CN1358783A - Method for covalence bonding chitosan and base material surface - Google Patents
Method for covalence bonding chitosan and base material surface Download PDFInfo
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- CN1358783A CN1358783A CN 02112539 CN02112539A CN1358783A CN 1358783 A CN1358783 A CN 1358783A CN 02112539 CN02112539 CN 02112539 CN 02112539 A CN02112539 A CN 02112539A CN 1358783 A CN1358783 A CN 1358783A
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Abstract
The method for covalent linkage of chitosan on surface of base material includes the following steps: bonding nitrine group on the chitosan, casting the triazo-containing chitosan solution on the base material to form chitosan coating layer containing nitrine group, under the irradiation of UV light photolyzing nitrine group into carbene, making carbene ane base material produce insertion reaction to make chitosan chain produce crosslinking reaction so as to make chitosan bond on the surface of base material. The preparation method of triazo-containing chitosan solution includes the following steps: adding chitosan powder, dicyclohexylcarbimide and 4-azidobenzoic acid into organic solvent stirring, filtering, washing with methyl alcohol or dichloromethane, filtering, drying to obtain triazochitosan, then dissolving it in mixed solvent of dilute acetic acid and glycol methyl ether.
Description
One, technical field
The present invention relates to a kind of substrate surface method of modifying, particularly a kind of method at substrate surface covalent bonding chitosan.
Two, background technology
Chitosan is the partially deacetylated derivative of chitin (extensively being present in the shell of shrimp crab); it is the random a kind of linear glycan of lining up of N-acetyl Portugal polyamine that the D-Portugal polyamine that linked to each other by β (1 → 4) and content do not wait; it is the unique alkaline polysaccharide of nature; existing hydrophilic group of its intramolecularly and hydrophobic group have amino and hydroxyl with coordination ability and derivative reaction again.These character have determined it to be with a wide range of applications in different field.
Important use is worth chitosan also having very aspect the water treatment with its exclusive biological activity, biocompatibility, good binding property, stronger one-tenth fibre and film forming ability and special absorption property.It can be used for the separation of clarification, Industrial Wastewater Treatment, aminoacid protein of the decolouring of waste water from dyestuff, the recovery of drinking water purification, heavy metal ion, water softening, molasses and fruit juice and recovery etc.Its application at gas-chromatography separating metal ions is also very extensive in addition.Chitosan can be used to measure Au, Pb, Pt, Pd, Ag, Cu in electroanalytical chemistry.
Because chitosan is nontoxic, have good biology performances such as biocompatibility, make film after, water white transparency has the better physical mechanical property, stable chemical properties.Abundant amino, can with the enzyme covalent coupling, chitosan has been widely used in immobilized enzyme and biosensor.With chitin immobilized enzyme, can adopt simple absorption method, gel embedding method, glutaraldehyde cross-linking method or the like.For enzyme is combined with chitosan by chemical bond, glutaraldehyde is the most frequently used linking agent.But the method for usefulness glutaraldehyde such as Krajewska is fixed in urokinase on the chitosan film, and this method is easy to make chitosan film flavescence, hardening, becomes fragile, and film is easy to fragmentation, is difficult for the preparation sensitive membrane.
Chitosan more and more is subjected to people's attention in the applied research of field of tissue engineering technology in recent years.Chitosan presents cationic property and high charge density in the aqueous solution, this is its major reason that is widely used in technical field of biological material.Because the surperficial and electronegative cell of positively charged chitosan easily produces electrostatic attraction, this helps the adhesion of cell.And high charge density makes chitosan and many water-soluble anionic polymers form insoluble polyelectrolyte title complex.For example, chitosan can form ionic complex (PEC) with negatively charged ion glycosaminoglycan (GAGs) and sodiun alginate and synthetic polyanion such as polyacrylic acid.Because chitosan electric density has the PH dependency, causes the polyanion of partial fixing to dissociate when these PEC being moved to physiology PH environment.This character can be used for original position load biological activity polyanion such as GAGs and DNA.For example, inflammatory cell discharged somatomedin near the heparin that discharges from chitosan-heparin PEC can promote graft, and the plasmid that the DNA that discharges from PEC can protect nuclease degradation to generate helps the cell migration relevant with cytolemma simultaneously.Chitosan has many biological activitys and is confirmed by numerous experiment.For