WO2024055284A1 - Ion cross-linked biomimetic membrane, preparation method therefor, and use thereof - Google Patents

Ion cross-linked biomimetic membrane, preparation method therefor, and use thereof Download PDF

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WO2024055284A1
WO2024055284A1 PCT/CN2022/119287 CN2022119287W WO2024055284A1 WO 2024055284 A1 WO2024055284 A1 WO 2024055284A1 CN 2022119287 W CN2022119287 W CN 2022119287W WO 2024055284 A1 WO2024055284 A1 WO 2024055284A1
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cross
membrane
buffer
amphiphilic block
block copolymer
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PCT/CN2022/119287
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French (fr)
Chinese (zh)
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王盛文
王照辉
云全新
曾涛
董宇亮
黎宇翔
章文蔚
滕波
徐讯
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深圳华大生命科学研究院
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Publication of WO2024055284A1 publication Critical patent/WO2024055284A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/02—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor characterised by their properties
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/12—Composite membranes; Ultra-thin membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/76—Macromolecular material not specifically provided for in a single one of groups B01D71/08 - B01D71/74
    • B01D71/80—Block polymers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G77/00—Macromolecular compounds obtained by reactions forming a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon in the main chain of the macromolecule
    • C08G77/42—Block-or graft-polymers containing polysiloxane sequences
    • C08G77/452—Block-or graft-polymers containing polysiloxane sequences containing nitrogen-containing sequences
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers

Definitions

  • the invention belongs to the field of membrane technology, and specifically relates to an ionically cross-linked bionic membrane, its preparation method and application.
  • the phospholipid bilayer is the main structure of most biological cell membranes.
  • the cell membranes of biological cells also contain transmembrane proteins with different functions. They play an important role in cell material exchange and information transmission.
  • biosensors based on membrane-protein systems. These sensors can be used to analyze various biological and chemical components, such as ions, small molecules, nucleic acids, amino acids, etc.
  • Phospholipid bimolecular membranes can be prepared using chemically extracted or synthesized phospholipids, but the low chemical stability and mechanical strength of phospholipid bimolecular membranes limit their application in biosensing.
  • the molecular membrane formed by the self-assembly of the synthesized amphiphilic polymer has high chemical stability and high mechanical strength, and is more resistant to external interference. It is an excellent substitute for phospholipid membranes.
  • the Chinese patent with publication number CN104936682B discloses a droplet interface. The patent discloses the technology of using amphiphilic block polymers to self-assemble at the interface of two polar solution droplets to form an amphiphilic molecular membrane.
  • Both di-block and tri-block amphiphilic block copolymers can self-assemble to form an amphiphilic molecular layer.
  • the di-block polymer contains a hydrophilic segment and a hydrophobic segment
  • the tri-block polymer has a hydrophobic middle segment. chain segment, with hydrophilic segments at both ends.
  • the hydrophobic segments of the block copolymer are arranged close to each other under the hydrophobic force to form a non-polar inner layer, and the hydrophilic segments are arranged toward the polar solvent side to form a hydrophilic outer layer. layer.
  • the object of the present invention is to provide an ionically cross-linked biomimetic membrane with strong mechanical strength and low leakage current, its preparation method and application.
  • the invention provides an ionically cross-linked bionic membrane, which is formed by self-assembly of film-forming molecules; the film-forming molecules include an amphiphilic block copolymer; between the hydrophilic chains of the amphiphilic block copolymer Cross-linking by a cross-linking agent; the hydrophilic segment of the amphiphilic block copolymer has opposite charge to that of the cross-linking agent in the aqueous solution.
  • the hydrophilic segment of the amphiphilic block copolymer is formed of hydrophilic monomers; the hydrophilic monomers include substituted and/or unsubstituted oxazoline monomers; the substituted oxazoline monomers
  • the substituents in the oxazoline monomer are selected from C1 to C10 alkyl groups.
  • the hydrophilic monomer includes one or more of oxazoline, 2-methyloxazoline and 2-ethyloxazoline.
  • the hydrophobic segment of the amphiphilic block copolymer is selected from polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxyethylene Butene, polyisoprene, polybutadiene, polyalkyl acrylate, polyacrylonitrile, polytetrahydrofuran, polymethacrylate, polyacrylate, polysulfone, polyvinyl ether, polypropylene oxide, poly Caprolactone and one or more of the above copolymers;
  • the degree of polymerization of the hydrophilic chain of the amphiphilic block polymer is 1-20, preferably 3-15, and most preferably 3-10; the degree of polymerization of the hydrophobic chain of the amphiphilic block polymer is 10-10. 60, preferably 20-40, most preferably 25-35.
  • the polymerization degree ratio of the hydrophilic segment and the hydrophobic segment of the amphiphilic block copolymer is (2-20): (20-60), preferably (2-18): (25-50), More preferably (2 to 16): (25 to 45), still more preferably (2 to 16): (28 to 42).
  • the cross-linking agent is an anionic compound; the anionic compound is selected from the group consisting of polyphosphate, polyphosphonic acid polymer, citrate, ethylenediaminetetraacetate, hexametaphosphate, and polyacrylate. , one or more of polysilicate and polyolefin sulfonate.
  • the invention also provides a method for preparing an ionically cross-linked biomimetic membrane, which includes:
  • the membrane solution is self-assembled under the condition that a first buffer and a second buffer are respectively provided on both sides to obtain an ionically cross-linked biomimetic membrane; the first buffer and/or the second buffer contain a cross-linking agent;
  • the hydrophilic segment of the amphiphilic block copolymer has an opposite charge to that of the cross-linking agent in the aqueous solution.
  • the concentration of the amphiphilic block copolymer in the membrane solution is 10-50 mg/mL; the concentration of the cross-linking agent in the first buffer and/or the second buffer is 0.1-100 mM.
  • the solvent is selected from alkanes and/or silicone oil; the first buffer and the second buffer are each independently selected from one of HEPES buffer, Tris buffer, and PBS buffer.
  • the present invention also provides a biosensor, which includes the above-mentioned ionically cross-linked biomimetic membrane.
  • the invention provides an ionically cross-linked bionic membrane, which is formed by self-assembly of an amphiphilic block copolymer; the hydrophilic chains of the amphiphilic block copolymer are cross-linked by a cross-linking agent; the amphiphilic block copolymer is cross-linked.
  • the hydrophilic segments of the block copolymer have opposite charges to the cross-linking agent in aqueous solution.
  • the present invention uses a cross-linking agent with an opposite charge to the amphiphilic block copolymer to generate a bridging effect through electrostatic adsorption to form cross-links between adjacent hydrophilic segments in the form of ionic bonds, thereby making the amphiphilic block copolymer
  • the self-assembly of segmented copolymers to form an amphiphilic molecular film reduces the intermolecular gaps, improves the stability of the film, and increases the resistance and mechanical strength of the film, allowing it to have lower leakage current and lower leakage when used in biosensing. Longer service life.
  • Figure 1 is a schematic diagram of the transmembrane current of the bionic membrane obtained in step 1.4 in Example 1 of the present invention
  • Figure 2 is a schematic diagram of the transmembrane current of the bionic membrane obtained in 1.6 in Example 1 of the present invention
  • Figure 3 is a schematic diagram of the transmembrane current of the bionic membrane obtained in 1.7 in Example 1 of the present invention
  • Figure 4 is a schematic diagram of the transmembrane current of the biomimetic membrane obtained in 2.4 in Example 2 of the present invention.
  • Figure 5 is a schematic diagram of the transmembrane current of the bionic membrane obtained in 2.6 in Example 2 of the present invention.
  • Figure 6 is a schematic diagram of the transmembrane current of the bionic membrane obtained in 2.7 in Example 2 of the present invention.
  • Figure 7 is a graph showing the changing trend of the sequencing channel ratio obtained in 3.6 with sequencing time in Example 3 of the present invention.
  • the invention provides an ionically cross-linked bionic membrane, which is formed by self-assembly of film-forming molecules; the film-forming molecules include an amphiphilic block copolymer; between the hydrophilic chains of the amphiphilic block copolymer Cross-linking by a cross-linking agent; the hydrophilic segment of the amphiphilic block copolymer has opposite charge to that of the cross-linking agent in the aqueous solution.
