CN100379761C - Titanium dioxide complex having molecule distinguishability - Google Patents
Titanium dioxide complex having molecule distinguishability Download PDFInfo
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- CN100379761C CN100379761C CNB2004800080532A CN200480008053A CN100379761C CN 100379761 C CN100379761 C CN 100379761C CN B2004800080532 A CNB2004800080532 A CN B2004800080532A CN 200480008053 A CN200480008053 A CN 200480008053A CN 100379761 C CN100379761 C CN 100379761C
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Abstract
A titanium dioxide complex capable of distinguishing molecules is obtained by modifying the surface of a fine titanium dioxide particle with a hydrophilic polymer in such a manner that titanium dioxide is bonded via an ester bond to a carboxyl group of the hydrophilic polymer and immobilizing a molecule having an ability to specifically bind to a target molecule to the carboxyl residue of the hydrophilic polymer. Due to the molecule distinguishability, this titanium dioxide complex can bind specifically to an endocrine disrupting chemical, a pathogenic factor, a cancer cell, etc. and decompose the same by a photocatalytic function.
Description
Technical field
The present invention relates to, can there be the molecule of special binding ability to fix to endocrine disruptor, virulence factor or cancer cells etc., by the Decomposition of demonstrations such as uviolizing, has the titanium dioxide complex of molecule distinguishability to these materials, molecule, cell.
Background technology
In recent years, the biomolecules such as DNA that disclose the molecule distinguishability by will having endocrine disruptor are fixed on supporting body, the material that will be endowed the selection associativity by this method is as surrounding purifying material (for example, with reference to TOHKEMY 2001-81098 communique).In addition, known anatase titanium dioxide has photocatalysis, decomposes organism such as microorganism, dirt, repugnant substance by its strong oxidizing power.Particularly, hardly-degradable substance as endocrine disruptor has also been shown powerful Decomposition, therefore expectation to environmental purification effectively (for example, with reference to Y.Ohko etc.: Environmental Science andTechnology, 35,2365-2368 (2001)).And then, at present by inorganic adsorbents such as titanium dioxide and activated carbon or zeolite carry out compoundization, carried out improving the effort (for example with reference to Japanese kokai publication hei 01-189322 communique) of titanium dioxide decomposition efficiency.Aspect the surface treatment of titanium dioxide, studied by making reduction reactions such as palladium promote catalytic metal to separate out in photocatalyst surface such as titanium dioxide, promote oxidation, the reduction reaction (for example, with reference to Japanese kokai publication sho 60-14940 communique) of photocatalyst.
But the selective binding material at the endocrine disruptor of DNA etc. does not have the practical means of removing or decomposing bonded endocrine disruptor etc., and has bottleneck aspect the detergent power from adsorbing saturated problem yet.In addition, for improving of the motion of above-mentioned titanium dioxide, not combination or the decomposition of pointing to predetermined substance as the ability of photocatalyst.Therefore, it is impossible for example only decomposing with the endocrine disruptor selective binding.In the field of environmental purification, still do not know a recognition objective material and a selective binding as mentioned above, its strong oxidizing power by photocatalyst is decomposed, promptly pass through the technology of titanium dioxide " combination of molecule distinguishability and photo-catalysis capability ".
On the other hand, in recent years as the administering mode of new medicament in field of medicaments, make medicine in vivo or the surface through the time slowly-releasing medicinal design system (drug delivery system: DDS) receive much concern.This is, when improving the drug effect of existing medicine to greatest extent, its side effect is controlled at system in the inferior limit.Carrier as medicament in DDS, (for example non-resolutive polymer or aminoacid polymers have been studied energetically, Japanese kokai publication hei 09-255590 communique), liposome (for example, with reference to TOHKEMY 2003-226638 communique) and protein hollow nanoparticle (for example, with reference to TOHKEMY 2003-286198 communique) etc.In the further progress of DDS notion, target (targeting) DDS is arranged.It is the system that medicine is sent into necessary position in the time of necessity with the amount of necessity, is purpose with guidance type (missile) medicine (guidance type therapy) of finally attacking focus effectively.
For guidance type medicine be, part is written in the DDS carrier, discern combination specifically, carry out target administration by the acceptor that the target cell surface is existed.As with the corresponding part of acceptor of the target administration target of this motility, can enumerate antigen, antibody, peptide, glycolipid or glycoprotein etc.Wherein, (for example play an important role as informational molecule in the iuntercellular information interchange that the sugar chain of cognitive gradually in recent years glycolipid or glycoprotein forms in the generation or the form of the propagation of cell or differentiation, tissue, organism defence or fertilization mechanism or canceration or its shift etc., with reference to N.Yamazaki etc.: Advanced Drug DeriveryReview, 43,225-244 (2000)).
In such DDS, the trial of having carried out using the titanium dioxide with high light catalytic decomposition ability is (with reference to N.Yamazaki etc.: Advanced Drug DeriveryReview, 43,225-244 (2000), TOHKEMY 2002-316950 communique, R.Cai etc.: Cancer Research, 52,2346-2348 (1992)) this be in as the cancer cells of target by after shooting the metallicss such as gold that are loaded with titanium dioxide and concentrating administration, light such as irradiation ultraviolet radiation are so that its kill cancer cell.Known titanium dioxide is in air or solution is medium is the material of stabilizer pole, and does not also have toxic safe material in (shading) animal body.And the activation of controlling titanium dioxide by the switch of light is possible, therefore expectation towards cancer therapy in the application aspect the DDS.