example, the chitosan oligopolymer has the effect to macrophage-stimulating, and this contains N-acetyl Portugal polyamine component with it closely related.Chitosan and chitin all have inside and outside neutrophilic granulocyte chemotactic effect.Many study tours the reaction of chitosan and derivative support thereof to tissue, generally speaking, the simplified reaction that these materials cause is little, does not almost observe the fiber capsule, formation be normal granular structure, accelerate vasculogenesis, present typical healing process, in short duration (less than 7 days), neutrophilic granulocyte is obviously piled up near graft, but disperse rapidly, chronic inflammatory reaction can not expanded.Component in chitosan above-mentioned and the chitosan can cause the original position cell migration to the hormesis of immunocyte, finally make timbering material with organize huge legendary turtle to close.Appear in the newspapers about chitosan and mammalian cell interact, the cell that relates to comprises scleroblast, fibroblast, scavenger cell, corneal cell and adrenal cells etc.From tissue repair and regenerated angle, in most cases chitosan is with the interaction of cell the time, and the simplified reaction that material causes is few.Report epithelial cells such as Popowicz can be grown on chitosan film, and inoblast can grow on collagen and chitosan complex film, introduce that chitosan can increase cell adhesion but the growth that can suppress cell.The snappiness hydrogel that polyvinyl alcohol (PVA) and chitosan blend form, when the component of chitosan surpasses 15wt%, the adherent rate of L929 cell on hydrogel increases with chitosan content, and when component reached 40wt%, the adhesion of cell surpassed collagen with growth; The composite membrane of PVA/ chitosan more is appropriate to cell cultures than PVA film, and the tenuigenin net is easier to sprawl, and cell is easier to adhere to and growth.The more important thing is on the pyranoid ring of chitosan chain to contain activated hydroxyl and amino, they can carry out derivative reaction, and the result not only can give chitosan special biological activity, and the mechanical property that can regulate chitosan.For example, by the primary amine in the chitosan molecule, it is carried out the side group ion modification, the chitosan crystalline structure is damaged, amorphous portion increases, and solvability is improved, and mechanical property is regulated.Certainly, specific character depends on the character of side group, can reduce greater than 5 N-alkyl derivative solvability as carbon atom, is easy to form micella in solution.Immobilizing biologically active molecule on chitosan surface institute deutero-group, the report of giving its biological nature is more, and as the compound fixedly microbiotic of porous chitosan-calcium phosphate powder, slowly the medicine that discharges can prevent osteomyelitis; Chitosan-GAG title complex is the somatomedin of bacterium colony emiocytosis fixedly, can suppress smooth muscle cell growth; Chitosan sponges bracket is Thr6 PDGF BB (PDGF-BB) fixedly, slowly releases somatomedin and can bring out or stimulate bone forming.In addition, chitosan sulfonyl derivative correspondingly is reversed into positively charged ion, hemostasis, the high pH value insoluble matter of chitosan negatively charged ion, is done blood coagulation and water-soluble characteristic.How chitosan can very not lifted by the deutero-group.Chitosan is easy to take place derivative reaction and provides unlimited potentiality for widening its application in every field.The chitosan character of more than enumerating has determined its using value in tissue engineering material field uniqueness, and well-known, the material surface chemistry is most important to the biocompatibility of its tissue.Therefore, the good chitosan of biocompatibility widely by people in order to modify substrate surface.Usually the method that adopts at present at substrate surface set casing glycan is that method is held back on surperficial precoating method and surface.The surface precoating method exists the stability of coating and permanence relatively poor, and the effect of chitosan precoating obviously depends on the character of substrate surface; And poor stability and the restricted defective of interception that there is coating equally in method held back on the surface.These two kinds of surperficial fixing meanss all lack chemical bonding, and come anchoring chitosan and base material with Methyl4-azidobenzoimidate (MABI) hydrochloride, and because synthesis material is rare, the synthesis technique complexity, preparation cycle is long, so be difficult to further apply.
Three, summary of the invention
1, goal of the invention: the purpose of this invention is to provide a kind of common photosensitive assorted-bifunctional cross-linker who is easy to get that adopts---4-azidobenzoic acid with chitosan by the method for chemical bonding at substrate surface, thereby improve stability, persistence, popularity, validity and the suitability of substrate surface set casing glycan.