  • the biomimetic membrane provided by the present invention is formed by the self-assembly of amphiphilic block copolymers; wherein, the amphiphilic block copolymers can be amphiphilic block copolymers well known to those skilled in the art, and there are no special restrictions; in the present invention , the amphiphilic block copolymer is selected from one or more of amphiphilic di-block copolymers, amphiphilic tri-block copolymers and amphiphilic penta-block copolymers; the configuration of the amphiphilic di-block copolymer is: Type A-B; the configuration of the amphiphilic triblock copolymer is type A-B-A1; the configuration of the amphiphilic pentablock copolymer is type B1-A1-B-A2-B2; where A, A1 and A2 are The hydrophilic segments may be the same or different; B, B1 and B2 are hydrophobic segments, and the three may be the same or different;
  • the hydrophobic segment is preferably polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyacetic acid Vinyl ester, polyoxybutylene, polyisoprene, polybutadiene, polyalkyl acrylate, polyacrylonitrile, polytetrahydrofuran, polymethacrylate, polyacrylate, polysulfone, polyvinyl ether, poly One or more of propylene oxide, polycaprolactone and the above-mentioned copolymers;
  • the hydrophilic chain polymerization degree of the amphiphilic block polymer is 1-20, preferably 3-15, most preferably 3-10 ;
  • the degree of polymerization of the hydrophobic chain of the amphiphilic block copolymer is 10-60, preferably 20-40, and most preferably 25-35; the polymerization of the hydrophilic segment and the hydrophobic
  • the hydrophilic chains of the amphiphilic block copolymer are cross-linked through a cross-linking agent; the hydrophilic segments of the amphiphilic block copolymer have opposite charges to those of the cross-linking agent in the aqueous solution; in the present invention, the The hydrophilic segment of the amphiphilic block copolymer is preferably positively charged in the aqueous solution, so the cross-linking agent is preferably an anionic compound, that is, a compound with a negative charge in the aqueous solution; further preferably, the anionic compound is selected from From one or more of polyphosphate, polyphosphonic acid polymer, citrate, ethylenediaminetetraacetate, hexametaphosphate, polyacrylate, polysilicate and polyolefin sulfonate species; in the present invention, the cross-linking agent is preferably in contact with the amphiphilic block copolymer in the buffer to cross-link its hydrophilic chain; the concentration of the cross-linking
  • porins can preferably be inserted into the amphipathic molecular membrane, so that the biomimetic membrane can carry transmembrane proteins for biosensing.
  • the channel proteins are preferably driven by voltage, autonomous fusion or protein synthesis.
  • the vesicles are inserted into biomimetic membranes through fusion and then used for biosensing.
  • the present invention uses a cross-linking agent with an opposite charge to the amphiphilic block copolymer to generate a bridging effect through electrostatic adsorption to form cross-links between adjacent hydrophilic segments in the form of ionic bonds, so that the amphiphilic block copolymer self-assembles to form an amphiphilic block copolymer.
  • the intermolecular gaps of the molecular film become smaller, which improves the stability of the film and increases the resistance and mechanical strength of the film, allowing it to have lower leakage current and longer service life when used for biosensing.
  • the invention also provides a method for preparing the above-mentioned ionically cross-linked biomimetic membrane, which includes: dissolving the amphiphilic block copolymer in a solvent to obtain a membrane solution; disposing a first buffer and a second buffer on both sides of the membrane solution. Self-assembly is performed under buffer conditions to obtain an ionically cross-linked biomimetic membrane; the first buffer and/or the second buffer include a cross-linking agent; the hydrophilic segment of the amphiphilic block copolymer is in the aqueous solution Opposite the charge of the cross-linking agent.
  • the present invention has no special restrictions on the sources of all raw materials, as long as they are commercially available; the types and proportions of the amphiphilic block copolymer and cross-linking agent are the same as mentioned above, and will not be described again here.
  • the amphiphilic block copolymer is dissolved in a solvent to obtain a membrane solution;
  • the present invention has no special restrictions on the type of the solvent, as long as it is a solvent well known to those skilled in the art.
  • it is preferably an alkane and/or Silicone oil;
  • the concentration of the amphiphilic block copolymer in the membrane solution is preferably 0.1 to 50 mg/mL, more preferably 1 to 40 mg/mL, and still more preferably 1 to 30 mg/mL.
  • the membrane solution is self-assembled under the condition that a first buffer and a second buffer are respectively provided on both sides to obtain an ionically cross-linked biomimetic membrane; in the present invention, this self-assembly process is preferably performed on an open-hole array chip or patch clamp.
  • the open-hole array chip Take the open-hole array chip as an example.
  • the polymer solution is painted on the open-hole array chip, and the first buffer and the second buffer can be self-assembled or sequentially passed through the channels of the open-hole array chip.
  • the first buffer, the membrane solution and the second buffer can self-assemble to form an ionic cross-linked biomimetic membrane; the first buffer and/or the second buffer include a cross-linking agent; the cross-linking agent is preferably anionic.
  • the anionic compound is selected from polyphosphate, polyphosphonic acid polymer, citrate, ethylenediaminetetraacetate, hexametaphosphate
  • concentration of the cross-linking agent in the first buffer and/or the second buffer is preferably 0.1 to 100 mmol/L , more preferably 0.1 ⁇ 50mmol/L, further preferably 1 ⁇ 30mmol/L, most preferably 5 ⁇ 30mmol/L; in the present invention, the first buffer and the second buffer can simultaneously contain a cross-linking agent Only one of them may contain a cross-linking agent.
  • the hydrophilic segments of the amphiphilic molecules on that side will be cross-linked; the cross-linking of the hydrophilic segments of the film-forming molecules of the biomimetic membrane provided by the invention
  • the cross-linking is reversible, that is, the degree of cross-linking of the biomimetic membrane can be adjusted by changing the content of the cross-linking agent in the first buffer and/or the second buffer on both sides of the membrane.
  • the cross-linking reaction balance between membrane molecules is determined by the content of the cross-linking agent in the first buffer and/or the second buffer on both sides of the membrane. Increase the content of the cross-linking agent in the first buffer and/or the second buffer.
  • the first buffer and the second buffer are buffers well known to those skilled in the art and are not particularly limited.
  • the first buffer and the second buffer in the present invention are each independently preferably a HEPES buffer, One of Tris buffer and PBS buffer; the concentration of the HEPES buffer is preferably 10 mmol/L; the concentration of the PBS buffer is specifically 10 mmol/L; the first buffer and the second buffer Preferably, it also includes a stabilizer; the stabilizer is preferably EDTA; the concentrations of the buffers in the first buffer and the second buffer are each independently 1 to 10 mmol/L, and more preferably each is independently 2 to 10 mmol/L.
  • the first buffer and the second buffer can also include other substances according to the purpose of the bionic membrane, such as The biomimetic membrane is used for biosensing.
  • the first buffer and the second buffer preferably further include potassium ferricyanide and potassium ferrocyanide; the potassium ferricyanide in the first buffer and the second buffer is preferably The concentration is preferably 10-1000mmol/L, more preferably 30-800mmol/L, still more preferably 40-600mmol/L, and most preferably 150mmol/L; ferrocyanation in the first buffer and the second buffer
  • concentration of potassium is preferably 10 to 1000 mmol/L, more preferably 30 to 800 mmol/L, further preferably 40 to 600 mmol/L, and most preferably 150 mmol/L.
  • the present invention also provides an application of the above-mentioned ionically cross-linked biomimetic membrane in carrying transmembrane proteins; it can then be used for biological sensing, such as nanopore gene sequencing, protein sequencing and detection of other components.
  • the present invention also provides a biosensor, which includes the above-mentioned ionically cross-linked biomimetic membrane; the biosensor is preferably a nanopore gene sequencer or a protein sequencer.
  • an ionically cross-linked biomimetic membrane provided by the present invention, its preparation method and application are described in detail below with reference to the examples.
  • the reagents used in the following examples are all commercially available; the data in the following examples are mainly based on using the biomimetic membrane of the present invention on a 256-channel nanopore sequencing system.
  • the system consists of an opening array microstructure chip, a control circuit system and control software. That is, the biomimetic membrane is formed in an open-hole array microstructure chip containing 256 channels.
  • polar solution 1 Use ultrapure water as the solvent to prepare polar solution 1 according to the following formula: 150mM potassium ferricyanide, 150mM potassium ferrocyanide, 5mM EDTA, 10mM HEPES, 5mM sodium citrate.
  • polar solution 2 150mM potassium ferricyanide, 150mM potassium ferrocyanide, 5mM EDTA, 10mM HEPES.
  • This cross-linking effect can reduce membrane leakage, and this cross-linking is reversible and controllable.
  • the degree of cross-linking of the membrane can be adjusted by adjusting the concentration of the cross-linking agent in the polar solution on both sides of the biomimetic membrane.
  • polar solution 2 Use ultrapure water as the solvent to prepare polar solution 2 according to the following formula: 150mM potassium ferricyanide, 150mM potassium ferrocyanide, 5mM EDTA, 10mM HEPES.
  • the transmembrane leakage current is the largest when both sides of the biomimetic membrane are polar solution two. .
  • the concentration of sodium citrate in the polar solution on one side of the membrane gradually increased, and the membrane molecules were gradually cross-linked, causing the transmembrane leakage current to gradually decrease. decrease.
  • sodium tripolyphosphate has a cross-linking effect on the biomimetic membrane formed by 8PEtOXZ-42PDMS-8PMOXZ. This cross-linking effect can reduce membrane leakage, and this cross-linking is controllable.
  • polar solution 2 150mM potassium ferricyanide, 150mM potassium ferrocyanide, 5mM EDTA, 10mM HEPES.
  • Example 3 has more embedded holes and less broken membranes. This shows that the ionically cross-linked biomimetic membrane has stronger mechanical strength and better stability.
  • the data in Figure 7 shows that the number of sequencing channels in Example 3 decreases more slowly than in Comparative Example 1, which means that the sequencing channels in Example 3 can work for a longer time.

