WO2020103422A1 - 一种用于捕获生物分子、细胞或细菌的捕获筛 - Google Patents

一种用于捕获生物分子、细胞或细菌的捕获筛

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WO2020103422A1
WO2020103422A1 PCT/CN2019/089945 CN2019089945W WO2020103422A1 WO 2020103422 A1 WO2020103422 A1 WO 2020103422A1 CN 2019089945 W CN2019089945 W CN 2019089945W WO 2020103422 A1 WO2020103422 A1 WO 2020103422A1
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capture screen
polymer layer
capture
layer
raw material
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PCT/CN2019/089945
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English (en)
French (fr)
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颜菁
方欣
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昆山汇先医药技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings

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  • the invention relates to the field of biotechnology, in particular to a capture screen for capturing biomolecules, cells or bacteria.
  • Microfluidic chip technology is to integrate the basic operating processes of biology, chemistry and other laboratories into a single chip. It has the advantages of integration, high throughput, fast detection, convenient operation, small sample size, and low energy consumption. Lai has more and more broad application prospects in many scientific research and life fields such as pharmaceutical research.
  • the methods used for cell separation and capture on the chip involve many fields such as light, electricity, sound, magnetism, hydrodynamics, mechanical processing, and chemical methods.
  • Micromachining technology combined with hydrodynamic control for the capture of whole and single cells and bacterial samples is currently the most effective fixation method. This technology often captures cells by processing geometric traps or obstacles such as microwells, micropores, microdams, microslits, and microchannels that match the size of the cells. It can not only form an open array system, but also be implemented in microchannels. Control of cells.
  • the advantages of microfluidic chips in bioanalysis have become more prominent, showing huge development potential and application value in many fields such as disease diagnosis, drug screening, and cell and molecular biology research.
  • the high-molecular polymers used in the capture sieve are mainly dimethyl siloxane (PDMS) and polyethylene (PC), etc., where PDMS has good biocompatibility, light permeability and easy processing
  • PDMS dimethyl siloxane
  • PC polyethylene
  • the production and other characteristics become one of the longest materials.
  • existing capture screens are prone to non-specific adsorption of biological macromolecules such as proteins, bacteria and cells in biological samples, affecting the bioanalysis efficiency of the chip.
  • the surface energy of the polymer is low, the active group required for the functionalization reaction is lacked, and the trapping efficiency is low.
  • the object of the present invention is to provide a capture screen for capturing biomolecules, cells or bacteria, which has a high capture efficiency.
  • a capture screen for capturing biomolecules, cells or bacteria including a base layer, a protective layer formed on the base layer, and a polymer layer formed on the protective layer, the raw materials of the polymer layer include : Polyvinyl alcohol and / or polyethylene glycol; hyaluronic acid; hydroxyethyl cellulose; agarose and / or dextran.
  • the raw material of the polymer layer further includes a degradation liquid.
  • the polymer layer added with the degradation liquid can be rapidly degraded to achieve "non-destructive" desorption and recultivation of the captured objects.
  • the raw material of the polymer layer further includes at least one of allyl alcohol, sodium alginate, polylactic acid, polyamide, and chitosan.
  • the degradation liquid is selected from one or a combination of hyaluronidase and cellulase.
  • the raw material of the polymer includes polyvinyl alcohol, polyethylene glycol and propylene alcohol with a mass ratio of 3-10: 8-15: 0.1-6: 4-12: 4-18: 0.5-5: 10-20
  • the raw materials of the polymer include polyvinyl alcohol and polyethylene with a mass ratio of 3-10: 8-15: 0.1-6: 4-12: 4-18: 0.5-5: 10-20: 0.1-6
  • a mixture of one or two of diol and propylene alcohol, hyaluronic acid, sodium alginate, hydroxyethyl cellulose, polylactic acid, polyamide, agarose and dextran and chitosan A mixture of species, a degradation solution selected from one or a combination of two selected from hyaluronidase and cellulase.
  • the raw material of the polymer layer is configured as a copolymer solution.
  • the copolymer solution contains 3-10% of a mixture of polyvinyl alcohol and one or two of polyethylene glycol and propylene alcohol.
  • Hyaluronic acid 8-15%, sodium alginate 0.1-6%, hydroxyethyl cellulose 4-12%, polylactic acid 4-18%, polyamide 0.5-5%, agarose and dextran and chitosan
  • One or two of the mixture is 10-20%, and the degradation solution is 0.1-6%.