But the iso-electric point of titanium dioxide is about pH6, has the problem points of TiO 2 particles cohesion under near the physiological condition the neutrality.Therefore, can not be with titanium dioxide self directly to intravascular administration, or directly use as the DDS carrier.Still do not know in addition that on titanium dioxide surface the molecule that above-mentioned part etc. is had the selective binding ability carries out the fixed technology, under present situation, sink into the situation of difficulty as the practicability of the DDS of titanium dioxide.That is, even in medical field, pass through " combination of molecule distinguishability and photo-catalysis capability " technology of titanium dioxide, because the problems referred to above point also is not developed.
Summary of the invention
The present inventor has carried out research with keen determination in order to solve above-mentioned problem, discovery is modified the titanium dioxide fine particles surface with hydrophilic macromolecule after, and then will carry out the fixed titanium dioxide complex to the molecule that target molecule has a specific binding capacity, can realize the unification of molecule distinguishability and photo-catalysis capability, thereby finish the present invention.
Promptly, titanium dioxide complex with molecule distinguishability of the present invention is, the titanium dioxide fine particles surface is modified by hydrophilic macromolecule, with the carboxyl of this hydrophilic macromolecule and titanium dioxide by the ester bond bonded simultaneously, the molecule that on the carboxyl residue of above-mentioned hydrophilic macromolecule target molecule is had a specific binding capacity is fixed.By this method, make to have on the TiO 2 particles of photocatalysis, the molecule that importing antibody etc. has specific binding capacity becomes possibility, can prepare the titanium dioxide complex with molecule distinguishability.
The titanium dioxide complex that as a result of obtains with molecule distinguishability, endocrine disruptor, cause of disease material, cancer cells etc. had molecule distinguishability, and, thereby provide titanium dioxide complex with molecule distinguishability by the decomposition reaction of photocatalysis demonstration to these materials.This complex body has the molecule of discerning specifically and catching as target in the water or the aqueous solution, by powerful abilities with its decomposition such as uviolizings.Particularly, can in containing the water solvent of this complex body, use, can correctly discern and catch target molecule, and showing powerful characteristics such as photo-catalysis capability, for example is being extremely useful in the disaggregating treatment of objectionable impurities of representative or the application at medical field such as destruction to specific virulence factor or cancer cells with the water system endocrine disruptor.
Description of drawings
Fig. 1 represents the mode chart with titanium dioxide complex of molecule distinguishability of the present invention.
Fig. 2 represents the titanium dioxide complex by the anti-α of immobilization of the present invention-fetoprotein antibody, to the degrading activity of antigen (α-fetoprotein).To antigenic degrading activity, represent with the minimizing of absorbancy.
Fig. 3 represent by the surface plasma body resonant vibration method estimate of the present invention with immobilization AHS albumin antibody titanium dioxide complex and the result of the associativity of antigen (human serum albumin).In contrast, use the titanium dioxide complex of immobilization streptavidin.
Fig. 4 represents the titanium dioxide complex by immobilization AHS's albumin antibody of the present invention, to the degrading activity result of antigen (human serum albumin).The antigen degrading activity is to be represented by the rate of decomposition (%) that the reduction of following the antigenic of decomposition with the antibodies amount (measuring by the surface plasma body resonant vibration method) is calculated.In contrast, use the titanium dioxide complex of immobilization streptavidin.
Embodiment
Specify embodiments of the present invention according to figure.Fig. 1 represents the mode chart with titanium dioxide complex of molecule distinguishability of the present invention.Promptly, titanium dioxide complex with molecule distinguishability of the present invention, be with titanium dioxide fine particles 1, be dispersed in the dimethyl formamide with hydrophilic macromolecule 2 with a plurality of carboxyls, under 90～180 ℃, carried out hydro-thermal reaction 1～12 hour, and with both by ester bond in conjunction with after, the molecule 3 that on the carboxyl residue of hydrophilic macromolecule 2 target molecule is had a specific binding ability carries out the fixed complex body.Herein, the ester bond of titanium dioxide and hydrophilic macromolecule is that the titanium oxide by particle surface is generated hydroxyl by the water hydration in the reaction system on its surface, and the carboxyl reaction formation ester bond of this hydroxyl and hydrophilic macromolecule forms.All can be suitable for as the various analytical procedures of the confirmation method of ester bond, for example can be by the absorption band 1700～1800cm of infrared spectrophotometry judgement at ester bond
-1Near having or not of infrared absorption confirmed.In addition, fixing for the molecule 3 with specific binding capacity, mainly is the amino that utilizes this molecule to have.In addition, even also be possible for there not being amino molecule to import amino by suitable modifying method, import amino in addition to carboxyl have reactive desired functional group or crosslinked also be possible.
