle, can be easy and isolation of target substances accurately, therefore can be used as the organism related industry that requires the high-purity separation target substance.
Claims (11)
1. affine particle, it is characterized in that: the surface of inorganic particle has the Phosphorylcholine base shown in the following formula (1) with the form of covalent bond, this Phosphorylcholine base is that the Si-OMe dehydration by compound shown in the hydroxyl on inorganic particle surface and following formula (3) or (4) forms chemical bond and imports, and the surface of inorganic particle has the reactive group or the adsorption group that can combine with the part that certain target substance is had pathoklisis with the form of covalent bond or absorption:
In the formula, m is 2-6,
OMe can be replaced by OEt or Cl, can be replaced by methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group for maximum two among OMe that combines with Si or OEt or the Cl.
2. affine particle, it is characterized in that: the surface of inorganic particle has the Phosphorylcholine base shown in the following formula (1) with the form of covalent bond, this Phosphorylcholine base is that the Si-OMe dehydration by compound shown in the hydroxyl on inorganic particle surface and following formula (3) or (4) forms chemical bond and imports, and the surface of inorganic particle has the part that has pathoklisis with certain target substance with the form of covalent bond or absorption:
In the formula, m is 2-6,
OMe can be replaced by OEt or Cl, can be replaced by methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group for maximum two among OMe that combines with Si or OEt or the Cl.
3. claim 1 or 2 affine particle, it is characterized in that: above-mentioned inorganic particle is one or more inorganic particles that are selected from silica, titanium oxide, zinc white, aluminium oxide, iron oxide, talcum powder, mica, collaurum, its average grain diameter is 20nm-500 μ m, and proportion is 1.0g/cm
3More than.
4. the affine particle of claim 3, wherein said mica is a sericite.
5. claim 1 or 2 affine particle, it is characterized in that: above-mentioned part is one or more parts that are selected from various antibody, antigen, enzyme, peptide, DNA, RNA, nucleic acid ligands, albumin A, Protein G, avidin, biotin.
6. claim 1 or 2 affine particle is characterized in that above-mentioned part is a substrate.
7. claim 1 or 2 affine particle is characterized in that above-mentioned part is an acceptor.
8. claim 1 or 2 affine particle is characterized in that above-mentioned part is an agglutinin.
9. claim 1 or 2 affine particle is characterized in that above-mentioned part is a chelate compound.
10. the affine separation method of a target substance that is undertaken by inorganic particle, it is characterized in that comprising following steps: the 1st step that (1) makes the affine particle of claim 1 combine with part arbitrarily, (2) make the affine particle of making in the 1st step be scattered in the 2nd step in the liquor sample, wherein liquor sample contains by part selectivity captured object material arbitrarily, and (3) reclaim the 3rd step by the target substance of affine particle capture.
11. the affine separation method of a target substance that is undertaken by inorganic particle, it is characterized in that comprising following steps: (1) makes the affine particle of claim 2 be scattered in the 1st step in the liquor sample, wherein said liquor sample contains by part selectivity captured object material arbitrarily, and (2) reclaim the 2nd step by the target substance of affine particle capture.
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| WO2010101126A1 (en) * | 2009-03-02 | 2010-09-10 | 株式会社資生堂 | Surface-modified base plate, biochip and process for producing the biochip |
| EP2541250A1 (en) * | 2011-06-30 | 2013-01-02 | Koninklijke Philips Electronics N.V. | Molecular architecture on magnetic particles for affinity assays with low non-specific binding |
| CN105148867A (en) * | 2015-09-15 | 2015-12-16 | 李云峰 | Graphene oxide-recombined streptococcal protein G non-covalent composite material as well as preparation method and application thereof |
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| CN1214771A (en) * | 1996-01-30 | 1999-04-21 | 阿比翁参股及管理股份有限公司 | Carrier material loadable by through flow for solid phase assays |
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2005
- 2005-05-18 CN CN2005800165535A patent/CN1956779B/en not_active Expired - Fee Related
- 2005-05-18 CN CNA2008101903039A patent/CN101513606A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1214771A (en) * | 1996-01-30 | 1999-04-21 | 阿比翁参股及管理股份有限公司 | Carrier material loadable by through flow for solid phase assays |
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| Publication number | Publication date |
|---|---|
| CN1956779A (en) | 2007-05-02 |
| CN101513606A (en) | 2009-08-26 |
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