  • the raw material of the polymer layer further includes a branched polymer, and the branched polymer forms a cross-linked network structure with other raw materials of the polymer layer, which further improves the capture efficiency. And can increase the types of capture, such as antibodies, antigens, aptamers, proteins or combinations thereof.
  • the branched polymer is selected from one of polyacrylamide, acrylamide, polyetheramine, polyamide-amine, polyesteramide, poly (amide-ester), polyphenylene ether, and polyethylene glycol One or more combinations.
  • the raw material of the polymer layer is configured as a copolymer solution.
  • the copolymer solution contains 3-10% of a mixture of polyvinyl alcohol and one or two of polyethylene glycol and propylene alcohol.
  • Hyaluronic acid 8-15%, sodium alginate 0.1-6%, hydroxyethyl cellulose 4-12%, polylactic acid 4-18%, polyamide 0.5-5%, agarose and dextran and chitosan
  • One or two of the mixture is 10-20%
  • the degradation solution is 0.1-6%
  • the branched polymer is 0.1-20%.
  • the capture screen further includes a branched polymer layer formed on the polymer layer.
  • a branched polymer layer formed on the polymer layer.
  • the raw material of the branched polymer layer is selected from polyacrylamide, acrylamide, polyetheramine, polyamide-amine, polyesteramide, poly (amide-ester), polyphenylene ether, polyethylene glycol One or more of the combination.
  • the raw material of the branched polymer layer is configured as a solution.
  • the solution includes polyacrylamide 5-20%, acrylamide 1-16%, polyetheramine 0.1-8%, Polyamide-amine 2-16%, polyester amide 2.5-10%, poly (amide-ester) 3-15%, polyphenylene ether 0.1-3%, polyethylene glycol 2-12%.
  • the material of the base layer is stainless steel; the material of the protective layer is a precious metal or its alloy.
  • the capture sieve provided by the present invention uses polyethylene glycol, polyvinyl alcohol, transparent grease, hyaluronic acid, sodium alginate, and hydroxyethyl cellulose by covering the surface of the protective layer with a degradable polymer layer.
  • Polylactic acid, polyamide, agarose, dextran, chitosan, etc. polymerize to activate the surface of the material, making the polymer material film have good hydrophilicity.
  • the degradation liquid is added to the copolymer, and the "non-destructive" elution or recultivation can be achieved in the stage of eluting the target molecule.
  • the capture efficiency of the target molecule is improved; on the other hand, the addition of degradation liquid to the polymer material improves the elution efficiency of the target capture.
  • the present invention adopts the above scheme, and has the following advantages compared with the prior art:
  • the surface of the protective layer By covering the surface of the protective layer with a polymer coating, the introduction of polyethylene glycol and / or polyvinyl alcohol, hyaluronic acid, hydroxyethyl cellulose, polylactic acid, polyamide, agarose and / or dextran After polymerization, the surface of the material is activated, making the polymer material film have good hydrophilicity and improving the capture efficiency of the target molecule (up to 93%).
  • the capture chip material made of polymer material is low in price and easy to process. It is suitable for industrial mass production, the sample reagent consumption is low, and various biochemical modifications can be performed according to the needs of biochemical analysis to meet the needs of biochemical analysis. The situation can meet the technological needs of the microfluidic chip.
  • FIG. 1 is a schematic cross-sectional view of a capture screen according to the present invention.
  • FIG. 2 is a schematic cross-sectional view of another capture screen according to the present invention.
  • FIG. 1 shows a partial cross-sectional view of the capture screen.
  • the capture screen is composed of a substrate layer 1, a protective layer 2, a polymer layer 3, a branched polymer layer 4, and a functional material layer (not shown in the figure).
  • the capture screen fixes the captured matter on the capture screen through physical action to achieve the purpose of capture.
  • the material of the base material layer 1 is stainless steel, and a plurality of micrometer-sized holes for fluid to pass through are formed on the hole.
  • the pore diameter of the holes is in the micrometer order, and the holes are parallel or parallel to each other.
  • the protective layer 2 is formed on the base material layer 1, for example, covering the outer surface of the base material layer 1, specifically covering the wall of each hole formed in each channel.
  • the protective layer 2 is a metal protective layer, and its material is a precious metal or its alloy, specifically a precious metal or its alloy such as gold, nickel, iron.
  • the polymer layer 3 is formed on the protective layer 2, such as covering the outer surface of the protective layer 2.