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Abstract

The present invention provides an ion cross-linked biomimetic membrane. The biomimetic membrane is formed by self-assembly of an amphiphilic block copolymer. Hydrophilic chains of the amphiphilic block copolymer are cross-linked by means of a cross-linking agent. The hydrophilic chain segments of the amphiphilic block copolymer have opposite charges to the cross-linking agent in an aqueous solution. Compared with the prior art, the present invention utilizes the cross-linking agent having a charge opposite to the charge of the amphiphilic block copolymer to generate a bridging effect by means of electrostatic adsorption to form, in the form of ionic bonds, cross-links from adjacent hydrophilic chain segments, such that intermolecular gaps of an amphiphilic molecular membrane formed by self-assembly of the amphiphilic block copolymer decreases, the stability of the membrane is enhanced, and the electrical resistance and the mechanical strength of the membrane are increased, such that the membrane has a lower leakage current and a longer service life when used for biosensing.

Description

一种离子交联的仿生膜、其制备方法及应用An ionically cross-linked biomimetic membrane, its preparation method and application 技术领域Technical field
本发明属于膜技术领域，具体涉及一种离子交联的仿生膜、其制备方法及应用。The invention belongs to the field of membrane technology, and specifically relates to an ionically cross-linked bionic membrane, its preparation method and application.
背景技术Background technique
磷脂双分子层是大多数生物细胞膜的主要结构，除此之外生物细胞的细胞膜中还含有具有不同功能的跨膜蛋白，它们为细胞的物质交换、信息传递起到了重要作用。受生物体的膜-蛋白系统的启发，人们设计了基于膜-蛋白系统的生物传感器，这些传感器可以用于分析各种生物、化学成分，如离子、小分子、核酸、氨基酸等。The phospholipid bilayer is the main structure of most biological cell membranes. In addition, the cell membranes of biological cells also contain transmembrane proteins with different functions. They play an important role in cell material exchange and information transmission. Inspired by the membrane-protein system of organisms, people have designed biosensors based on membrane-protein systems. These sensors can be used to analyze various biological and chemical components, such as ions, small molecules, nucleic acids, amino acids, etc.
利用化学提取或合成的磷脂可以制备出磷脂双分子膜，但磷脂双分子膜较低的化学稳定性和机械强度限制了其在生物传感方面的应用。而合成的双亲高分子聚合物自组装形成的分子膜具有较高的化学稳定性和较高的机械强度，耐受外界干扰的能力更强，是磷脂膜的优良替代物。公开号为CN104936682B的中国专利公开了一种微滴界面，该专利中公开了利用双亲嵌段聚合物在两个极性溶液液滴界面处发生自组装形成双亲分子膜的技术。两嵌段和三嵌段的双亲嵌段共聚物都可以自组装形成双亲分子层，其中两嵌段聚合物含有一条亲水链段和一条疏水链段，三嵌段聚合物中间链段是疏水链段，两端为亲水链段。双亲嵌段共聚物在形成双亲分子膜的过程中，在疏水作用力下嵌段共聚物的疏水段相互靠拢排列形成非极性内层，亲水段朝向极性溶剂一侧排列形成亲水外层。虽然高分子聚合物膜的稳定性优于磷脂双分子膜，但是其稳定性依然有很大的提升空间。此外，很多基于膜-蛋白系统设计的生物传感器都需要膜具有很高的阻抗以获得更低噪音的电信号，但很多聚合物自组装成膜时分子间的空隙较大，导致膜分子堆积不够紧密，膜的机械强度较低，且在膜两侧施加电压时存在较大程度的漏电，即膜的阻抗不够大。Phospholipid bimolecular membranes can be prepared using chemically extracted or synthesized phospholipids, but the low chemical stability and mechanical strength of phospholipid bimolecular membranes limit their application in biosensing. The molecular membrane formed by the self-assembly of the synthesized amphiphilic polymer has high chemical stability and high mechanical strength, and is more resistant to external interference. It is an excellent substitute for phospholipid membranes. The Chinese patent with publication number CN104936682B discloses a droplet interface. The patent discloses the technology of using amphiphilic block polymers to self-assemble at the interface of two polar solution droplets to form an amphiphilic molecular membrane. Both di-block and tri-block amphiphilic block copolymers can self-assemble to form an amphiphilic molecular layer. The di-block polymer contains a hydrophilic segment and a hydrophobic segment, and the tri-block polymer has a hydrophobic middle segment. chain segment, with hydrophilic segments at both ends. In the process of forming an amphiphilic molecular membrane from an amphiphilic block copolymer, the hydrophobic segments of the block copolymer are arranged close to each other under the hydrophobic force to form a non-polar inner layer, and the hydrophilic segments are arranged toward the polar solvent side to form a hydrophilic outer layer. layer. Although the stability of polymer membranes is better than that of phospholipid bimolecular membranes, there is still a lot of room for improvement in its stability. In addition, many biosensors designed based on membrane-protein systems require the membrane to have high impedance to obtain lower noise electrical signals. However, when many polymers self-assemble into membranes, the gaps between molecules are large, resulting in insufficient accumulation of membrane molecules. Tight, the mechanical strength of the membrane is low, and there is a large degree of leakage when voltage is applied on both sides of the membrane, that is, the impedance of the membrane is not large enough.
发明内容Contents of the invention
本发明的目的在于提供一种具有较强机械强度与较低漏电流的离子交联的仿生膜、其制备方法及应用。The object of the present invention is to provide an ionically cross-linked biomimetic membrane with strong mechanical strength and low leakage current, its preparation method and application.
本发明提供了一种离子交联的仿生膜，所述仿生膜由成膜分子自组装形成；所述成膜分子包括双亲嵌段共聚物；所述双亲嵌段共聚物的亲水链之间通过交联剂交联；所述双亲嵌段共聚物的亲水链段在水溶液中与交联剂的电荷性相反。The invention provides an ionically cross-linked bionic membrane, which is formed by self-assembly of film-forming molecules; the film-forming molecules include an amphiphilic block copolymer; between the hydrophilic chains of the amphiphilic block copolymer Cross-linking by a cross-linking agent; the hydrophilic segment of the amphiphilic block copolymer has opposite charge to that of the cross-linking agent in the aqueous solution.
优选的，所述双亲嵌段共聚物的亲水链段由亲水性单体形成；所述亲水性单体包括取 代和/或未取代的恶唑啉类单体；所述取代的恶唑啉类单体中的取代基选自C1～C10的烷基。Preferably, the hydrophilic segment of the amphiphilic block copolymer is formed of hydrophilic monomers; the hydrophilic monomers include substituted and/or unsubstituted oxazoline monomers; the substituted oxazoline monomers The substituents in the oxazoline monomer are selected from C1 to C10 alkyl groups.
优选的，所述亲水性单体包括恶唑啉、2-甲基恶唑啉与2-乙基恶唑啉中的一种或多种。Preferably, the hydrophilic monomer includes one or more of oxazoline, 2-methyloxazoline and 2-ethyloxazoline.
优选的，所述双亲嵌段共聚物的疏水链段选自聚硅氧烷、聚烯烃、全氟聚醚、全氟烃基聚醚、聚苯乙烯、聚氧丙烯、聚醋酸乙烯酯、聚氧丁烯、聚异戊二烯、聚丁二烯、聚烷基丙烯酸酯、聚丙烯腈、聚四氢呋喃、聚甲基丙烯酸酯、聚丙烯酸酯、聚砜、聚乙烯醚、聚环氧丙烷、聚己内酯与上述共聚物中的一种或多种；Preferably, the hydrophobic segment of the amphiphilic block copolymer is selected from polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxyethylene Butene, polyisoprene, polybutadiene, polyalkyl acrylate, polyacrylonitrile, polytetrahydrofuran, polymethacrylate, polyacrylate, polysulfone, polyvinyl ether, polypropylene oxide, poly Caprolactone and one or more of the above copolymers;
优选的，所述双亲嵌段聚合物的亲水链聚合度为1-20，优选为3-15，最优选为3-10；所述双亲嵌段聚合物的疏水链的聚合度为10-60，优选为20-40，最优选为25-35。Preferably, the degree of polymerization of the hydrophilic chain of the amphiphilic block polymer is 1-20, preferably 3-15, and most preferably 3-10; the degree of polymerization of the hydrophobic chain of the amphiphilic block polymer is 10-10. 60, preferably 20-40, most preferably 25-35.
优选的，所述双亲嵌段共聚物的亲水链段与疏水链段的聚合度比为(2～20)：(20～60)，优选为(2～18)：(25～50)，更优选为(2～16)：(25～45)，再优选为(2～16)：(28～42)。Preferably, the polymerization degree ratio of the hydrophilic segment and the hydrophobic segment of the amphiphilic block copolymer is (2-20): (20-60), preferably (2-18): (25-50), More preferably (2 to 16): (25 to 45), still more preferably (2 to 16): (28 to 42).
优选的，所述交联剂为阴离子化合物；所述阴离子化合物选自多聚磷酸盐、多聚膦酸聚合物、柠檬酸盐、乙二胺四乙酸盐、六偏磷酸盐、聚丙烯酸盐、聚硅酸盐与聚烯烃磺酸盐中的一种或多种。Preferably, the cross-linking agent is an anionic compound; the anionic compound is selected from the group consisting of polyphosphate, polyphosphonic acid polymer, citrate, ethylenediaminetetraacetate, hexametaphosphate, and polyacrylate. , one or more of polysilicate and polyolefin sulfonate.