2, technical scheme: for achieving the above object, the method at substrate surface covalent bonding chitosan of the present invention is characterized in that this method at first is that azido group is bonded on the chitosan; Cast on the base material with containing the azido-chitosan solution then, make it to form the chitosan coat that contains the phenylazide group; Under ultra violet lamp, making the azido group photodissociation is carbene with above-mentioned coating; Carbene and base material generation insertion reaction make the chitosan chain crosslinked simultaneously, and the result makes chitosan be bonded in substrate surface.The described preparation method who contains the azido-chitosan solution joins chitosan powder, dicyclohexyl carbimide (DCC) and 4-azidobenzoic acid in the organic solvent, stirring at room then, filter, with methyl alcohol or methylene dichloride thorough washing, filter, be drying to obtain and contain the azido-chitosan, then it is dissolved in the mixed solvent of dilute acetic acid and ethylene glycol monomethyl ether, to form the azido-chitosan solution.Photosensitive assorted-bifunctional cross-linker such as 4-azidobenzoic acid that the present invention selects for use, its acidic group and the amino in the chitosan can form the chitosan of the azido group that contains 0.5%～0.6% molar content by interacting.Contain the azido-chitosan coat under ultra violet lamp, the azido group photodissociation is a carbene, active carbon rare can with base material generation insertion reaction, simultaneously also can make the chitosan body crosslinked, the result by photosensitive assorted-bifunctional cross-linker is bonded in substrate surface with chitosan.
3, beneficial effect: 4-azidobenzoic acid (add dewatering agent, room temperature was reacted 8 hours) under certain condition can generate amido linkage with the amino interaction in the chitosan, thereby the UV photosensitive azido group is incorporated in the chitosan.Because reaction can be carried out in heterogeneous, so product is easily separated.And the 4-azidobenzoic acid be easy to the preparation, cheap.Chitosan modified solution, as long as be coated in substrate surface in advance earlier, after the airing, ultraviolet lighting can be bonded in substrate surface with chitosan.Operability of the present invention is strong especially, whether base material is had active group do not have particular requirement, both can be on glass-carbon electrode the bonding chitosan fix enzyme and biomacromolecule is prepared into biosensor, also can prepare spherical resin and be used for water conditioner; More outstanding application is that this method can be used for the modified biological material, thereby gives biomaterial special biological activity, also can further fix other biomacromolecule simultaneously.
Four, description of drawings
Fig. 1 is the UV spectrum of Az-CS in the acetum of 0.1M.
Fig. 2 is the Fu Li leaf infrared spectrum (FTIR) of Az-CS powder.
Fig. 3, Fig. 4 are the total reflection infrared spectrums (ATR-FTIR) of poly(lactic acid) (PLA) and PLA/CS.
Fig. 5, Fig. 6 are the stereoscan photographs (SEM) of blank PU sheet and PU/CS/Hp.
Fig. 7 is the circulation-voltammogram of modified glassy carbon electrode, and wherein: 1-CS modifies, and 2-CS/DNA modifies.
Five, embodiment
The preparation of azido-chitosan (Az-CS) solution: 1g chitosan powder, 0.48gDCC and 0.05～0.5g4-azidobenzoic acid are joined in the 100mL solvent, at room temperature stir 8h then, filter, use the methyl alcohol thorough washing, filter, vacuum-drying promptly gets Az-CS.Then it is dissolved in the mixed solvent of 1% dilute acetic acid and ethylene glycol monomethyl ether (80/20 V/V), to form 0.1% Az-CS solution.
1, in the method for polylactic acid membrane surface covalent bonding chitosan: chitosan is fixed on poly(lactic acid) (PLA) film surface, Az-CS solution-cast with 0.1% is on PLA film surface, airing in brown moisture eliminator, film is apart from the ultraviolet lamp 10cm of 8W, illumination 10min, then with the film order with 5% acetic acid, 0.05NaOH solution and water washing, last vacuum-drying.
2, in the method for polyurethane surface covalent bonding chitosan: chitosan is fixed on urethane (PU) surface (fixing means is with 1), then heparin (Hp) is fixed on chitosan-modified PU sheet surface with ionic complex and forms the PU/CS/Hp surface, test the anticoagulation function of checking PU surface modification front and back according to platelet adhesion reaction then.
3, in the method for glass-carbon electrode surface covalent bonding chitosan: chitosan is fixed on the glass-carbon electrode surface by 1 method, then DNA is fixed on chitosan-modified glass-carbon electrode surface with ionic complex.
4, characterize:
The absorption of 272.6nm is the characteristic absorbance of azido group in the Az-CS molecule among Fig. 1, compares with the characteristic absorbance (268.4nm) of azido group in the 4-azidobenzoic acid, and absorption peak obviously moves to high wavelength, and this is because the environmental change of azido group causes.
As seen from Figure 2, compare, obviously increased 2127cm in the spectrogram of Az-CS with the FTIR spectrogram of chitosan
-1, this is the characteristic infrared absorption of azido group.Fig. 1, Fig. 2 explanation has been bonded on the CS molecule by generating the amido linkage azido group.
By Fig. 3, Fig. 4 as seen, compare, on the PLA/CS spectrogram, obviously increased by 3363 and 1594cm with the PLA spectrogram
-1The characteristic absorbance at place, this explanation chitosan has been fixed on the surface of PLA film.