  • the raw materials of the polymer layer 3 include: a mixture of one or more of polyvinyl alcohol, polyethylene glycol, and propylene alcohol, hyaluronic acid, sodium alginate, hydroxyethyl cellulose, polylactic acid, polyamide, and agar A combination of one or more of sugar, dextran, chitosan, etc., which is a mixture of one or more of the above materials.
  • the raw material of the polymer layer further includes a degradation liquid, and the degradation liquid is selected from one or a combination of two of hyaluronidase and cellulase.
  • the branched polymer layer 4 is formed on the polymer layer 3 such as covering the outer surface of the polymer layer 3.
  • the raw material of the branched polymer layer 4 is selected from one or more of polyacrylamide, acrylamide, polyetheramine, polyamide, polyesteramide, poly (amide-ester), polyphenylene ether, and polyethylene glycol The combination.
  • the functional material layer is formed on the branched polymer layer 4 and specifically includes capture objects such as antibodies, bacteriophages, etc. coupled to the branched polymer layer 4.
  • the preparation method of the above capture sieve is as follows:
  • Step 1 Preparation of the substrate layer: provide stainless steel, and form a plurality of parallel micron-level fluid channels on the stainless steel to form a sieve-like structure to obtain a stainless steel substrate layer with micron-level fluid channels;
  • Step 2 Preparation of the plating layer (that is, the protective layer): physical vapor deposition (PVD) or chemical vapor deposition (CVD) method is used to plate a metal or metal alloy on the walls of each channel of the base layer, and the The wall is covered with a metal protective layer;
  • PVD physical vapor deposition
  • CVD chemical vapor deposition
  • Step 3 Encapsulation of the biological macromolecular layer (that is, the polymer layer): coupling the raw materials of the polymer layer to the surface of the metal protective layer in the pores by physical adsorption or chemical reaction;
  • Step 4 Preparation of the branched polymer layer: the raw material of the branched polymer layer is coupled to the surface of the polymer layer in the pores by chemical or physical methods.
  • Step 5 Preparation of functional material layer: the capture substance (such as antibody, bacteriophage, etc.) is coupled to the branched substance layer by physical or chemical methods
  • step 3 is as follows:
  • Step 3-1 Mix polyvinyl alcohol and a mixture of polyethylene glycol and propylene alcohol, hyaluronic acid, sodium alginate, hydroxyethyl cellulose, polylactic acid, polyamide, agarose, and dextran A mixture of one or two of chitosan and a degradation solution (hyaluronidase and / or cellulase) are dissolved in a PBS-EDTA solution with a pH of 8 and configured as solution A. The quality of each component The percentages are 3-10%, 8-15%, 0.1-6%, 4-12%, 4-18%, 0.5-5%, 10-20%, 0.1-6%.
  • Step 3-2 Prepare a 0.8 mM DTT (dithiothreitol) or TECP (tris (2-carboxyethyl) phosphine) solution B using a PBS-EDTA solution with a pH of 8.
  • Step 3-3 Mix solution A and solution B at a volume ratio of 1: 1 to obtain solution C.
  • Step 3-4 Place the cleaned capture sieve prepared in Step 2 in solution C and incubate on a shaker for 12 hours;
  • Step 3-5 The capture sieve is washed with a PBS-EDTA solution to obtain a capture sieve wrapped in a layer of biological macromolecules.
  • step 4 is as follows:
  • Step 4-1 Preparation of branched polymer: dissolve a mixture of polyacrylamide, acrylamide, polyetheramine, polyamide-amine, polyesteramide, poly (amide-ester), polyphenylene ether, and polyethylene glycol In a PBS-EDTA buffer solution with a pH of 8, it is configured as solution D, and the mass percentage of each component is 5-20%, 1-16%, 0.1-8%, 2-16%, 2.5-10%, 3 -15%, 0.1-3%, 2-12%.
  • Step 4-2 A 0.8 mM DTT (dithiothreitol) or TECP (tris (2-carboxyethyl) phosphine) solution E is prepared using a PBS-EDTA solution with a pH of 8.
  • Step 4-3 Using solution E, prepare a 1 mM biotin PEG solution F. Mix solutions D and F according to 1: 1 to obtain solution G. Solution G is incubated at room temperature for 12 hours.
  • Step 4-4 Add streptavidin to the solution G and incubate at room temperature for 1 hour to obtain the solution H;
  • Step 4-5 Place the semi-finished capture sieve prepared in step 3-5 in solution H and incubate in a shaker for 3 hours;
  • Step 4-6 The capture sieve is washed with PBS-EDTA solution to obtain the capture sieve wrapped by the branched polymer layer.
