CN101955377B - Method for modifying air stability phospholipid membrane on solid surface - Google Patents
Method for modifying air stability phospholipid membrane on solid surface Download PDFInfo
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- CN101955377B CN101955377B CN200910088949A CN200910088949A CN101955377B CN 101955377 B CN101955377 B CN 101955377B CN 200910088949 A CN200910088949 A CN 200910088949A CN 200910088949 A CN200910088949 A CN 200910088949A CN 101955377 B CN101955377 B CN 101955377B
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Abstract
The invention relates to the field of surface chemistry and applied chemistry, in particular to a method for modifying an air stability phospholipid membrane on a solid surface. The method comprises the following steps of: 1) preparing small single-wall phospholipid vesicle solution of poly(ethylene glycol) (PEG) modified phospholipid molecules, wherein the mole percentage of the PEG modified phospholipid molecules is no more than 5 mol percent; 2) performing standing reaction at room temperature between the small single-wall phospholipid vesicle solution obtained in the step 1) and the chitosan modified solid surface; and 3) removing unbonded phospholipid vesicle. The method can solve the problem of air instability when the phospholipid membrane is modified by the conventional method; and due to the addition of the PEG modified phospholipid, the preparation time for the small single-wall phospholipid vesicle is greatly shortened and the stability of the phospholipid vesicle is improved. The phospholipid bilayer formed by the method has the advantages of good air stability, convenient operation and good repeatability. If 2 to 3 mol percent PEG modified phospholipid molecules are added, the single phospholipid bilayer with the air stability can be modified.
Description
Technical field
The present invention relates to surface chemistry and applied chemistry field, particularly, the present invention relates to the method that a kind of solid surface is modified the air stability immobilized artificial membrane.
Background technology
Biochip and application thereof are the present focuses of research, because biomolecules is after chip surface is fixing, and the possibility that often exists biological activity to reduce even to lose; Chip surface also usually causes proteic non-specific adsorption in addition, and these problems are the significant obstacle in the biochip applications.And microbial film is the composition structure of natural maintenance biomolecule activity, and has the characteristics of the protein adsorption of preventing; If employing bionic principle; Modify artificial membrane in biochip surface, promptly modify similar biomembranous phosphatide rete, then can provide biomolecules to keep and bring into play the environment of BA; Can also effectively prevent simultaneously the non-specific adsorption of other biomolecules; Therefore, immobilized artificial membrane has a good application prospect in research fields such as biochips.
The modifying method of immobilized artificial membrane mainly contains two kinds at present; A kind of is the Langmuir-Blodgett/Shaefer method; This method is in special device; Utilize phospholipid molecule to be arranged in unimolecular layer, transfer to solid to be finished surface by certain way then, form unimolecular layer or polymolecular tunic in the liquid-gas interface self-assembly.Second method is preparation little unilamellar vesicle of uniform size (small unilaminar vesicles; SUVs); Vesica direct absorption from liquid phase is fused to solid substrate surface and forms the phosphatide rete, and the phosphatide rete of these methods preparations all need remain on ability stable existence in the water surrounding always, in case run into air; Immobilized artificial membrane will peel off from stayed surface; Thereby brought certain influence for the practical application of phosphatide rete, especially for some instruments that need under air ambient, detect and analyze, this air unstable of immobilized artificial membrane has limited the application of this quasi-instrument.
There is report to adopt methene acrylic amide-sorb acyl Yelkin TTS (bis-SorbPC) to form the phospholipid bilayer film earlier at silica sphere; Make phospholipid molecule side chain generation photopolymerization through uviolizing then; Form the phosphatide rete of polymerization; Though this rete has air stability, lost the flowability of immobilized artificial membrane.Other has report that protein molecular is encapsulated the immobilized artificial membrane surface, makes immobilized artificial membrane be able in air, keep stable, but need be coated with very fine and close egg white layer.Also have to be reported in and add the phospholipid molecule that is modified with the PEG molecule in the immobilized artificial membrane, can make that immobilized artificial membrane has air stability.Utilize PEG-SUV molecule as the bolt mould material in addition, can realize the air stability of immobilized artificial membrane, but these methods are when preparation air stability immobilized artificial membrane, or operational requirement is harsh, or have problem such as poor repeatability, can't realize effective application.
Summary of the invention
The objective of the invention is to solve to have now and can't modify the technological deficiency of air stability immobilized artificial membrane, a kind of method of modifying the air stability immobilized artificial membrane in solid substrate surface is provided in solid substrate surface.
Method according to solid surface of the present invention is modified the air stability immobilized artificial membrane is characterized in that, said method comprising the steps of:
1) preparation contain the Pegylation phospholipid molecule, the little single wall phospholipid capsule bubble of big or small homogeneous;
2) little single wall phospholipid capsule bubble that step 1) is obtained and chitin modified solid surface room temperature standing and reacting; And
3) remove unconjugated phospholipid capsule bubble.