As the titanium dioxide fine particles 1 that uses in the present invention, from coherent problem or using aspect the intravital suitability etc., from the preferred 2～200nm of angular separation particle diameter of the degree of freedom of its use-pattern as being used for cancer therapy.And then, as the titanium dioxide that the present invention uses, can preferably use any one of anatase titanium dioxide and rutile-type.Even this is because different its chemical property of titanium dioxide of crystal type is essentially identical, no matter be that any crystal type can be modified and the molecule with specific binding capacity is fixed with water-soluble polymer.For example, for strong anatase titanium dioxide of destruction of cancer cells selective light catalytic activity etc., can select to use the titanium dioxide of desired crystal type according to purposes.
And then, as long as titanium dioxide is present in the part of particle surface at least, even for example as the matrix material of magneticsubstance and titanium dioxide, the characteristic of the titanium dioxide of particle surface is close approximate, and it promptly is possible therefore by carboxyl the molecule with specific binding capacity being fixed.Therefore, even the matrix material of magneticsubstance and titanium dioxide can be by the time with single TiO 2 particles identical method, preparation has the titanium dioxide complex of molecule distinguishability.
As the hydrophilic macromolecule 2 of the present invention's use, owing to anticipation is used with the state that this titanium dioxide complex is dispersed in the aqueous solution, so preferably water capacitive polymer.Water-soluble polymer as the present invention's use, so long as it is any all applicable to contain the polymer of a plurality of carboxyls, for example, can enumerate carboxymethyl starch, Sensor Chip CM 5, carboxymethyl cellulose, polycarboxylic acid and have the unitary interpolymer of carboxyl (multipolymer) etc.Particularly,, more preferably use polycarboxylic acids such as polyacrylic acid, polymaleic acid, and vinylformic acid/toxilic acid or vinylformic acid/sulfonic acid are monomeric interpolymer (multipolymer) from the angle of the water-disintegrable and solubleness of water-soluble polymer.By with these hydrophilic macromolecule modified titanic oxides, making on the carboxyl residue of hydrophilic macromolecule fixedly becomes possibility to the molecule 3 with desired specific binding capacity.And then even molecule 3 is being fixed the back by the electrostatic repulsion between remaining carboxyl, titanium dioxide complex of the present invention can be kept dispersive state equably near the broad pH scope comprising neutrality.
In the present invention, as titanium dioxide complex being given molecule distinguishability, the molecule 3 with specific binding capacity so long as can get final product with target molecule specificity bonded molecule, is not limited only to the molecule of following qualification.Knowing so intermolecular specificity combination gradually, is diversified in vivo.Therein, can enumerate protein as most important molecule.According to the present invention, can be aptly to as proteinic antibody, part, acceptor, number peptide, also have amino acid to fix.In addition, simple protein is carried out on titanium dioxide complex fixing in can be aldehyde radical with amino and thiol group, for protein with sugar, the target functional group during as immobilization.In addition, also can on the carboxyl of the titanium dioxide that water-soluble polymer is modified, import vitamin H (or avidin) in advance, by making protein and avidin (or biotinyl) crosslinked, utilize vitamin H: the interaction of avidin is fixed.
And then titanium dioxide complex of the present invention can be pointed out the specific factor or part on particle surface.Therefore, the cell as cancer cells for for example having expressed special receptor, pass through part: the specificity of acceptor is in conjunction with this complex body being imported in the cell.As these factors or acceptor, except enumerating except that epidermal growth factor (EGF), transforming the proliferate factor such as multiplicaiton factor, thrombocyte origin multiplicaiton factor, the bone forming factor, nerve growth factor or form the factor, can also enumerate hormone such as Interferon, rabbit, interleukin-, colony stimulating factor, tumour necrosis factor, erythropoietin, Fas antigen, Nrolone Phenylpropionate or part etc.These protein also can be fixed same as described abovely.That is, to finding the specific cells of their corresponding acceptors specifically, the guidance type medicine that structure can the target administration possibility that becomes.
In recent years, receive publicity with particular proteins specificity bonded aptamer.This is fit, also can be used as the molecule 3 with the specificity associativity of giving molecule distinguishability of the present invention and is utilized.To nucleic acid fixedly the time, when carrying out DNA cloning by polymerase chain reaction (PCR), by with amination primer, biotinylation primer, mercaptan primer synthetic modification DNA, can adopt uses the same method fixes on modified titanic oxide.For example, with being used for fixing of aminated dna the time, on the carboxyl of the titanium dioxide of modifying in advance, import ester, can make aminated dna on modified titanic oxide, carry out covalent bonds by nucleophilic substitution reaction as N-hydroxy-succinamide (NHS) and so on.When using mercaptan DNA,, similarly can on modified titanic oxide, fix mercaptan DNA by making it carry out making 2-(2-pyridyl dimercapto) ethamine effect after the NHS reaction on the carboxyl.
When use is fixed the aldehyde radical of molecule, also can make carboxyl and NHS reaction after, be attached on the modified titanic oxide by make the molecule that is fixed with hydrazine, with amino boronation sodium reduction.In addition, if with vitamin H hydrazides or amination vitamin H with carboxybiotinization, just can be easily the molecule that is fixed of avidin 9 albefaction be imported on the modified titanic oxide.Need only suitably selective reagents, modification and cross-linking method as mentioned above, immobilization easily has the molecule 3 of diversified specific binding capacity to the carboxyl residue that imports on modified titanic oxide.