  • FIG. 2 shows a partial cross-sectional view of the capture screen.
  • the difference between the capture screen and the capture screen of Example 1 is that the capture screen is composed of a substrate layer 1, a protective layer 2, a polymer layer 3 ', and a functional material layer (not shown in the figure)
  • the branched polymer is added to the raw material of the polymer layer 3 '.
  • the base layer 1 and the protective layer 2 are the same as those in Example 1.
  • the raw material of the polymer layer 3 'further includes a degradation liquid selected from one or a combination of hyaluronidase and cellulase.
  • the raw material of the polymer layer 3 'further includes a branched polymer selected from polyacrylamide, acrylamide, polyetheramine, polyamide, polyesteramide, poly (amide-ester), polyphenylene ether, A combination of one or more of polyethylene glycol.
  • the functional material layer is formed on the polymer layer 3 ', and specifically includes capture objects such as antibodies, phages, etc. coupled to the polymer layer 3'.
  • the preparation method of the above capture sieve is as follows:
  • Step 1 Preparation of the substrate layer: provide stainless steel, and form a plurality of parallel micron-level fluid channels on the stainless steel to form a sieve-like structure to obtain a stainless steel substrate layer with micron-level fluid channels;
  • Step 2 Preparation of the plating layer (that is, the protective layer): physical vapor deposition (PVD) or chemical vapor deposition (CVD) method is used to plate a metal or metal alloy on the walls of each channel of the base layer, and the The wall is covered with a metal protective layer;
  • PVD physical vapor deposition
  • CVD chemical vapor deposition
  • Step 3 Preparation of the biomacromolecule layer and the branched polymer layer (that is, the polymer layer): the raw materials of the polymer layer are coupled to the surface of the metal protective layer in the pores through physical adsorption or chemical reaction.
  • Step 4 Preparation of functional material layer: the capture substance (such as antibody, bacteriophage, etc.) is coupled to the branched substance layer by physical or chemical methods
  • step 3 is as follows:
  • Step 3-1 Mix polyvinyl alcohol and one or two of polyethylene glycol and propylene alcohol, hyaluronic acid, sodium alginate, hydroxyethyl cellulose, polylactic acid, polyamide, agarose and dextran Mixture of one or two of sugar and chitosan, degradation solution (hyaluronidase and / or cellulase), branched polymer (polyacrylamide, acrylamide, polyetheramine) at pH 8
  • the PBS-EDTA solution is stirred and dissolved, and is configured as copolymer solution A.
  • the mass percentage of each component is 3-10%, 8-15%, 0.1-6%, 4-12%, 4-18%, 0.5- 5%, 10-20%, 0.1-6%, 0.1-20%;
  • Step 3-2 Prepare a 0.8 mM DTT (dithiothreitol) or TECP (tris (2-carboxyethyl) phosphine) solution B using a PBS-EDTA solution with a pH of 8;
  • Step 3-3 Using solution B, prepare a 1 mM biotin PEG solution C. Mix solution A and solution C at 1: 1 to obtain solution D. Solution D is incubated at room temperature for 12 hours;
  • Step 3-4 Add streptavidin to solution D and incubate at room temperature for 1 hour to obtain solution E;
  • Step 3-5 Place the cleaned semi-finished capture sieve prepared in Step 2 in solution E and incubate for 3 hours in a shaker;
  • Steps 3-6 The capture sieve is washed with a PBS-EDTA solution to obtain a biological macromolecular layer and a branched wrapped capture sieve.
  • the capture screen provided by the present invention is coated with a degradable polymer coating on the surface of the metal protective layer, using polyethylene glycol, polyvinyl alcohol, transparent grease, hyaluronic acid, sodium alginate and hydroxyethyl fiber
  • Polysaccharide, polylactic acid, polyamide, agarose, dextran or chitosan etc. polymerize to activate the surface of the material and make the polymer material film have good hydrophilicity.
  • the degradation liquid is added to the copolymer, and the "non-destructive" elution or recultivation can be achieved in the stage of eluting the target molecule.
  • the capture efficiency of the target molecule is improved; on the other hand, the addition of degradation liquid to the polymer material improves the elution efficiency of the target capture.
  • the surface of the polymer layer is covered with a branched polymer coating, or a branched polymer is added to the polymer layer, and the functional groups on the surface of the branched polymer increase with the relative molecular mass and molecular size, which improves the nanoparticles.