本发明还提供了一种离子交联的仿生膜的制备方法，包括：The invention also provides a method for preparing an ionically cross-linked biomimetic membrane, which includes:
将双亲嵌段共聚物溶解于溶剂中，得到膜溶液；Dissolve the amphiphilic block copolymer in the solvent to obtain a membrane solution;
将膜溶液在两侧分别设置第一缓冲液与第二缓冲液的条件下进行自组装，得到离子交联的仿生膜；所述第一缓冲液和/或第二缓冲液含有交联剂；所述双亲嵌段共聚物的亲水链段在水溶液中与交联剂的电荷性相反。The membrane solution is self-assembled under the condition that a first buffer and a second buffer are respectively provided on both sides to obtain an ionically cross-linked biomimetic membrane; the first buffer and/or the second buffer contain a cross-linking agent; The hydrophilic segment of the amphiphilic block copolymer has an opposite charge to that of the cross-linking agent in the aqueous solution.
优选的，所述膜溶液中双亲嵌段共聚物的浓度为10～50mg/mL；所述第一缓冲液和/或第二缓冲液中交联剂的浓度为0.1～100mM。Preferably, the concentration of the amphiphilic block copolymer in the membrane solution is 10-50 mg/mL; the concentration of the cross-linking agent in the first buffer and/or the second buffer is 0.1-100 mM.
优选的，所述溶剂选自烷烃和/或硅油；所述第一缓冲液与第二缓冲液各自独立地选自HEPES缓冲液、Tris缓冲液、PBS缓冲液中的一种。Preferably, the solvent is selected from alkanes and/or silicone oil; the first buffer and the second buffer are each independently selected from one of HEPES buffer, Tris buffer, and PBS buffer.
本发明还提供了一种生物传感器，包括上述离子交联的仿生膜。The present invention also provides a biosensor, which includes the above-mentioned ionically cross-linked biomimetic membrane.
本发明提供了一种离子交联的仿生膜，所述仿生膜由双亲嵌段共聚物自组装形成；所述双亲嵌段共聚物的亲水链之间通过交联剂交联；所述双亲嵌段共聚物的亲水链段在水溶液中与交联剂的电荷性相反。与现有技术相比，本发明采用与双亲嵌段共聚物电荷相反的交联剂通过静电吸附产生桥连作用将相邻的亲水链段以离子键的形式形成交联，从而使双亲嵌段共聚物自组装形成双亲分子膜的分子间空隙变小，提高了膜的稳定性，同时增大了膜的电阻和机械强度，使其在用于生物传感时具有更低的漏电流和更长的使用寿命。The invention provides an ionically cross-linked bionic membrane, which is formed by self-assembly of an amphiphilic block copolymer; the hydrophilic chains of the amphiphilic block copolymer are cross-linked by a cross-linking agent; the amphiphilic block copolymer is cross-linked. The hydrophilic segments of the block copolymer have opposite charges to the cross-linking agent in aqueous solution. Compared with the existing technology, the present invention uses a cross-linking agent with an opposite charge to the amphiphilic block copolymer to generate a bridging effect through electrostatic adsorption to form cross-links between adjacent hydrophilic segments in the form of ionic bonds, thereby making the amphiphilic block copolymer The self-assembly of segmented copolymers to form an amphiphilic molecular film reduces the intermolecular gaps, improves the stability of the film, and increases the resistance and mechanical strength of the film, allowing it to have lower leakage current and lower leakage when used in biosensing. Longer service life.
附图说明Description of drawings
图1为本发明实施例1中1.4得到的仿生膜的跨膜电流示意图；Figure 1 is a schematic diagram of the transmembrane current of the bionic membrane obtained in step 1.4 in Example 1 of the present invention;
图2为本发明实施例1中1.6得到的仿生膜的跨膜电流示意图；Figure 2 is a schematic diagram of the transmembrane current of the bionic membrane obtained in 1.6 in Example 1 of the present invention;
图3为本发明实施例1中1.7得到的仿生膜的跨膜电流示意图；Figure 3 is a schematic diagram of the transmembrane current of the bionic membrane obtained in 1.7 in Example 1 of the present invention;
图4为本发明实施例2中2.4得到的仿生膜的跨膜电流示意图；Figure 4 is a schematic diagram of the transmembrane current of the biomimetic membrane obtained in 2.4 in Example 2 of the present invention;
图5为本发明实施例2中2.6得到的仿生膜的跨膜电流示意图；Figure 5 is a schematic diagram of the transmembrane current of the bionic membrane obtained in 2.6 in Example 2 of the present invention;
图6为本发明实施例2中2.7得到的仿生膜的跨膜电流示意图；Figure 6 is a schematic diagram of the transmembrane current of the bionic membrane obtained in 2.7 in Example 2 of the present invention;
图7为本发明实施例3中3.6得到的测序通道比例随测序时间的变化趋势图。Figure 7 is a graph showing the changing trend of the sequencing channel ratio obtained in 3.6 with sequencing time in Example 3 of the present invention.
具体实施方式Detailed ways
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.
本发明提供了一种离子交联的仿生膜，所述仿生膜由成膜分子自组装形成；所述成膜分子包括双亲嵌段共聚物；所述双亲嵌段共聚物的亲水链之间通过交联剂交联；所述双亲嵌段共聚物的亲水链段在水溶液中与交联剂的电荷性相反。The invention provides an ionically cross-linked bionic membrane, which is formed by self-assembly of film-forming molecules; the film-forming molecules include an amphiphilic block copolymer; between the hydrophilic chains of the amphiphilic block copolymer Cross-linking by a cross-linking agent; the hydrophilic segment of the amphiphilic block copolymer has opposite charge to that of the cross-linking agent in the aqueous solution.
本发明提供的仿生膜由双亲嵌段共聚物自组装形成；其中，所述双亲嵌段共聚物为本领域技术人员熟知的双亲嵌段共聚物即可，并无特殊的限制；在本发明中，所述双亲嵌段共聚物选自双亲两嵌段共聚物、双亲三嵌段共聚物与双亲五嵌段共聚物中的一种或多种；所述双亲两嵌段共聚物的构型为A-B型；所述双亲三嵌段共聚物的构型为A-B-A1型；所述双亲五嵌段共聚物的构型为B1-A1-B-A2-B2型；其中A、A1与A2为亲水链段，可以相同也可不同；B、B1与B2为疏水链段，三者可以相同，也可不同；在本发明中，所述双亲嵌段共聚物的亲水链段由亲水性单体形成；所述亲水性单体优选包括取代和/或未取代的恶唑啉类单体；所述取代的恶唑啉类单体中的取代基优选为C1～C10的烷基，更优选为C1～C8的烷基，再优选为C1～C6的烷基，再优选为C1～C4的烷基，最优选为甲基或乙基；最优选的，所述亲水性单体包括恶唑啉、2-甲基恶唑啉与2-乙基恶唑啉中的一种或多种；所述双亲嵌段共聚物的疏水链段为本领域技术人员熟知的疏水链段即可，并无特殊的限制，在本发明中，所述疏水链段优选为聚硅氧烷、聚烯烃、全氟聚醚、全氟烃基聚醚、聚苯乙烯、聚氧丙烯、聚醋酸乙烯酯、聚氧丁烯、聚异戊二烯、聚丁二烯、聚烷基丙烯酸酯、聚 丙烯腈、聚四氢呋喃、聚甲基丙烯酸酯、聚丙烯酸酯、聚砜、聚乙烯醚、聚环氧丙烷、聚己内酯与上述共聚物中的一种或多种；所述双亲嵌段聚合物的亲水链聚合度为1-20，优选为3-15，最优选为3-10；所述双亲嵌段聚合物的疏水链的聚合度为10-60，优选为20-40，最优选为25-35；所述双亲嵌段共聚物的亲水链段与疏水链段的聚合度比优选为(2～20)：(20～60)，更优选为(2～18)：(25～50)，再优选为(2～16)：(25～45)，最优选为(2～16)：(28～42)。The biomimetic membrane provided by the present invention is formed by the self-assembly of amphiphilic block copolymers; wherein, the amphiphilic block copolymers can be amphiphilic block copolymers well known to those skilled in the art, and there are no special restrictions; in the present invention , the amphiphilic block copolymer is selected from one or more of amphiphilic di-block copolymers, amphiphilic tri-block copolymers and amphiphilic penta-block copolymers; the configuration of the amphiphilic di-block copolymer is: Type A-B; the configuration of the amphiphilic triblock copolymer is type A-B-A1; the configuration of the amphiphilic pentablock copolymer is type B1-A1-B-A2-B2; where A, A1 and A2 are The hydrophilic segments may be the same or different; B, B1 and B2 are hydrophobic segments, and the three may be the same or different; in the present invention, the hydrophilic segment of the amphiphilic block copolymer is composed of hydrophilic segments Hydrophilic monomers are formed; the hydrophilic monomers preferably include substituted and/or unsubstituted oxazoline monomers; the substituents in the substituted oxazoline monomers are preferably C1 to C10 alkyl groups , more preferably a C1-C8 alkyl group, further preferably a C1-C6 alkyl group, even more preferably a C1-C4 alkyl group, most preferably a methyl or ethyl group; most preferably, the hydrophilic monomer The body includes one or more of oxazoline, 2-methyloxazoline and 2-ethyloxazoline; the hydrophobic segment of the amphiphilic block copolymer is a hydrophobic segment well known to those skilled in the art. That is, there is no special limitation. In the present invention, the hydrophobic segment is preferably polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyacetic acid Vinyl ester, polyoxybutylene, polyisoprene, polybutadiene, polyalkyl acrylate, polyacrylonitrile, polytetrahydrofuran, polymethacrylate, polyacrylate, polysulfone, polyvinyl ether, poly One or more of propylene oxide, polycaprolactone and the above-mentioned copolymers; the hydrophilic chain polymerization degree of the amphiphilic block polymer is 1-20, preferably 3-15, most preferably 3-10 ; The degree of polymerization of the hydrophobic chain of the amphiphilic block copolymer is 10-60, preferably 20-40, and most preferably 25-35; the polymerization of the hydrophilic segment and the hydrophobic segment of the amphiphilic block copolymer The degree ratio is preferably (2 to 20): (20 to 60), more preferably (2 to 18): (25 to 50), further preferably (2 to 16): (25 to 45), most preferably ( 2～16): (28～42).