As mentioned above, as long as have can with the carboxyl residue bonded functional group that on modified titanic oxide, imports, and known this combining method except protein, nucleic acid or carbohydrate, also can preferably utilize lipid or various physiologically active substances etc. as the molecule with specific binding capacity (3).
On the other hand, it is physiological condition in the organism that the titanium dioxide complex with molecule distinguishability of the present invention is handled when medical or medical in the objectionable impurities of desiring to be applied to water system, therefore requires dispersion equably in the neutral water solvent.As mentioned above, the titanium dioxide complex with molecule distinguishability of the present invention has remaining carboxyl residue, and therefore the repulsion that is produced by the negative charge of carboxyl in water solvent acts between complex body.Therefore, even in the aqueous solution in the pH field of the broad range of pH3～13, this complex body still can keep not condensing ground homogeneous dispersion state.Therefore, the titanium dioxide complex with molecule distinguishability of the present invention is distributed in water, various pH buffered soln, transfusion or the physiological saline, making provides homogeneous, stable dispersion liquid to become possibility.Can prepare the ointment that contains this dispersion liquid or sprays etc. in addition.This characteristic, especially extremely useful in the DDS that titanium dioxide is applied to the inside and outside time.That is, though also not cohesion under near the physiological condition of the dispersion liquid of the titanium dioxide complex with molecule distinguishability of the present invention neutral, so make and carry out target administration in affected tissue direct injection or intravenous injection and become possibility.In addition, the ointment or the sprays that will contain this dispersion liquid directly are coated on affected parts such as skin, and making by the treatment of enforcement light such as sunlight or UV-lamp becomes possibility.
And then, the titanium dioxide complex with molecule distinguishability of the present invention to be utilized from being possible needless to say as DDS individually, a kind of mode that also can be used as DDS is included in other the carrier and is utilized.Carrier as this moment does not have particular restriction, can preferably use liposome, virion, hollow nanoparticle etc.
For for the titanium dioxide complex with molecule distinguishability of the present invention is activated, the activatory light supply apparatus is not special necessary, is below the 400nm but fasten preferred its wavelength in the pass of the wavelength band gap of titanium dioxide.In external application purposes such as skin, can preferably use sunlight or common UV-lamp or black lamp.In addition, preferably pass through on endoscope, to install ultraviolet optical fiber irradiation ultraviolet radiation for intravital affected part.And then, particularly when anticipation destroys the actinotherapy of diseased region with near the ultraviolet partial irradiation affected part the 280nm, also can preferably use the titanium dioxide complex with molecule distinguishability of the present invention as its potentiator.
Below, describe the present invention in detail according to embodiment.But the present invention is not limited only to these embodiment.
Polyacrylic importing on the TiO 2 particles
The 3.6g tetraisopropoxy titanium is mixed with the 3.6g Virahol, and the ultrapure water that drips 60mL under ice bath is hydrolyzed.At room temperature stirred after the dropping 30 minutes.After the stirring, drip 1mL12N nitric acid, stirred 8 hours down, carry out peptization at 80 ℃.After peptization finishes, with 0.45 μ m membrane filtration, with desalination chromatographic column (PD10; Amersham Pharmacia Bioscience company) carries out the anatase titanium dioxide colloidal sol that solution exchanged and prepared 1% solids component.This dispersion liquid is joined in the phial of volume 100mL, under 200Hz, carried out supersound process 30 minutes.The average mark shot that carries out the supersound process front and back directly is respectively 36.4nm, 20.2nm.After supersound process, concentrated solution is prepared the TiO 2 sol (anatase titanium dioxide) of 20% solids component.
The 0.75mL TiO 2 sol that obtains is dispersed in the dimethyl formamide (DMF) of 20mL, add the DMF that 10mL dissolved 0.2g polyacrylic acid (molecular-weight average: 5000 and the pure medicine of light) after, mix.Solution is transferred in the hydro-thermal reaction container, under 180 ℃, carried out hydro-thermal synthetic 6 hours.After reaction finished, the reaction vessel temperature was cooled to below 50 ℃, added 80mL water behind the taking-up solution and mixed.After removing DMF and water with evaporimeter, add 20mL water again and make it become the polyacrylic acid modified titanic oxide aqueous solution.Add 1mL 2N hydrochloric acid and make the TiO 2 particles precipitation, separate unreacted polyacrylic acid by removing supernatant liquor after centrifugal.Add water once more and wash, centrifugal back is removed and is anhydrated.After adding 10mL50mM phosphoric acid buffer (pH7.0), under 200Hz, carried out ultrasonication 30 minutes, TiO 2 particles is disperseed.After the ultrasonication,, obtain the polyacrylic acid modified titanic oxide colloidal sol of 1.5% solids component with 0.45 μ m membrane filtration.Measure the dispersion particle diameter of prepared polyacrylic acid modified titanic oxide particulate (anatase titanium dioxide), the result is 45.5nm.