  • the dispersibility in the polymer increases the interfacial binding force between the nanoparticle and the polymer, reduces the surface energy of the particle, eliminates the surface charge of the particle, improves the affinity of the particle and the organic phase, weakens the surface polarity of the particle, and introduces support After polymerizing, it shows better toughness than modified organosilicate.
  • the core or shell of the "core-shell" structure of the branched polymer acts as a site for small molecule reactions to improve the performance of the capture screen.
  • branched polymers including polyacrylamide, acrylamide, polyetheramine and other materials
  • the non-specific adsorption and toxicity of the material are reduced, and the hydrophilicity and modification of the material are improved.
  • the material and the liquid are easily separated without centrifugation The operation can efficiently capture cells, bacteria and target biomolecules, and there is no obvious damage to the cell activity after capture.
  • the capture sieve of the present invention has the following characteristics: (1) The capture sieve of the present invention is to covalently connect target molecules (small biological molecules, DNA, RNA, antibody protein, etc.) to the polymer capture sieve through photocrosslinking. Compared with the conventional capture sieve, its anti-non-specific ability on the carrier has been greatly improved, and the success rate of drug target capture has been greatly improved, reaching 93%. (2) The high-molecular copolymers and branched polymers used in the present invention can increase the types of capture objects, such as antibodies, antigens, aptamers, proteins, or combinations thereof. (3) The non-specific adsorption is small during the entire hybridization process, and there is no obvious cross-interference.