所述双亲嵌段共聚物的亲水链之间通过交联剂交联；所述双亲嵌段共聚物的亲水链段在水溶液中与交联剂的电荷性相反；在本发明中，所述双亲嵌段共聚物的亲水链段优选在水溶液中带正电荷，因此所述交联剂优选为阴离子化合物，即在水溶液中带有负电荷的化合物；进一步优选的，所述阴离子化合物选自多聚磷酸盐、多聚膦酸聚合物、柠檬酸盐、乙二胺四乙酸盐、六偏磷酸盐、聚丙烯酸盐、聚硅酸盐与聚烯烃磺酸盐中的一种或多种；在本发明中，所述交联剂优选在缓冲液中与双亲嵌段共聚物接触从而使其亲水链交联；所述交联剂在缓冲液中的浓度优选为0.1-100mM，更优选为1-50mM，最优选为5-30mM。The hydrophilic chains of the amphiphilic block copolymer are cross-linked through a cross-linking agent; the hydrophilic segments of the amphiphilic block copolymer have opposite charges to those of the cross-linking agent in the aqueous solution; in the present invention, the The hydrophilic segment of the amphiphilic block copolymer is preferably positively charged in the aqueous solution, so the cross-linking agent is preferably an anionic compound, that is, a compound with a negative charge in the aqueous solution; further preferably, the anionic compound is selected from From one or more of polyphosphate, polyphosphonic acid polymer, citrate, ethylenediaminetetraacetate, hexametaphosphate, polyacrylate, polysilicate and polyolefin sulfonate species; in the present invention, the cross-linking agent is preferably in contact with the amphiphilic block copolymer in the buffer to cross-link its hydrophilic chain; the concentration of the cross-linking agent in the buffer is preferably 0.1-100mM, More preferably, it is 1-50mM, and most preferably, it is 5-30mM.
在本发明中，所述双亲分子膜中优选还可插入孔蛋白，从而使仿生膜承载跨膜蛋白用于生物传感，在本发明中，所述通道蛋白优选通过电压驱动、自主融合或蛋白囊泡融合的方式插入至仿生膜中，进而用于生物传感。In the present invention, porins can preferably be inserted into the amphipathic molecular membrane, so that the biomimetic membrane can carry transmembrane proteins for biosensing. In the present invention, the channel proteins are preferably driven by voltage, autonomous fusion or protein synthesis. The vesicles are inserted into biomimetic membranes through fusion and then used for biosensing.
本发明采用与双亲嵌段共聚物电荷相反的交联剂通过静电吸附产生桥连作用将相邻的亲水链段以离子键的形式形成交联，从而使双亲嵌段共聚物自组装形成双亲分子膜的分子间空隙变小，提高了膜的稳定性，同时增大了膜的电阻和机械强度，使其在用于生物传感时具有更低的漏电流和更长的使用寿命。The present invention uses a cross-linking agent with an opposite charge to the amphiphilic block copolymer to generate a bridging effect through electrostatic adsorption to form cross-links between adjacent hydrophilic segments in the form of ionic bonds, so that the amphiphilic block copolymer self-assembles to form an amphiphilic block copolymer. The intermolecular gaps of the molecular film become smaller, which improves the stability of the film and increases the resistance and mechanical strength of the film, allowing it to have lower leakage current and longer service life when used for biosensing.
本发明还提供了一种上述离子交联的仿生膜的制备方法，包括：将双亲嵌段共聚物溶解于溶剂中，得到膜溶液；将膜溶液在两侧分别设置第一缓冲液与第二缓冲液的条件下进行自组装，得到离子交联的仿生膜；所述第一缓冲液和/或第二缓冲液包括交联剂；所述双亲嵌段共聚物的亲水链段在水溶液中与交联剂的电荷性相反。The invention also provides a method for preparing the above-mentioned ionically cross-linked biomimetic membrane, which includes: dissolving the amphiphilic block copolymer in a solvent to obtain a membrane solution; disposing a first buffer and a second buffer on both sides of the membrane solution. Self-assembly is performed under buffer conditions to obtain an ionically cross-linked biomimetic membrane; the first buffer and/or the second buffer include a cross-linking agent; the hydrophilic segment of the amphiphilic block copolymer is in the aqueous solution Opposite the charge of the cross-linking agent.
其中，本发明对所有原料的来源并没有特殊的限制，为市售即可；所述双亲嵌段共聚物与交联剂的种类及比例均同上所述，在此不再赘述。Among them, the present invention has no special restrictions on the sources of all raw materials, as long as they are commercially available; the types and proportions of the amphiphilic block copolymer and cross-linking agent are the same as mentioned above, and will not be described again here.
将双亲嵌段共聚物溶于溶剂中，得到膜溶液；本发明对所述溶剂的种类并没有特殊的限制，为本领域技术人员熟知的溶剂即可，在本发明中优选为烷烃和/或硅油；所述膜溶液中双亲嵌段共聚物的浓度优选为0.1～50mg/mL，更优选为1～40mg/mL，再优选为1～30 mg/mL。The amphiphilic block copolymer is dissolved in a solvent to obtain a membrane solution; the present invention has no special restrictions on the type of the solvent, as long as it is a solvent well known to those skilled in the art. In the present invention, it is preferably an alkane and/or Silicone oil; the concentration of the amphiphilic block copolymer in the membrane solution is preferably 0.1 to 50 mg/mL, more preferably 1 to 40 mg/mL, and still more preferably 1 to 30 mg/mL.
将膜溶液在两侧分别设置第一缓冲液与第二缓冲液的条件下进行自组装，得到离子交联的仿生膜；在本发明中此自组装过程优选在开孔阵列芯片或膜片钳中进行；以开孔阵列芯片为例，将聚合物溶液涂刷在开孔阵列芯片中，配合第一缓冲液与第二缓冲液即可自组装或在开孔阵列芯片的通道内依次通入第一缓冲液、膜溶液与第二缓冲液即可自组装形成离子交联的仿生膜；所述第一缓冲液和/或第二缓冲液包括交联剂；所述交联剂优选为阴离子化合物，即在水溶液中带有负电荷的化合物；进一步优选的，所述阴离子化合物选自多聚磷酸盐、多聚膦酸聚合物、柠檬酸盐、乙二胺四乙酸盐、六偏磷酸盐、聚丙烯酸盐、聚硅酸盐与聚烯烃磺酸盐中的一种或多种；所述第一缓冲液和/或第二缓冲液中交联剂的浓度优选为0.1～100mmol/L，更优选为0.1～50mmol/L，再优选为1～30mmol/L，最优选为5～30mmol/L；在本发明中，所述第一缓冲液与第二缓冲液可同时包含交联剂也可只有其中的一个包含交联剂，交联剂添加在哪侧则该侧的双亲分子的亲水链段被交联；本发明提供的仿生膜的成膜分子的亲水链段的交联是可逆的，即该仿生膜的交联程度可通过改变膜两侧第一缓冲液和/或第二缓冲液中交联剂的含量来调节。膜分子间的交联反应平衡由膜两侧的第一缓冲液和/或第二缓冲液中交联剂的含量决定，提高第一缓冲液和/或第二缓冲液中交联剂的含量，则膜的交联程度增大，膜越稳定，漏电越小，反之，降低第一缓冲液和/或第二缓冲液中交联剂含量，则膜越不稳定，漏电越大；所述第一缓冲液与第二缓冲液为本领域技术人员熟知的缓冲液即可，并无特殊的限制，本发明中所述第一缓冲液与第二缓冲液各自独立地优选为HEPES缓冲液、Tris缓冲液、PBS缓冲液中的一种；所述HEPES缓冲液的浓度优选具体为10mmol/L；所述PBS缓冲液的浓度具体为10mmol/L；所述第一缓冲液与第二缓冲液中优选还包括稳定剂；所述稳定剂优选具体为EDTA；所述第一缓冲液与第二缓冲液中缓冲剂的浓度各自独立地为1～10mmol/L，更优选各自独立地为2～8mmol/L，再优选各自独立地为4～6mmol/L，最优选为5mmol/L；所述第一缓冲液与第二缓冲液可根据仿生膜的用途不同还可包括其他的物质，如将仿生膜用于生物传感，所述第一缓冲液与第二缓冲液中优选还包括铁氰化钾与亚铁氰化钾；所述第一缓冲液与第二缓冲液中铁氰化钾的浓度优选为10～1000mmol/L，更优选为30～800mmol/L，再优选为40～600mmol/L，最优选为150mmol/L；所述第一缓冲液与第二缓冲液中亚铁氰化钾的浓度优选为10～1000mmol/L，更优选为30～800mmol/L，再优选为40～600mmol/L，最优选为150mmol/L。The membrane solution is self-assembled under the condition that a first buffer and a second buffer are respectively provided on both sides to obtain an ionically cross-linked biomimetic membrane; in the present invention, this self-assembly process is preferably performed on an open-hole array chip or patch clamp. Take the open-hole array chip as an example. The polymer solution is painted on the open-hole array chip, and the first buffer and the second buffer can be self-assembled or sequentially passed through the channels of the open-hole array chip. The first buffer, the membrane solution and the second buffer can self-assemble to form an ionic cross-linked biomimetic membrane; the first buffer and/or the second buffer include a cross-linking agent; the cross-linking agent is preferably anionic. compound, that is, a compound with a negative charge in an aqueous solution; further preferably, the anionic compound is selected from polyphosphate, polyphosphonic acid polymer, citrate, ethylenediaminetetraacetate, hexametaphosphate One or more of salt, polyacrylate, polysilicate and polyolefin sulfonate; the concentration of the cross-linking agent in the first buffer and/or the second buffer is preferably 0.1 to 100 mmol/L , more preferably 0.1~50mmol/L, further preferably 1~30mmol/L, most preferably 5~30mmol/L; in the present invention, the first buffer and the second buffer can simultaneously contain a cross-linking agent Only one of them may contain a cross-linking agent. On which side the cross-linking agent is added, the hydrophilic segments of the amphiphilic molecules on that