The immobilization of anti-AFP antibody molecule on the titanium dioxide fine particles that polyacrylic acid is modified
1mL is carried out the solution exchange by the polyacrylic acid modified titanic oxide colloidal sol (anatase titanium dioxide) that embodiment 1 obtains with desalination chromatographic column PD10, obtain dispersive polyacrylic acid modified titanic oxide colloidal sol 3mL in water.In this solution of 1.5mL, the mixed solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide of interpolation 0.1mL 200mM and the N-hydroxy-succinamide (NHS) of 50mM stirred 10 minutes, made activated carboxylic.After stir finishing, carry out the solution exchange, obtain the polyacrylic acid modified titanic oxide colloidal sol that 3mL is dispersed in the activated carboxylic in the 10mM hac buffer (pH5.0) in order to the PD10 of 10mM hac buffer (pH5.0) equilibration.Add anti-α-fetoprotein (anti-AFP) polyclonal antibody (goat 1gG, SC-8108 with same damping fluid preparation; Cosmobio company) so that its concentration is 0.05mg/mL.After at room temperature stirring 15 minutes, add the ethanolamine hydrochloric salt aqueous solution (pH8.5) so that its concentration is 0.5M.After stirring 10 fens kinds, add 1mL 2N hydrochloric acid and make the TiO 2 particles precipitation, remove supernatant liquor after centrifugal.Add water once more and wash, centrifugal back is removed and is anhydrated.After adding 2.5mL 50mM phosphoric acid buffer (pH7.0), under 200Hz, carried out ultrasonication 30 minutes, TiO 2 particles is disperseed.After the ultrasonication,, make the anti-AFP antibody immobilization titanium dioxide complex colloidal sol that becomes 0.3% solids component with 0.45 μ m membrane filtration.Measure the dispersion particle diameter of prepared anti-AFP antibody immobilization titanium dioxide complex (anatase titanium dioxide), the result is 52.8nm.
The immobilization of anti-HSA antibody molecule on the titanium dioxide fine particles that polyacrylic acid is modified
1mL is carried out the solution exchange by the polyacrylic acid modified titanic oxide colloidal sol (anatase titanium dioxide) that embodiment 1 obtains with desalination chromatographic column PD10, obtain dispersive polyacrylic acid modified titanic oxide colloidal sol 3mL in water.The mixed solution 0.1mL of the N-hydroxy-succinamide (NHS) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide of interpolation 200mM and 50mM stirred 10 minutes in this solution of 1.5mL, made activated carboxylic.After stir finishing, carry out the solution exchange, obtain the polyacrylic acid modified titanic oxide colloidal sol that 3mL is dispersed in the activated carboxylic in the 10mM hac buffer (pH5.0) in order to the PD10 of 10mM hac buffer (pH5.0) equilibration.Add AHS's albumin (anti-HSA) monoclonal antibody (mouse 1gG, MSU-304 with same damping fluid preparation; Cosmobio company) so that its concentration is 0.05mg/mL.After at room temperature stirring 15 minutes, add the ethanolamine hydrochloric salt aqueous solution (pH8.5) so that its concentration is 0.5M.After stirring 10 fens kinds, NaCl, 20% (w/v) polyoxyethylene glycol that equivalent is added 2.5M makes the TiO 2 particles precipitation, and centrifugal back is removed and anhydrated.Add water once more and wash, centrifugal back is removed and is anhydrated.Add 2.5mL PBS damping fluid (pH7.0: contain 100mM NaCl, Japan Gene), TiO 2 particles is disperseed.With 0.45 μ m membrane filtration, make the anti-HSA antibody immobilization titanium dioxide complex colloidal sol that becomes 0.3% solids component.Measure the dispersion particle diameter of prepared anti-HSA antibody immobilization titanium dioxide complex (anatase titanium dioxide), the result is 52.8nm.
Embodiment 4
The immobilization of streptavidin molecule on the titanium dioxide fine particles that polyacrylic acid is modified
1mL is carried out the solution exchange by the polyacrylic acid modified titanic oxide colloidal sol (anatase titanium dioxide) that embodiment 1 obtains with desalination chromatographic column PD10, obtain 3mL dispersive polyacrylic acid modified titanic oxide colloidal sol in water.The mixed solution 0-1mL of the N-hydroxy-succinamide (NHS) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide of interpolation 200mM and 50mM stirred 10 minutes in this solution of 1.5mL, made activated carboxylic.After stir finishing, carry out the solution exchange, obtain the polyacrylic acid modified titanic oxide colloidal sol that 3mL is dispersed in the activated carboxylic in the 10mM hac buffer (pH5.0) in order to the PD10 of 10mM hac buffer (pH5.0) equilibration.Add streptavidin (Pierce Bioisystech Co., Ltd, numbering: 21126) so that its concentration is 0.05mg/mL.After at room temperature stirring 15 minutes, add the ethanolamine hydrochloric salt aqueous solution (pH8.5) so that its concentration is 0.5M.After stirring 10 fens kinds, NaCl, 20% (w/v) polyoxyethylene glycol that equivalent is added 2.5M makes the TiO 2 particles precipitation, removes supernatant liquor after centrifugal.Add water once more and wash, centrifugal back is removed and is anhydrated.Add 2.5mL PBS damping fluid (pH7.0, Japan Gene), TiO 2 particles is disperseed.With 0.45 μ m membrane filtration, making becomes the streptavidin of 0.3% solids component immobilized titanium dioxide composite sol.Measure the dispersion particle diameter of prepared streptavidin immobilized titanium dioxide complex body (anatase titanium dioxide), the result is 50.5nm.