Fig. 5, Fig. 6 show that after whole blood fully contacted, the blank PU sheet of non-modified adhere to more thrombocyte, and the surface of modified mistake did not observe any hematoblastic vestige.This proves absolutely that heparin is fixed on the PU surface by chitosan with ionic complex, thereby shows excellent anticoagulant coatings.
Fig. 7 (sweep limit :-0.2～0.6V, scanning speed: 100V/s, Co (phen)
3 3+Concentration: 4 * 10
-4Mol/L, pH:7.04, end liquid: show that Tris-KCl-NaCL) compare with CS, the glass-carbon electrode of CS/DNA modified has bigger peak current, promptly show better electrode reversibility, this explanation DNA is fixed on the glass-carbon electrode surface by the CS bonded layer.
Enumerated more than that with present method CS is chemically bound on three kinds of different base materials (PLA, PU and glass-carbon electrode), accompanying drawing shows, the validity of this method.
Claims (2)
1, a kind of method at substrate surface covalent bonding chitosan is characterized in that this method is:
(1) azido group is bonded on the chitosan;
(2) cast on the base material with containing the azido-chitosan solution, make it to form the chitosan coat that contains azido group;
(3) with above-mentioned coating under ultra violet lamp, making the azido group photodissociation is carbene;
(4) carbene and base material generation insertion reaction make the chitosan chain crosslinked simultaneously, and the result makes chitosan be bonded in substrate surface.
2, the method at substrate surface covalent bonding chitosan according to claim 1, it is characterized in that the described preparation method who contains the azido-chitosan solution joins chitosan powder, dicyclohexyl carbimide (DCC) and 4-azidobenzoic acid in the organic solvent, stirring at room then, filter, with methyl alcohol or methylene dichloride thorough washing, filter, be drying to obtain the azido-chitosan, then it is dissolved in the mixed solvent of dilute acetic acid and ethylene glycol monomethyl ether, to form the azido-chitosan solution.
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| CNB021125392A CN1158343C (en) | 2002-01-15 | 2002-01-15 | Method for covalence bonding chitosan and base material surface |
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| CNB021125392A CN1158343C (en) | 2002-01-15 | 2002-01-15 | Method for covalence bonding chitosan and base material surface |
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| CN1158343C CN1158343C (en) | 2004-07-21 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102516568A (en) * | 2011-11-23 | 2012-06-27 | 天津大学 | Preparation method of chitosan-base photocrosslinked aquagel |
| CN105189625A (en) * | 2013-01-18 | 2015-12-23 | 赛尔格有限责任公司 | Surface modifying agents, modified materials and methods |
| CN108341976A (en) * | 2017-01-25 | 2018-07-31 | 四川大学 | The derivative and synthetic method of methyl-prop alkylene host material based on click chemistry |
| CN109895468A (en) * | 2019-03-20 | 2019-06-18 | 广州江美印刷有限公司 | The degradable easy printing packaging film of one kind and its production method |
| CN112175106A (en) * | 2020-10-13 | 2021-01-05 | 湖南洋韬材料科技有限公司 | High-activity azide chitosan antibacterial agent and preparation method thereof |
| CN112194814A (en) * | 2020-10-13 | 2021-01-08 | 湖南洋韬材料科技有限公司 | Medical polyurethane azide antibacterial modification method |
-
2002
- 2002-01-15 CN CNB021125392A patent/CN1158343C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102516568A (en) * | 2011-11-23 | 2012-06-27 | 天津大学 | Preparation method of chitosan-base photocrosslinked aquagel |
| CN105189625A (en) * | 2013-01-18 | 2015-12-23 | 赛尔格有限责任公司 | Surface modifying agents, modified materials and methods |
| CN108341976A (en) * | 2017-01-25 | 2018-07-31 | 四川大学 | The derivative and synthetic method of methyl-prop alkylene host material based on click chemistry |
| CN108341976B (en) * | 2017-01-25 | 2021-06-29 | 四川大学 | Derivative of methacrylated matrix material based on click chemistry and synthesis method |
| CN109895468A (en) * | 2019-03-20 | 2019-06-18 | 广州江美印刷有限公司 | The degradable easy printing packaging film of one kind and its production method |
| CN112175106A (en) * | 2020-10-13 | 2021-01-05 | 湖南洋韬材料科技有限公司 | High-activity azide chitosan antibacterial agent and preparation method thereof |
| CN112194814A (en) * | 2020-10-13 | 2021-01-08 | 湖南洋韬材料科技有限公司 | Medical polyurethane azide antibacterial modification method |
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| CN1158343C (en) | 2004-07-21 |
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