side will be cross-linked; the cross-linking of the hydrophilic segments of the film-forming molecules of the biomimetic membrane provided by the invention The cross-linking is reversible, that is, the degree of cross-linking of the biomimetic membrane can be adjusted by changing the content of the cross-linking agent in the first buffer and/or the second buffer on both sides of the membrane. The cross-linking reaction balance between membrane molecules is determined by the content of the cross-linking agent in the first buffer and/or the second buffer on both sides of the membrane. Increase the content of the cross-linking agent in the first buffer and/or the second buffer. , then the degree of cross-linking of the membrane increases, the more stable the membrane, the smaller the leakage, conversely, if the content of the cross-linking agent in the first buffer and/or the second buffer is reduced, the more unstable the membrane, the greater the leakage; The first buffer and the second buffer are buffers well known to those skilled in the art and are not particularly limited. The first buffer and the second buffer in the present invention are each independently preferably a HEPES buffer, One of Tris buffer and PBS buffer; the concentration of the HEPES buffer is preferably 10 mmol/L; the concentration of the PBS buffer is specifically 10 mmol/L; the first buffer and the second buffer Preferably, it also includes a stabilizer; the stabilizer is preferably EDTA; the concentrations of the buffers in the first buffer and the second buffer are each independently 1 to 10 mmol/L, and more preferably each is independently 2 to 10 mmol/L. 8mmol/L, more preferably each independently 4-6mmol/L, most preferably 5mmol/L; the first buffer and the second buffer can also include other substances according to the purpose of the bionic membrane, such as The biomimetic membrane is used for biosensing. The first buffer and the second buffer preferably further include potassium ferricyanide and potassium ferrocyanide; the potassium ferricyanide in the first buffer and the second buffer is preferably The concentration is preferably 10-1000mmol/L, more preferably 30-800mmol/L, still more preferably 40-600mmol/L, and most preferably 150mmol/L; ferrocyanation in the first buffer and the second buffer The concentration of potassium is preferably 10 to 1000 mmol/L, more preferably 30 to 800 mmol/L, further preferably 40 to 600 mmol/L, and most preferably 150 mmol/L.
本发明还提供了一种上述离子交联的仿生膜在承载跨膜蛋白中的应用；进而可用于进 行生物传感，如纳米孔基因测序、蛋白质测序及其他成分的检测。The present invention also provides an application of the above-mentioned ionically cross-linked biomimetic membrane in carrying transmembrane proteins; it can then be used for biological sensing, such as nanopore gene sequencing, protein sequencing and detection of other components.
本发明还提供了一种生物传感器，包括上述的离子交联的仿生膜；所述生物传感器优选为纳米孔基因测序仪、蛋白质测序仪。The present invention also provides a biosensor, which includes the above-mentioned ionically cross-linked biomimetic membrane; the biosensor is preferably a nanopore gene sequencer or a protein sequencer.
为了进一步说明本发明，以下结合实施例对本发明提供的一种离子交联的仿生膜、其制备方法及应用进行详细描述。In order to further illustrate the present invention, an ionically cross-linked biomimetic membrane provided by the present invention, its preparation method and application are described in detail below with reference to the examples.
以下实施例中所用的试剂均为市售；以下实施例中的数据主要基于将本发明所述仿生膜用在256通道的纳米孔测序系统上所得。该系统由开孔阵列微结构芯片、控制电路系统及控制软件组成。即仿生膜是在含有256个通道的开孔阵列微结构芯片中形成的。The reagents used in the following examples are all commercially available; the data in the following examples are mainly based on using the biomimetic membrane of the present invention on a 256-channel nanopore sequencing system. The system consists of an opening array microstructure chip, a control circuit system and control software. That is, the biomimetic membrane is formed in an open-hole array microstructure chip containing 256 channels.
实施例1Example 1
1.1将三嵌段共聚物(6PMOXZ-33PDMS-6PMOXZ)溶于硅油AR-20中得到20mg/ml的膜溶液一。1.1 Dissolve the triblock copolymer (6PMOXZ-33PDMS-6PMOXZ) in silicone oil AR-20 to obtain a 20 mg/ml membrane solution.
1.2用超纯水作溶剂按照如下配方配制极性溶液一：150mM铁氰化钾、150mM亚铁氰化钾、5mM EDTA、10mM HEPES、5mM柠檬酸酸钠。1.2 Use ultrapure water as the solvent to prepare polar solution 1 according to the following formula: 150mM potassium ferricyanide, 150mM potassium ferrocyanide, 5mM EDTA, 10mM HEPES, 5mM sodium citrate.
1.3依次往芯片通道内通入极性溶液一、膜溶液一、极性溶液一即在芯片阵列上形成了离子交联的仿生膜。1.3 Pour polar solution 1, membrane solution 1, and polar solution 1 into the chip channel in sequence to form an ionically cross-linked biomimetic membrane on the chip array.
1.4通过测序仪向膜两侧施加0.18V电压，得到跨膜电流如图1所示，跨膜电流集中在20PA以下。1.4 Apply a voltage of 0.18V to both sides of the membrane through the sequencer, and obtain the transmembrane current as shown in Figure 1. The transmembrane current is concentrated below 20PA.
1.5用超纯水作溶剂按照如下配方配制极性溶液二：150mM铁氰化钾、150mM亚铁氰化钾、5mM EDTA、10mM HEPES。1.5 Use ultrapure water as the solvent to prepare polar solution 2 according to the following formula: 150mM potassium ferricyanide, 150mM potassium ferrocyanide, 5mM EDTA, 10mM HEPES.
1.6向测序芯片的流体槽内通入200μl极性溶液二，通过测序仪向膜两侧施加0.18V电压，得到跨膜电流如图2所示，跨膜电流集中在35PA以下。1.6 Pour 200 μl of polar solution II into the fluid tank of the sequencing chip, apply a voltage of 0.18V to both sides of the membrane through the sequencer, and obtain the transmembrane current as shown in Figure 2. The transmembrane current is concentrated below 35PA.
1.7再次向测序芯片的流体槽内通入400μl极性溶液二，通过测序仪向膜两侧施加0.18V电压，得到跨膜电流如图3所示，跨膜电流集中在68PA以下。1.7 Pour 400 μl of polar solution 2 into the fluid tank of the sequencing chip again, apply a voltage of 0.18V to both sides of the membrane through the sequencer, and obtain the transmembrane current as shown in Figure 3. The transmembrane current is concentrated below 68PA.
通过对比步骤1.4、1.6、1.7的跨膜电流大小可以发现，由于极性溶液一中含有柠檬酸酸钠，在仿生膜的两侧均为极性溶液一的情况下，跨膜漏电流最小。而当仿生膜上层被逐步替换为不含柠檬酸酸钠的极性溶液二后，仿生膜上层极性溶液中的柠檬酸钠浓度逐渐降低，跨膜漏电流随之逐渐增大。因此通过本实施例的结果对比可以证明：柠檬酸酸钠对膜溶液一形成的仿生膜具有交联作用，这种交联作用可以减小膜漏电，且这种交联是可逆和可调控的，可以通过调整仿生膜两侧的极性溶液中的交联剂浓度来调整膜的交联程度。By comparing the magnitude of the transmembrane current in steps 1.4, 1.6, and 1.7, it can be found that since polar solution one contains sodium citrate, the transmembrane leakage current is minimal when both sides of the biomimetic membrane are polar solution one. When the upper layer of the biomimetic membrane was gradually replaced with the polar solution 2 without sodium citrate, the concentration of sodium citrate in the polar solution of the upper layer of the biomimetic membrane gradually decreased, and the transmembrane leakage current gradually increased. Therefore, the comparison of the results of this example can prove that sodium citrate has a cross-linking effect on the biomimetic membrane formed by membrane solution 1. This cross-linking effect can reduce membrane leakage, and this cross-linking is reversible and controllable. , the degree of cross-linking of the membrane can be adjusted by adjusting the concentration of the cross-linking agent in the polar solution on both sides of the biomimetic membrane.