Embodiment 5
Synthesizing of the magneticsubstance composite titanium dioxide particulate that polyacrylic acid is modified
Dissolving 45.16g polyoxyethylene (15) cetyl ether in separating flask (C-15: Japanese Surfactant industrial) after 5 minutes, adds 75mL tetrahydrobenzene solution (with the pure medicine of light) with the nitrogen displacement, adds the FeCl of 3.6mL 0.67M
2(with the pure medicine of light) aqueous solution on one side stir with 250rpm, Yi Bian add 30% ammonia soln 5.4mL, makes its reaction 1 hour.Then, the Dropwise 5 0mM tetraethyl orthosilicate aqueous solution (with the pure medicine of light) 0.4mL makes its reaction 1 hour.Then, add tetraisopropoxy titanium (with the pure medicine of light) so that ultimate density is 0.005M.With 10 minutes each at interval 50% (w/v) aqueous ethanolic solution 10mL that add 1mL.With this reaction soln centrifugation, with throw out 350 ℃ of following sintering 2 hours.Behind the sintering, it is disperseed in the 10mM aqueous nitric acid, after the ultrasonication, with the membrane filtration of 0.1 μ m.With the 0.75mL magneticsubstance that obtains: composite titanium dioxide colloidal sol is dispersed in the dimethyl formamide (DMF) of 20mL, add the DMF that 10mL dissolved 0.3g polyacrylic acid (molecular-weight average: 5000 and the pure medicine of light) after, mix.Solution is transferred in the hydro-thermal reaction container (HU-50, three possess a fondness for science), under 180 ℃, synthesized 6 hours.After reaction finished, the reaction vessel temperature was cooled to below 50 ℃, behind the taking-up solution, added 10mL water and mixed in separating funnel.Then, add the 40mL chloroform and mix the back, reclaim the upper strata except that sub-cloud.This step is repeated 2 times, remove DMF.NaCl, 20% (w/v) polyethylene glycol 6000 (with the pure medicine of light) that adds 10mL 1.5M in this solution of 10mL is removed supernatant liquor after centrifugal.In precipitation, add 2.5mL water, carry out the dispersion liquid that gel-filtration obtains polyacrylic acid modified magnetic material composite titanium dioxide particulate (anatase titanium dioxide) with Sephadex G-25 post.
Embodiment 6
The immobilization of anti-HSA antibody molecule on the magneticsubstance composite titanium dioxide particulate that polyacrylic acid is modified
The mixed solution 0.1mL of the N-hydroxy-succinamide (NHS) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide of interpolation 200mM and 50mM in the dispersion liquid of the magneticsubstance composite titanium dioxide particulate that the polyacrylic acid that 1.5mL is obtained by embodiment 5 is modified, stirred 10 minutes, and made activated carboxylic.After stir finishing, carry out the solution exchange, obtain the magneticsubstance composite titanium dioxide particulate colloidal sol that polyacrylic acid that 3mL is dispersed in the activated carboxylic in the 10mM hac buffer (pH5.0) is modified in order to the PD10 of 10mM hac buffer (pH5.0) equilibration.Add AHS's albumin (anti-HSA) monoclonal antibody (mouse 1gG, MSU-304 with same buffer preparation; Cosmobio company) so that its concentration is 0.05mg/mL.After at room temperature stirring 15 minutes, add the ethanolamine hydrochloric salt aqueous solution (pH8.5) so that its concentration is 0.5M.After stirring 10 fens kinds, NaCl, 20% (w/v) polyoxyethylene glycol that equivalent is added 2.5M makes magneticsubstance composite titanium dioxide particle precipitation, removes supernatant liquor after centrifugal.Add water once more and wash, centrifugal back is removed and is anhydrated.Add 2.5mL PBS (Japan Gene), magneticsubstance dioxide composite titanium particle is disperseed.With 0.45 μ m membrane filtration, make the anti-HSA antibody immobilization magneticsubstance composite titanium dioxide composite sol that becomes 0.3% solids component.Measure the dispersion particle diameter of prepared anti-HSA antibody immobilization magneticsubstance composite titanium dioxide complex body (anatase titanium dioxide), the result is 105nm.
Embodiment 7
Vinylformic acid/sulfonic acid is the importing of interpolymer on the TiO 2 particles
The TiO 2 sol (anatase titanium dioxide) of 0.75mL 20% solids component that will obtain in embodiment 1 is dispersed in the dimethyl formamide (DMF) of 20mL, it is monomer interpolymers (molecular-weight average: 5000 that interpolation 10mL has dissolved 0.3g vinylformic acid/sulfonic acid, lyophilize standard substance after the proton displacement, Japan's catalysis) behind the DMF, mix.Solution is transferred in the hydro-thermal reaction container (HU-50, three possess a fondness for science), reacted 5 hours down at 150 ℃.After reaction finished, the reaction vessel temperature was cooled to room temperature, added the Virahol (with the pure medicine of light) with respect to 2 times of amounts of reaction soln.After at room temperature leaving standstill more than 30 minutes, carried out centrifugation 20 minutes, reclaim throw out with 4000 * g.With this throw out with 70% washing with alcohol after, add 2.5mL water, obtaining vinylformic acid/sulfonic acid is interpolymer modified titanic oxide colloidal sol (anatase titanium dioxide).