  • the capture chip material made of polymer material is low in price and easy to process. It is suitable for industrial mass production, the sample reagent consumption is low, and various biochemical modifications can be performed according to the needs of biochemical analysis to meet the needs of biochemical analysis and cost. Low-volume manufacturing, the composition ratio of polymer materials is not a single material, which can meet the process requirements of microfluidic chips according to the ratio.
  • the polymer layer added with the degradation liquid used in the capture sieve of the present invention can be rapidly degraded, to achieve "non-destructive" desorption and recultivation of the captured object, and the elution efficiency is over 85%.

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Abstract

一种用于捕获生物分子、细胞或细菌的捕获筛，该捕获筛具有较高的捕获效率。一种用于捕获生物分子、细胞或细菌的捕获筛，包括基体层、形成于所述基体层上的保护层及形成于所述保护层上的高分子层，包括基体层、形成于所述基体层上的保护层及形成于所述保护层上的高分子层，所述高分子层的原料包括：聚乙烯醇和／或聚乙二醇；透明质酸；羟乙基纤维素；琼脂糖和／或葡聚糖。

Description

一种用于捕获生物分子、细胞或细菌的捕获筛
相关申请的交叉引用
本申请要求2018年11月19日提交的申请号为CN201811375355.3的中国专利申请的优先权，其全部内容通过引用的方式并入本发明中。
技术领域
本发明涉及生物技术领域，特别涉及一种用于捕获生物分子、细胞或细菌的捕获筛。
背景技术
微流控芯片技术是把生物学、化学等实验室的基本操作过程集成到一块芯片上，具有集成性、高通量、检测快速、操作便利、所需样本量少、低耗能优点，近年来在药物研究等众多科研与生活领域拥有越来越多的广阔应用前景。用于芯片上细胞分离和捕获的手段涉及光、电、声、磁、流体力学、机械加工以及化学方法等众多领域。微机械加工技术结合流体力学控制用于整体及单个细胞、细菌样本的捕获是目前最有效的固定方式。这种技术往往通过加工尺寸与细胞相匹配的微井、微孔、微坝、微狭缝及微管道等几何陷阱或障碍来捕获细胞，不仅可形成开放阵列体系，还可以在微通道中实现对细胞的控制。
随着微加工技术的不断进步，微流控芯片在生物分析中的优势愈显突出，在疾病诊断、药物筛选和细胞分子生物学研究等多个领域里显示出巨大的发展潜力和应用价值。在生物分析领域，捕获筛所采用的高分子聚合物主要有二甲基硅氧烷(PDMS)和聚乙烯(PC)等，其中PDMS因具有良好的生物相容性、光透性和易加工制作等特点，成为最长用的材料之一。然而，现有的捕获筛对生物样本中的蛋白质等生物大分子、细菌和细胞等物质易发生非特异性吸附，影响芯片的生物分析效能。且聚合物表面能量低，缺乏功能化反应所需要的活性基团，捕获效率较低。
发明内容
本发明的目的是提供一种用于捕获生物分子、细胞或细菌的捕获筛，该捕获筛具有较高的捕获效率。
为达到上述目的，本发明采用的技术方案为：
一种用于捕获生物分子、细胞或细菌的捕获筛，包括基体层、形成于所述基体层上的保护层及形成于所述保护层上的高分子层，所述高分子层的原料包括：聚乙烯醇和/或聚乙二醇；透明质酸；羟乙基纤维素；琼脂糖和/或葡聚糖。
进一步地，所述高分子层的原料还包括降解液。添加有降解液的高分子层能够进行快速降解，实现捕获物的“无损”脱附及再培养。
进一步地，所述高分子层的原料还包括丙烯醇、海藻酸钠、聚乳酸、聚酰胺、壳聚糖中的至少一种。
更进一步地，所述降解液选自透明质酸酶、纤维素酶中的一种或两种的组合。
优选地，所述高分子的原料包括质量比例为3-10:8-15:0.1-6:4-12:4-18:0.5-5:10-20的聚乙烯醇和聚乙二醇和丙烯醇中的一种或两种的混合物、透明质酸、海藻酸钠、羟乙基纤维素、聚乳酸、聚酰胺、琼脂糖和葡聚糖和壳聚糖中的一种或两种的混合物。