实施例2Example 2
2.1将三嵌段共聚物(8PEtOXZ-42PDMS-8PMOXZ)溶于硅油AR-20中得到20mg/ml的膜溶液一。2.1 Dissolve the triblock copolymer (8PEtOXZ-42PDMS-8PMOXZ) in silicone oil AR-20 to obtain a 20 mg/ml membrane solution.
2.2用超纯水作溶剂按照如下配方配制极性溶液二：150mM铁氰化钾、150mM亚铁氰化钾、5mM EDTA、10mM HEPES。2.2 Use ultrapure water as the solvent to prepare polar solution 2 according to the following formula: 150mM potassium ferricyanide, 150mM potassium ferrocyanide, 5mM EDTA, 10mM HEPES.
2.3依次往芯片通道内通入极性溶液一、膜溶液一、极性溶液一即在芯片阵列上形成了未交联的仿生膜。2.3 Pour polar solution 1, membrane solution 1, and polar solution 1 into the chip channel in sequence to form an uncross-linked bionic membrane on the chip array.
2.4通过测序仪向膜两侧施加0.18V电压，得到跨膜电流如图4所示，跨膜电流集中在55PA以下。2.4 Apply a voltage of 0.18V to both sides of the membrane through the sequencer, and obtain the transmembrane current as shown in Figure 4. The transmembrane current is concentrated below 55PA.
2.5用超纯水作溶剂按照如下配方配制极性溶液三：150mM铁氰化钾、150mM亚铁氰化钾、5mM EDTA、10mM HEPES、10mM三聚磷酸钠。2.5 Use ultrapure water as the solvent to prepare polar solution three according to the following formula: 150mM potassium ferricyanide, 150mM potassium ferrocyanide, 5mM EDTA, 10mM HEPES, and 10mM sodium tripolyphosphate.
2.6向测序芯片的流体槽内通入200μl极性溶液三，通过测序仪向膜两侧施加0.18V电压，得到跨膜电流如图5所示，跨膜电流集中在6～27PA范围内。2.6 Pour 200 μl of polar solution III into the fluid tank of the sequencing chip, apply a voltage of 0.18V to both sides of the membrane through the sequencer, and obtain the transmembrane current as shown in Figure 5. The transmembrane current is concentrated in the range of 6 to 27PA.
2.7再次向测序芯片的流体槽内通入400μl极性溶液三，通过测序仪向膜两侧施加0.18V电压，得到跨膜电流如图6所示，跨膜电流集中在0～25PA范围内。2.7 Pour 400 μl of polar solution 3 into the fluid tank of the sequencing chip again, apply a voltage of 0.18V to both sides of the membrane through the sequencer, and obtain the transmembrane current as shown in Figure 6. The transmembrane current is concentrated in the range of 0 to 25PA.
通过对比步骤2.4、2.6、2.7的跨膜电流大小可以发现，由于极性溶液二中不含有柠檬酸酸钠，在仿生膜的两侧均为极性溶液二的情况下，跨膜漏电流最大。而当仿生膜上层被逐步替换为含有三聚磷酸钠的极性溶液三后，膜一侧的极性溶液中的柠檬酸钠浓度逐渐提高，膜分子逐渐被交联，导致跨膜漏电流逐渐减小。因此通过本实施例的结果可以证明：三聚磷酸钠对8PEtOXZ-42PDMS-8PMOXZ形成的仿生膜具有交联作用，这种交联作用可以减小膜漏电，且这种交联是可调控的。By comparing the magnitude of the transmembrane current in steps 2.4, 2.6, and 2.7, it can be found that since polar solution two does not contain sodium citrate, the transmembrane leakage current is the largest when both sides of the biomimetic membrane are polar solution two. . When the upper layer of the biomimetic membrane was gradually replaced with a polar solution containing sodium tripolyphosphate, the concentration of sodium citrate in the polar solution on one side of the membrane gradually increased, and the membrane molecules were gradually cross-linked, causing the transmembrane leakage current to gradually decrease. decrease. Therefore, the results of this example can prove that sodium tripolyphosphate has a cross-linking effect on the biomimetic membrane formed by 8PEtOXZ-42PDMS-8PMOXZ. This cross-linking effect can reduce membrane leakage, and this cross-linking is controllable.
实施例3Example 3
3.1将三嵌段共聚物(6PMOXA-33PDMS-6PMOXA)溶于硅油AR-20中得到20mg/ml的膜溶液一。3.1 Dissolve the triblock copolymer (6PMOXA-33PDMS-6PMOXA) in silicone oil AR-20 to obtain a 20 mg/ml membrane solution.
3.2用超纯水作溶剂按照如下配方配制极性溶液三：150mM铁氰化钾、150mM亚铁氰化钾、5mM EDTA、10mM HEPES、10mM三聚磷酸钠。3.2 Use ultrapure water as the solvent to prepare polar solution three according to the following formula: 150mM potassium ferricyanide, 150mM potassium ferrocyanide, 5mM EDTA, 10mM HEPES, and 10mM sodium tripolyphosphate.
3.3依次往芯片通道内通入极性溶液三、膜溶液一、极性溶液三即在芯片阵列上形成了离子交联的仿生膜。3.3 Pour polar solution three, membrane solution one, and polar solution three into the chip channel in sequence to form an ionically cross-linked biomimetic membrane on the chip array.
3.4用极性溶液三稀释hemolysin蛋白得到200μl的0.01μg/ml的hemolysin蛋白溶液 一并将其注入到测序芯片的流体槽内。3.4 Dilute the hemolysin protein with polar solution 3 to obtain 200 μl of 0.01 μg/ml hemolysin protein solution and inject it into the fluid tank of the sequencing chip.
3.5通过测序仪向膜两侧施加0.3V电压，施加电压30min后停止施加电压并统计嵌孔数和破膜数，数据记录在表1中。3.5 Apply a voltage of 0.3V to both sides of the membrane through the sequencer. After applying the voltage for 30 minutes, stop applying the voltage and count the number of embedded holes and the number of broken membranes. The data are recorded in Table 1.
3.6往测序buffer(480mM KCl，25mM HEPES，5mM ATP，25mM MgCl 2，1mM EDTA，PH 8.0)中加入10mM三聚磷酸钠，再加入文库开始测序，统计测序通道数随测序时间的变化情况，以测序通道数占开始测序时总通道数的比例记录数据，开始时测序通道比例记为100％，数据作图如图7所示。 3.6 Add 10mM sodium tripolyphosphate to the sequencing buffer (480mM KCl, 25mM HEPES, 5mM ATP, 25mM MgCl 2 , 1mM EDTA, pH 8.0), then add the library to start sequencing, and count the changes in the number of sequencing channels with sequencing time to determine Data were recorded as the ratio of the number of sequencing channels to the total number of channels at the beginning of sequencing. The proportion of sequencing channels at the beginning was recorded as 100%. The data plot is shown in Figure 7.
对比例1Comparative example 1
1.1将三嵌段共聚物(6PMOXZ-33PDMS-6PMOXZ)溶于硅油AR-20中得到20mg/ml的膜溶液一。1.1 Dissolve the triblock copolymer (6PMOXZ-33PDMS-6PMOXZ) in silicone oil AR-20 to obtain a 20 mg/ml membrane solution.
1.2用超纯水作溶剂按照如下配方配制极性溶液二：150mM铁氰化钾、150mM亚铁氰化钾、5mM EDTA、10mM HEPES。1.2 Use ultrapure water as the solvent to prepare polar solution 2 according to the following formula: 150mM potassium ferricyanide, 150mM potassium ferrocyanide, 5mM EDTA, 10mM HEPES.
1.3依次往芯片通道内通入极性溶液二、膜溶液一、极性溶液二即在芯片阵列上形成了离子交联的仿生膜。1.3 Pour polar solution 2, membrane solution 1, and polar solution 2 into the chip channel in sequence to form an ionically cross-linked biomimetic membrane on the chip array.
1.4用极性溶液二稀释hemolysin蛋白得到200μl的0.01μg/ml的hemolysin蛋白溶液一并将其注入到测序芯片的流体槽内。1.4 Dilute the hemolysin protein with polar solution 2 to obtain 200 μl of 0.01 μg/ml hemolysin protein solution and inject it into the fluid tank of the sequencing chip.
1.5通过测序仪向膜两侧施加0.3v电压，施加电压30min后停止施加电压并统计嵌孔数和破膜数，数据记录在表1中。1.5 Apply a voltage of 0.3v to both sides of the membrane through the sequencer. After applying the voltage for 30 minutes, stop applying the voltage and count the number of embedded holes and the number of broken membranes. The data are recorded in Table 1.