Embodiment 8
Vinylformic acid/sulfonic acid is the immobilization of anti-DR4 antibody molecule on the interpolymer modified titanic oxide particulate
At 1.5mL is the mixed solution 0.1mL that adds the N-hydroxy-succinamide (NHS) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide of 200mM and 50mM in the interpolymer modified titanic oxide colloidal sol by the vinylformic acid/sulfonic acid of embodiment 7 preparation, stirred 10 minutes, and made activated carboxylic.After stir finishing, carry out the solution exchange in order to the PD10 of 10mM hac buffer (pH5.0) equilibration, obtaining vinylformic acid/sulfonic acid that 3mL is dispersed in the activated carboxylic in the 10mM hac buffer (pH5.0) is interpolymer modified titanic oxide colloidal sol.Add anti-DR4 monoclonal antibody (Anti-TRAIL acceptor 1, mouse, numbering: SA-225, Funakoshi company) therein so that its concentration is 0.05mg/mL.After at room temperature stirring 1 minute, add the ethanolamine hydrochloric salt aqueous solution (pH8.5) so that its concentration is 0.5M.After at room temperature stirring 10 fens kinds, NaCl, 20% (w/v) polyoxyethylene glycol that equivalent is added 2.5M makes the TiO 2 particles precipitation, removes supernatant liquor by centrifugation.Wash the back centrifugation with water and reclaim precipitation, add 2.5mL PBS buffered soln (pH7.0, Japan Gene) TiO 2 particles is disperseed.With 0.45 μ m membrane filtration, obtain the anti-DR4 antibody immobilization titanium dioxide complex colloidal sol (anatase titanium dioxide) of 0.3% solids component.
Embodiment 9
The decomposition of the antigen A FP that causes by anti-AFP antibody immobilization titanium dioxide complex
With α-fetoprotein (AFP, Cosmobio company) the PBS buffered soln (pH7.0 of usefulness 50mM, Japan Gene) dilution is so that its concentration is 1 μ g/mL, and the anti-AFP antibody immobilization titanium dioxide complex that is added on preparation among the embodiment 2 becomes 0.01% so that its concentration is solids component.Then, under 37 ℃, leave standstill and made it form aggregate by antigen antibody reaction in 3 hours.Form aggregate by AFP and anti-AFP antibody immobilization titanium dioxide complex, anti-as can be known AFP antibody immobilization titanium dioxide complex is discerned AFP and combination specifically.Shine the UV-light of 340nm so that its dosage is 1mW/cm while stirring this aggregate
2, with the absorption (turbidity of aggregate) of spectrophotometric determination wavelength under 600nm.The result as shown in Figure 2.Only confirmed when ultraviolet ray (UV) irradiation, to follow the reduction absorbancy of aggregate concentration to reduce.That is, known photocatalysis by anti-AFP antibody immobilization titanium dioxide complex, antigen A FP is decomposed.
Embodiment 10
The affirmation of the antigen-antibody reaction that produces by anti-HSA antibody immobilization titanium dioxide complex
Use the PBS buffered soln (pH7.0, Japan Gene) of 50mM to be diluted to 250 μ g/mL human serum albumin (HSA, Cosmobio company).In addition, the induction chip (BIACORE company) of the mixed solution activating surface plasma resonance inductor block of the N-hydroxy-succinamide (NHS) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide of usefulness 400mM and 100mM.This induction chip is installed to surface plasmon resonance measurement decides device: BIACORE1000 (BIACORE company).With above-mentioned HSA solution with the logical liquid of flow velocity 10 μ L/min after, carry out the sealing of active group with the 0.1M thanomin, prepare HSA immobilization induction chip.Carry by 0.01% anti-HSA antibody immobilization titanium dioxide complex colloidal sol of embodiment 3 preparations with by 0.01% streptavidin immobilized titanium dioxide composite sol of embodiment 4 preparations to this HSA immobilization induction chip, confirmed antigen-antibody reaction.The result as shown in Figure 3.For HSA immobilization induction chip, anti-HSA antibody immobilization titanium dioxide complex reacts and is combined on the chip, and streptavidin immobilized titanium dioxide complex body does not have reaction and combination does not take place.That is, confirmed the anti-HSA monoclonal antibody on the hydrophilic macromolecule of being fixed on the titanium dioxide, even after being fixed, still kept activity effectively as antibody.
Embodiment 11
The decomposition of the antigen HSA that produces by anti-HSA antibody immobilization titanium dioxide complex
HSA is diluted so that its concentration is 20ng/mL with PBS buffered soln (pH7.0, Japan Gene), and the anti-HSA antibody immobilization titanium dioxide complex that is added on preparation among the embodiment 3 is so that solids component becomes 0.01%.Then, at room temperature place 30 minutes after, the UV-light of illumination wavelength 340nm is so that dosage is 1mW/cm
2, sampled 90 minutes every 15 minutes.The streptavidin immobilized titanium dioxide complex body of embodiment 4 preparations is handled too.In addition, according to the method for embodiment 8, the preparation surface plasmon resonance measurement is decided anti-HSA polyclonal antibody (rabbit) the immobilization induction chip of usefulness.Use BIACORE1000 similarly to Example 8, in anti-HAS antibody immobilization induction chip, carry each through the time sample 20 μ L, carry out sandwich assay as 2 logical liquid 10 μ L anti-HSA polyclonal antibodies of 50 μ g/mL (rabbit) of antibody then, the RU value (suitable) behind the logical humoral antibody of mensuration after 10 seconds with binding capacity.The HSA rate of decomposition of being calculated as 100% relative value by the RU value when not shining UV as shown in Figure 4.By the result of Fig. 4, show that anti-HSA antibody immobilization titanium dioxide complex compares with streptavidin immobilized titanium dioxide complex body, the decomposition rate of HSA is exceedingly fast.