According to the method for the invention, wherein, said little single wall phospholipid capsule bubble solution prepares through following steps:
A) use dissolved in chloroform phosphatide;
B) nitrogen dries up, and vacuum-drying to chloroform volatilizees fully;
C) with the damping fluid aquation;
D) water-bath supersound process to solution is clarified; And
E) pushed film, wherein, extruding uses the aperture of filter membrane to be 50nm, extruding number of times at least 11 times.
According to the method for the invention, wherein, said Pegylation phospholipid molecule is that polyethylene glycol polymer and phospholipid molecule form through chemical covalent bonding.
According to the method for the invention, wherein, the molar content of said Pegylation phospholipid molecule is no more than 5mol%.
According to the method for the invention, wherein, in step 2) in, said vesica solution and solid surface room temperature standing and reacting 5～10 minutes.
The inventive method can be modified the immobilized artificial membrane with air stability at solid surface.The PEG molecule can promote absorption and the fusion of vesica in solid substrate; Earlier form single phospholipid bilayer film in solid substrate; Because the PEG molecule of film surface has the air conservation effect to the upper strata immobilized artificial membrane; The chitosan of solid surface can be protected the rock steady structure of lower floor's immobilized artificial membrane, in air, keeps stable purpose thereby reach the phosphatide rete.
In sum, according to the invention provides a kind of vesica that is mixed with a certain amount of PEGization phosphatide that on chitin modified solid substrate, utilizes, modify the method that forms immobilized artificial membrane with air stability.Air instability problem when this method can solve ordinary method decorated phospholipid film.Owing to the interpolation of PEGization phosphatide, the preparation time of little single wall phospholipid capsule bubble is shortened greatly, and improved the stability of phospholipid capsule bubble in addition.The phospholipid bilayer film that utilizes this method to form; Has good air stability; Easy to operate, good reproducibility, and through regulating the content of PEG molecule; Can form the single phospholipid bilayer film of even compact, the related application research field of modifying the air stability immobilized artificial membrane for needs provides an effective means foundation.
Description of drawings
Fig. 1: the formation mechanism synoptic diagram of air stability phospholipid bilayer film of the present invention;
Fig. 2: chitin modified solid surface of the present invention forms the ellipsometry imaging figure of air stability immobilized artificial membrane;
Fig. 3: of the present invention through regulating the ellipsometry imaging figure that PEG content forms single phospholipid bilayer film;
Fig. 4: the present invention forms the AFM height and the phase analysis figure of air stability immobilized artificial membrane at chitin modified solid surface.
Embodiment
The present invention proposes to form at solid surface the method for single phospholipid bilayer film, only needs preparation to contain the uniform little single wall phospholipid capsule bubble solution of size of finite concentration PEGization phosphatide, can form with chitin modified solid surface room temperature standing and reacting then.
As shown in Figure 1; Use earlier dissolved in chloroform phosphatide; Blow with vacuum drying treatment with nitrogen then chloroform is volatilized totally, on wall of container, form phospholipid membrane, add a certain amount of damping fluid phospholipid membrane is carried out aquation; Water-bath is ultrasonic after clear, pushes film with squeezer and obtains the uniform little single wall phospholipid capsule bubble solution of size.With little single wall phospholipid capsule bubble solution and chitin modified solid surface room temperature standing and reacting, get final product with the remaining vesica of deionized water flush away at last.
Embodiment 1:
Carry out the amination modified of silicon chip surface with ordinary method; With the silicon chip that cleans up with the vitriol oil, hydrogen peroxide (volume ratio 3: 1) oscillation treatment 30 minutes; After water cleans; Change over to and contained in 10% aminopropyltriethoxywerene werene (APTES) ethanolic soln oscillation treatment 2 hours, after rinsing well, promptly get amino modified surface.Handle amino modified silicon chip with 5% glutaraldehyde solution, 1 hour post-flush of room temperature oscillation treatment is clean, again with treated fixed low-molecular weight chitoglycan solution reaction 10 minutes, the washing, nitrogen dries up subsequent use.
With above little single wall phospholipid capsule bubble and the silica sphere standing and reacting for preparing, behind the room temperature 5-10min, get final product with the unconjugated unnecessary vesica of deionized water flush away.
Adopt the ellipse inclined to one side detection technique of imaging to above three kinds of different DSPE-PEG
200The immobilized artificial membrane of concentration detects, and is as shown in Figure 2, and its thickness of ellipsometer measurement is respectively 7.3nm, 6.2nm and 4.9nm (from left to right), and shown in Figure 3 is AFM analytical results to immobilized artificial membrane under air condition.
The preparation process of " chitin modified glass surface " is with embodiment 1.
With the above little single wall phospholipid capsule bubble for preparing with through chitin modified glass surface standing and reacting, behind the room temperature 5-10min, get final product with the unconjugated unnecessary vesica of deionized water flush away.
The preparation process of " chitin modified silica sphere " is with embodiment 1.
With above little single wall phospholipid capsule bubble for preparing and chitin modified silica sphere standing and reacting, behind the room temperature 5-10min, get final product with the unconjugated unnecessary vesica of deionized water flush away.