1.6往测序buffer(480mM KCl，25mM HEPES，5mM ATP,25mM MgCl2，1mM EDTA，PH 8.0)中加入10mM三聚磷酸钠，再加入文库开始测序，统计测序通道数随测序时间的变化情况，以测序通道数占开始测序时总通道数的比例记录数据，开始时测序通道比例记为100％，数据作图如图7所示。1.6 Add 10mM sodium tripolyphosphate to the sequencing buffer (480mM KCl, 25mM HEPES, 5mM ATP, 25mM MgCl2, 1mM EDTA, PH 8.0), then add the library to start sequencing, count the changes in the number of sequencing channels with sequencing time, and use sequencing Data were recorded as the ratio of the number of channels to the total number of channels at the beginning of sequencing. The proportion of sequencing channels at the beginning was recorded as 100%. The data plot is shown in Figure 7.
通过对比表1中的数据，很明显可以发现，与对比例1相比，实施例3的嵌孔数量更多、破膜数量更少。这说明，离子交联的仿生膜具有更强的机械强度，具有更好的稳定性。By comparing the data in Table 1, it is obvious that compared with Comparative Example 1, Example 3 has more embedded holes and less broken membranes. This shows that the ionically cross-linked biomimetic membrane has stronger mechanical strength and better stability.
图7中的数据表明实施例3中的测序通道数比对比例1减少得慢，也就是说实施例3中的测序通道可以工作更长的时间。这说明交联剂通过静电吸附产生桥连作用将相邻的亲水链段以离子键的形式形成交联后，双亲嵌段共聚物自组装形成双亲分子膜的稳定性得到了提高，使其在用于生物传感时具有了更长的使用寿命。The data in Figure 7 shows that the number of sequencing channels in Example 3 decreases more slowly than in Comparative Example 1, which means that the sequencing channels in Example 3 can work for a longer time. This shows that after the cross-linking agent creates a bridging effect through electrostatic adsorption and forms cross-links between adjacent hydrophilic segments in the form of ionic bonds, the stability of the amphiphilic molecular membrane formed by the self-assembly of the amphiphilic block copolymer is improved, making it It has a longer service life when used in biosensing.
表1实施例3和对比例1的嵌孔数和破膜数Table 1 Number of embedded holes and number of film breaks in Example 3 and Comparative Example 1
实验名称Experiment name 嵌孔数Number of embedded holes 破膜数Number of membrane ruptures
实施例3Example 3 160160 24twenty four
对比例1Comparative example 1 6565 113113

Claims (11)

  1. 一种离子交联的仿生膜，其特征在于，所述仿生膜由成膜分子自组装形成；所述成膜分子包括双亲嵌段共聚物；所述双亲嵌段共聚物的亲水链之间通过交联剂交联；所述双亲嵌段共聚物的亲水链段在水溶液中与交联剂的电荷性相反。An ionically cross-linked bionic membrane, characterized in that the bionic membrane is formed by self-assembly of film-forming molecules; the film-forming molecules include an amphiphilic block copolymer; between the hydrophilic chains of the amphiphilic block copolymer Cross-linking by a cross-linking agent; the hydrophilic segment of the amphiphilic block copolymer has opposite charge to that of the cross-linking agent in the aqueous solution.
  2. 根据权利要求1所述的仿生膜，其特征在于，所述双亲嵌段共聚物的亲水链段由亲水性单体形成；所述亲水性单体包括取代和/或未取代的恶唑啉类单体；所述取代的恶唑啉类单体中的取代基选自C1～C10的烷基。The biomimetic membrane according to claim 1, characterized in that the hydrophilic segment of the amphiphilic block copolymer is formed of hydrophilic monomers; the hydrophilic monomers include substituted and/or unsubstituted oxalic acid monomers. Oxazoline monomers; the substituents in the substituted oxazoline monomers are selected from C1 to C10 alkyl groups.
  3. 根据权利要求2所述的仿生膜，其特征在于，所述亲水性单体包括恶唑啉、2-甲基恶唑啉与2-乙基恶唑啉中的一种或多种。The biomimetic membrane according to claim 2, wherein the hydrophilic monomer includes one or more of oxazoline, 2-methyloxazoline and 2-ethyloxazoline.
  4. 根据权利要求1所述的仿生膜，其特征在于，所述双亲嵌段共聚物的疏水链段选自聚硅氧烷、聚烯烃、全氟聚醚、全氟烃基聚醚、聚苯乙烯、聚氧丙烯、聚醋酸乙烯酯、聚氧丁烯、聚异戊二烯、聚丁二烯、聚烷基丙烯酸酯、聚丙烯腈、聚四氢呋喃、聚甲基丙烯酸酯、聚丙烯酸酯、聚砜、聚乙烯醚、聚环氧丙烷、聚己内酯与上述共聚物中的一种或多种。The biomimetic membrane according to claim 1, wherein the hydrophobic segment of the amphiphilic block copolymer is selected from the group consisting of polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, Polyoxypropylene, polyvinyl acetate, polyoxybutylene, polyisoprene, polybutadiene, polyalkyl acrylate, polyacrylonitrile, polytetrahydrofuran, polymethacrylate, polyacrylate, polysulfone , polyvinyl ether, polypropylene oxide, polycaprolactone and one or more of the above copolymers.
  5. 根据权利要求1所述的仿生膜，其特征在于，所述双亲嵌段聚合物的亲水链聚合度为1-20，优选为3-15，最优选为3-10；所述双亲嵌段聚合物的疏水链的聚合度为10-60，优选为20-40，最优选为25-35。The biomimetic membrane according to claim 1, characterized in that the hydrophilic chain polymerization degree of the amphiphilic block polymer is 1-20, preferably 3-15, most preferably 3-10; the amphiphilic block polymer The degree of polymerization of the hydrophobic chains of the polymer is 10-60, preferably 20-40, most preferably 25-35.
  6. 根据权利要求1所述的仿生膜，其特征在于，所述双亲嵌段共聚物的亲水链段与疏水链段的聚合度比为(2～20)：(20～60)，优选为(2～18)：(25～50)，更优选为(2～16)：(25～45)，再优选为(2～16)：(28～42)。The biomimetic membrane according to claim 1, characterized in that the polymerization degree ratio of the hydrophilic segment and the hydrophobic segment of the amphiphilic block copolymer is (2-20): (20-60), preferably ( 2-18): (25-50), more preferably (2-16): (25-45), even more preferably (2-16): (28-42).
  7. 根据权利要求1所述的仿生膜，其特征在于，所述交联剂为阴离子化合物；所述阴离子化合物选自多聚磷酸盐、多聚膦酸聚合物、柠檬酸盐、乙二胺四乙酸盐、六偏磷酸盐、聚丙烯酸盐、聚硅酸盐与聚烯烃磺酸盐中的一种或多种。The biomimetic membrane according to claim 1, characterized in that the cross-linking agent is an anionic compound; the anionic compound is selected from polyphosphate, polyphosphonic acid polymer, citrate, ethylenediaminetetraethyl One or more of acid salt, hexametaphosphate, polyacrylate, polysilicate and polyolefin sulfonate.
  8. 一种离子交联的仿生膜的制备方法，其特征在于，包括：A method for preparing an ionically cross-linked biomimetic membrane, which is characterized by including:
    将双亲嵌段共聚物溶解于溶剂中，得到膜溶液；Dissolve the amphiphilic block copolymer in the solvent to obtain a membrane solution;
    将膜溶液在两侧分别设置第一缓冲液与第二缓冲液的条件下进行自组装，得到离子交联的仿生膜；所述第一缓冲液和/或第二缓冲液含有交联剂；所述双亲嵌段共聚物的亲水链段在水溶液中与交联剂的电荷性相反。The membrane solution is self-assembled under the condition that a first buffer and a second buffer are respectively provided on both sides to obtain an ionically cross-linked biomimetic membrane; the first buffer and/or the second buffer contain a cross-linking agent; The hydrophilic segment of the amphiphilic block copolymer has an opposite charge to that of the cross-linking agent in the aqueous solution.
  9. 根据权利要求8所述的制备方法，其特征在于，所述膜溶液中双亲嵌段共聚物的浓度为0.1～50mg/mL；所述第一缓冲液和/或第二缓冲液中交联剂的浓度为0.1～100mM。The preparation method according to claim 8, characterized in that the concentration of the amphiphilic block copolymer in the membrane solution is 0.1-50 mg/mL; the cross-linking agent in the first buffer and/or the second buffer The concentration is 0.1~100mM.
  10. 根据权利要求8所述的制备方法，其特征在于，所述溶剂选自烷烃和/或硅油；所述第一缓冲液与第二缓冲液各自独立地选自HEPES缓冲液、Tris缓冲液或PBS缓冲液中的一种。The preparation method according to claim 8, wherein the solvent is selected from alkanes and/or silicone oil; the first buffer and the second buffer are each independently selected from HEPES buffer, Tris buffer or PBS. One of the buffer solutions.
  11. 一种生物传感器，其特征在于，包括权利要求1～7任意一项所述的离子交联的仿生膜或权利要求8～10任意一项制备方法所制备的离子交联的仿生膜。A biosensor, characterized by comprising the ionically cross-linked biomimetic membrane described in any one of claims 1 to 7 or the ionically cross-linked biomimetic membrane prepared by the preparation method of any one of claims 8 to 10.
PCT/CN2022/119287 2022-09-16 2022-09-16 Ion cross-linked biomimetic membrane, preparation method therefor, and use thereof WO2024055284A1 (en)

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