优选地，所述高分子的原料包括质量比例为3-10:8-15:0.1-6:4-12:4-18:0.5-5:10-20:0.1-6的聚乙烯醇和聚乙二醇和丙烯醇中的一种或两种的混合物、透明质酸、海藻酸钠、羟乙基纤维素、聚乳酸、聚酰胺、琼脂糖和葡聚糖和壳聚糖中的一种或两种的混合物、降解液，所述降解液选自透明质酸酶、纤维素酶中的一种或两种的组合。具体地，所述高分子层的原料被配置为共聚物溶液，按质量百分比计，所述共聚物溶液包含聚乙烯醇和聚乙二醇和丙烯醇中一种或两种的混合物3-10％、透明质酸8-15％、海藻酸钠0.1-6％、羟乙基纤维素4-12％、聚乳酸4-18％、聚酰胺0.5-5％、琼脂糖和葡聚糖和壳聚糖中的一种或两种的混合物10-20％、降解液0.1-6％。
进一步地，所述高分子层的原料还包括支化聚合物，支化聚合物与高分子层的其他原料形成交联网络结构，进一步提高了捕获效率。且能够增加捕获物的种类，如抗体、抗原、适配体、蛋白质或其组合。
更进一步地，所述支化聚合物选自聚丙烯酰胺、丙烯酰胺、聚醚胺、聚酰胺-胺、聚酯酰胺、聚(酰胺-酯)、聚苯醚、聚乙二醇中的一种或多种的组合。
具体地，所述高分子层的原料被配置为共聚物溶液，按质量百分比计，所述共聚物溶液包含聚乙烯醇和聚乙二醇和丙烯醇中一种或两种的混合物3-10％、透明质酸8-15％、海藻酸钠0.1-6％、羟乙基纤维素4-12％、聚乳酸4-18％、聚酰胺0.5-5％、琼脂糖和葡聚糖和壳聚糖中 的一种或两种的混合物10-20％、降解液0.1-6％、支化聚合物0.1-20％。
进一步地，所述捕获筛还包括形成于所述高分子层上的支化聚合物层。能够增加捕获物的种类，如抗体、抗原、适配体、蛋白质或其组合。
更进一步地，所述支化聚合物层的原料选自聚丙烯酰胺、丙烯酰胺、聚醚胺、聚酰胺-胺、聚酯酰胺、聚(酰胺-酯)、聚苯醚、聚乙二醇中的一种或多种的组合。
具体地，所述支化聚合物层的原料被配置成溶液，按质量百分比计，所述溶液包含将聚丙烯酰胺5-20％、丙烯酰胺1-16％、聚醚胺0.1-8％、聚酰胺-胺2-16％、聚酯酰胺2.5-10％、聚(酰胺-酯)3-15％、聚苯醚0.1-3％、聚乙二醇2-12％。
进一步地，所述基体层的材料为不锈钢；所述保护层的材料为贵金属或其合金。
本发明提供的捕获筛，通过在保护层表面覆着上可降解高分子聚合物层，采用聚乙二醇、聚乙烯醇、透明脂、透明质酸、海藻酸钠、羟乙基纤维素、聚乳酸、聚酰胺、琼脂糖或葡聚糖或壳聚糖等进行聚合，活化材料表面，使得高分子材料膜具有很好的亲水性。本发明在共聚物中加入降解液，在洗脱目标分子阶段能够实现“无损”洗脱或再培养。一方面提高了目标分子的捕获效率，另一方面在高分子材料中添加降解液，提高了目标捕获物的洗脱效率。
本发明采用以上方案，相比现有技术具有如下优点：
通过在保护层表面覆着上高分子聚合物涂层，引入聚乙二醇和/或聚乙烯醇、透明质酸、羟乙基纤维素、聚乳酸、聚酰胺、琼脂糖和/或葡聚糖等进行聚合，活化材料表面，使得高分子材料膜具有很好的亲水性提高了目标分子的捕获效率(可达93％以上)。高分子材料制成的捕获芯片材料价格低、易加工，适于工业大量生产，样品试剂消耗少，可以根据生化分析的需要进行各种生物化学改性，满足生化分析的需要，能够根据配比情况能够满足微流控芯片的工艺需求。
附图说明
为了更清楚地说明本发明的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是根据本发明的一种捕获筛的截面示意图；
图2是根据本发明的另一种捕获筛的截面示意图。
具体实施方式
下面结合附图对本发明的较佳实施例进行详细阐述，以使本发明的优点和特征能更易于被本领域的技术人员理解。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以互相结合。
实施例1
本实施例提供一种捕获筛，其能够用于微流控芯片中捕获生物样本中的分子(尤其是生物大分子)、细胞和细菌等物质。图1示出了该捕获筛的局部截面图。参照图1所示，所述捕获筛由基材层1、保护层2、高分子层3、支化聚合物层4及功能材料层(图中未示出)组成。当包含生物分子、细菌或细胞的流体流过捕获筛时，捕获筛将捕获物通过物理作用固定在捕获筛上，达到捕获的目的。
基材层1的材料为不锈钢，其上形成有多个微米级的供流体通过的孔道，孔道的孔径为微米级，各孔道相互平行或并列。
保护层2形成于基材层1上，如覆盖在基材层1的外表面上，具体为覆盖形成于各孔道的孔壁上。保护层2为金属保护层，其材料为贵金属或其合金，具体为金、镍、铁等贵金属或其合金。
高分子层3形成于保护层2上，如覆盖在保护层2的外表面上。高分子层3的原料包括：聚乙烯醇、聚乙二醇、丙烯醇中的一种或多种的混合物，透明质酸，海藻酸钠，羟乙基纤维素，聚乳酸，聚酰胺，琼脂糖、葡聚糖、壳聚糖中的一种或多种的组合等，其由上述材料的一种或多种的混合物。该高分子层的原料还包括降解液，降解液选自透明质酸酶、纤维素酶中的一种或两种的组合。