Industrial applicibility
By the present invention, can provide and endocrine disruptor, virulence factor, cancer cell etc. Specific binding, and by the decomposition of photocatalysis demonstration to them, and have The titanium dioxide complex of molecule distinguishability.
Claims (16)
1. titanium dioxide complex with molecule distinguishability, be that titanium dioxide surface is that modify with the hydrophilic macromolecule with a plurality of bases, particulate titanium dioxide complex, it is characterized by, the carboxyl of this hydrophilic macromolecule and particle diameter are that the hydroxyl of the titanium dioxide surface of 2～200nm passes through the ester bond bonded simultaneously, on the carboxyl residue of this hydrophilic macromolecule, the molecule that has a specific binding capacity with target molecule is fixed.
2. the described titanium dioxide complex with molecule distinguishability of claim 1, above-mentioned titanium dioxide is anatase titanium dioxide or rutile-type.
3. claim 1 or 2 described titanium dioxide complexes with molecule distinguishability is characterized by, the composite titanium dioxide of above-mentioned titanium dioxide for being made of titanium dioxide and magneticsubstance.
4. any described titanium dioxide complex with molecule distinguishability of claim 1～3 is characterized by, and above-mentioned hydrophilic macromolecule is a water-soluble polymer.
5. the described titanium dioxide complex with molecule distinguishability of claim 4 is characterized by, and above-mentioned water-soluble polymer contains poly carboxylic acid.
6. the described titanium dioxide complex with molecule distinguishability of claim 4 is characterized by, and above-mentioned water-soluble polymer contains in molecule and has the unitary interpolymer of a plurality of carboxyls.
7. any described titanium dioxide complex with molecule distinguishability of claim 1～6 is characterized by, and the molecule that above-mentioned target molecule is had specific binding capacity is amino acid, peptide, simple protein, complex proteins and antibody.
8. any described titanium dioxide complex with molecule distinguishability of claim 1～6 is characterized by, and the molecule that above-mentioned target molecule is had specific binding capacity is nucleosides, Nucleotide, nucleic acid and peptide nucleic acid(PNA).
9. any described titanium dioxide complex with molecule distinguishability of claim 1～6 is characterized by, and the molecule that above-mentioned target molecule is had specific binding capacity is monose, sugar chain, polysaccharide and compounding sugar.
10. any described titanium dioxide complex with molecule distinguishability of claim 1～6 is characterized by, and the molecule that above-mentioned target molecule is had specific binding capacity is lipid acid, derivative of fatty acid, simple lipid, compound lipid.
11. any described titanium dioxide complex with molecule distinguishability of claim 1～6 is characterized by, the molecule that above-mentioned target molecule is had specific binding capacity is a physiologically active substance.
12. the dispersion liquid with titanium dioxide complex of molecule distinguishability is characterized by, and in allowing the aqueous solution that is directed into organism, contains any described titanium dioxide complex with molecule distinguishability of claim 7～11.
13. the described dispersion liquid with titanium dioxide complex of molecule distinguishability of claim 12 is characterized by, the above-mentioned aqueous solution is pH buffered soln.
14. the described dispersion liquid with titanium dioxide complex of molecule distinguishability of claim 12 is characterized by, the above-mentioned aqueous solution is physiological saline.
15. any described dispersion liquid with titanium dioxide complex of molecule distinguishability of claim 12～14 is characterized by, and in allowing the inclusion body that is directed into organism, comprises the titanium dioxide complex with this molecule distinguishability.
16. the described dispersion liquid with titanium dioxide complex of molecule distinguishability of claim 15 is characterized by, above-mentioned inclusion body is, any one of liposome, virus particle, hollow nanoparticle.
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| CN1038073A (en) * | 1988-03-30 | 1989-12-20 | 罗纳·布朗克化学公司 | The preparation method of titanium dioxide |
| JPH07236690A (en) * | 1994-02-25 | 1995-09-12 | Unitika Ltd | Method for fixing fibrinogenolysis active material |
| WO2002034301A1 (en) * | 2000-10-20 | 2002-05-02 | Noritake Co., Limited | Material for treating harmful substance, and method and apparatus for treating harmful substance |
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| CN1038073A (en) * | 1988-03-30 | 1989-12-20 | 罗纳·布朗克化学公司 | The preparation method of titanium dioxide |
| JPH07236690A (en) * | 1994-02-25 | 1995-09-12 | Unitika Ltd | Method for fixing fibrinogenolysis active material |
| WO2002034301A1 (en) * | 2000-10-20 | 2002-05-02 | Noritake Co., Limited | Material for treating harmful substance, and method and apparatus for treating harmful substance |
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