Embodiment 4
The chloroformic solution of preparation Yelkin TTS (egg phosphocholine); Wherein contain 1 of 3mol%; 2-two myristoyls-glycerine-3-phosphatidylethanolamine-N-carboxyl (Macrogol 2000) 1; 2-Dimyristoylphosphatidylethanolamine-N-[Methoxy (Polyethylene glycol)-2000], DMPE-PEG
2000), most of chloroform drying up earlier with nitrogen, vacuum-drying 2h at least to remove residual chloroform, forms phospholipid membrane simultaneously on wall of container then; Add a certain amount of phosphate buffered saline buffer (pH 7.4) with the film aquation, and to make the final concentration of Yelkin TTS be 1mM.Water-bath supersound process 30-60min is the clarification shape to solution; Adopt the MiniExtruder of Avanti company to push solution then, selecting the aperture for use is the polycarbonate membrane of 50nm, and the extruding number of times is no less than 11 times.
The preparation process of " chitin modified silica sphere " is with embodiment 1.
With above little single wall phospholipid capsule bubble for preparing and chitin modified silica sphere standing and reacting, behind the room temperature 5-10min, get final product with the unconjugated unnecessary vesica of deionized water flush away.
Embodiment 5
The chloroformic solution of preparation DMPC; Wherein contain 1 of 3mol%, 5mol% respectively; 2-two myristoyls-glycerine-3-phosphatidylethanolamine-N-carboxyl (cetomacrogol 1000) 1; 2-Dimyristoylphosphatidylethanolamine-N-[Methoxy (Polyethylene glycol)-1000], DMPE-PEG
1000), most of chloroform drying up earlier with nitrogen, vacuum-drying 2h at least to remove residual chloroform, forms phospholipid membrane simultaneously on wall of container then; Add a certain amount of phosphate buffered saline buffer (pH 7.4) with the film aquation, and to make the final concentration of Yelkin TTS be 1mM.Water-bath supersound process 30-60min is the clarification shape to solution; Adopt the MiniExtruder of Avanti company to push solution then, selecting the aperture for use is the polycarbonate membrane of 50nm, and the extruding number of times is no less than 11 times.
The preparation process of " chitin modified silica sphere " is with embodiment 1.
With above little single wall phospholipid capsule bubble for preparing and chitin modified silica sphere standing and reacting, behind the room temperature 5-10min, get final product with the unconjugated unnecessary vesica of deionized water flush away.
Claims (6)
1. a method of modifying the air stability immobilized artificial membrane at chitin modified solid surface is characterized in that, said method comprising the steps of:
1) preparation contain the Pegylation phospholipid molecule, the little single wall phospholipid capsule bubble of big or small homogeneous;
2) little single wall phospholipid capsule bubble that step 1) is obtained and chitin modified solid surface room temperature standing and reacting 5～10 minutes; And
3) remove unconjugated phospholipid capsule bubble.
2. method according to claim 1 is characterized in that, in step 1), the molar content of said Pegylation phosphatide is no more than 5mol%.
3. method according to claim 1 is characterized in that, said little single wall phospholipid capsule bubble prepares through following steps:
A) use dissolved in chloroform phosphatide;
B) nitrogen dries up, and vacuum-drying to chloroform volatilizees fully;
C) with the damping fluid aquation;
D) water-bath supersound process to solution is clarified; And
E) pushed film.
4. method as claimed in claim 3 is characterized in that, in step e), extruding uses the aperture of filter membrane to be 50nm, extruding number of times at least 11 times.
5. the method for claim 1 is characterized in that, said Pegylation phospholipid molecule is that polyethylene glycol polymer and phospholipid molecule form through chemical covalent bonding.
6. the method for claim 1; It is characterized in that, in step 2) in, described chitin modified solid surface obtains with following method: the silicon chip that cleans up was carried out oscillation treatment 30 minutes with 3: 1 the vitriol oil of volume ratio, hydrogen peroxide; After water cleans; Change in the ethanolic soln that contains 10% aminopropyltriethoxywerene werene oscillation treatment over to 2 hours, and after rinsing well, obtained amino modified silicon chip; Handle said amino modified silicon chip with 5% glutaraldehyde solution, 1 hour post-flush of room temperature oscillation treatment is clean, again with treated fixed low-molecular weight chitoglycan solution reaction 10 minutes, the washing, nitrogen dries up subsequent use.
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| CN110013770B (en) * | 2018-01-10 | 2021-05-25 | 中国科学院苏州纳米技术与纳米仿生研究所 | Capsule-like lipid solid support membrane and construction method thereof |
| CN108827817A (en) * | 2018-07-05 | 2018-11-16 | 华南理工大学 | A kind of preparation method of the bionic phospholipid membrane of morphology controllable |
| CN113599861B (en) * | 2021-08-13 | 2023-06-27 | 广东石油化工学院 | Method for separating cholesterol in phospholipid membrane by using modified substrate |
| CN114094182A (en) * | 2021-11-03 | 2022-02-25 | 珠海冠宇电池股份有限公司 | Secondary battery |
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Non-Patent Citations (1)
| Title |
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| 张义浜等.PEG化磷脂膜在蛋白质芯片表面修饰中的应用.《材料工程》.2008,(第10期),第208-210页. * |
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