支化聚合物层4形成于高分子层3上，如覆盖在高分子层3的外表面上。支化聚合物层4的原料选自聚丙烯酰胺、丙烯酰胺、聚醚胺、聚酰胺、聚酯酰胺、聚(酰胺-酯)、聚苯醚、聚乙二醇中的一种或多种的组合。
功能材料层形成于支化聚合物层4上，具体包括耦合至支化聚合物层4上的捕获物，如抗体、噬菌体等。
上述捕获筛的制备方法如下：
步骤1、基体层制备：提供不锈钢，在不锈钢上形成多个平行的微米级流体孔道，形成筛状结构，得到具有微米级流体孔道的不锈钢基体层；
步骤2、镀层(即所述保护层)制备：采用物理气相沉积(PVD)或化学气相沉积(CVD)方法在基体层的各孔道的壁上镀金属或金属合金，在基体层的各孔道的壁上覆盖形成金属保护层；
步骤3、生物大分子层(即所述高分子层)包裹：将上述高分子层的原料通过采用物理吸附或化学反应方法耦合到孔道中金属保护层的表面；
步骤4、支化聚合物层制备：将上述支化聚合物层的原料通过化学或物理方法耦合到孔道中高分子层的表面。
步骤5、功能材料层制备：将捕获物(如抗体、噬菌体等)通过物理或化学方法耦合到支化物层
上述步骤3具体如下：
步骤3-1、将聚乙烯醇和聚乙二醇和丙烯醇中一种或两种的混合物、透明质酸、海藻酸钠、羟乙基纤维素、聚乳酸、聚酰胺、琼脂糖和葡聚糖和壳聚糖中的一种或两种的混合物、降解液(透明质酸酶和/或纤维素酶)溶解于pH为8的PBS-EDTA溶液中，配置成溶液A，各组分的质量百分比分别为3-10％、8-15％、0.1-6％、4-12％、4-18％、0.5-5％、10-20％、0.1-6％。
步骤3-2、采用pH为8的PBS-EDTA溶液配制0.8mM的DTT(二硫苏糖醇)或者TECP(三(2-羧乙基)膦)溶液B。
步骤3-3、将溶液A和溶液B按照体积比1:1混合得到溶液C。
步骤3-4、将清洁后的由步骤2制得的捕获筛放置于溶液C中，在摇床上孵育12h；
步骤3-5、采用PBS-EDTA溶液清洗捕获筛，获得生物大分子层包裹的捕获筛。
上述步骤4具体如下：
步骤4-1、支化聚合物制备：将聚丙烯酰胺、丙烯酰胺、聚醚胺、聚酰胺-胺、聚酯酰胺、聚(酰胺-酯)、聚苯醚、聚乙二醇的混合物溶解于pH为8的PBS-EDTA缓冲溶液中，配置成溶液D，各组分的质量百分比为5-20％、1-16％、0.1-8％、2-16％、2.5-10％、3-15％、0.1-3％、2-12％。
步骤4-2、采用pH为8的PBS-EDTA溶液配制0.8mM的DTT(二硫苏糖醇)或者TECP(三(2-羧乙基)膦)溶液E。
步骤4-3、采用溶液E配制1mM的生物素PEG溶液F，将溶液D和F按照1:1混合得到溶液G，溶液G室温孵育12h。
步骤4-4、向溶液G中加入链霉亲和素，室温孵育1h，得到溶液H；
步骤4-5、将由步骤3-5制得的捕获筛半成品放置于溶液H中，摇床中孵育3h；
步骤4-6、采用PBS-EDTA溶液清洗捕获筛，获得支化聚合物层包裹的捕获筛。
实施例2
本实施例提供一种捕获筛，其能够用于微流控芯片中捕获生物样本中的分子(尤其是生物大分子)、细胞和细菌等物质。图2示出了该捕获筛的局部截面图。参照图2所示，该捕获筛与实施例1的捕获筛的区别在于：该捕获筛由基材层1、保护层2、高分子层3’及功能材料层(图中未示出)组成，支化聚合物被加入在高分子层3’的原料中。其中，基材层1和保护层2同实施例1。
高分子层3’的原料包括：聚乙烯醇、聚乙二醇、丙烯醇中的一种或多种的组合，透明质酸，海藻酸钠，羟乙基纤维素，聚乳酸，聚酰胺，琼脂糖、葡聚糖、壳聚糖中的一种或多种的组合等，其由上述材料的一种或多种的组合形成。该高分子层3’的原料还包括降解液，降解液选自透明质酸酶、纤维素酶中的一种或两种的组合。该高分子层3’的原料还包括支化聚合物，支化聚合物选自聚丙烯酰胺、丙烯酰胺、聚醚胺、聚酰胺、聚酯酰胺、聚(酰胺-酯)、聚苯醚、聚乙二醇中的一种或多种的组合。
功能材料层形成于高分子层3’上，具体包括耦合至高分子层3’上的捕获物，如抗体、噬菌体等。
上述捕获筛的制备方法如下：
步骤1、基体层制备：提供不锈钢，在不锈钢上形成多个平行的微米级流体孔道，形成筛状结构，得到具有微米级流体孔道的不锈钢基体层；
步骤2、镀层(即所述保护层)制备：采用物理气相沉积(PVD)或化学气相沉积(CVD)方法在基体层的各孔道的壁上镀金属或金属合金，在基体层的各孔道的壁上覆盖形成金属保护层；
步骤3、生物大分子层及支化聚合物层(即所述高分子层)制备：将上述高分子层的原料通过物理吸附或化学反应耦合到孔道中金属保护层的表面。
步骤4、功能材料层制备：将捕获物(如抗体、噬菌体等)通过物理或化学方法耦合到支化物层
上述步骤3具体如下：
步骤3-1、将聚乙烯醇和聚乙二醇和丙烯醇中的一种或两种的混合物、透明质酸、海藻酸钠、羟乙基纤维素、聚乳酸、聚酰胺、琼脂糖和葡聚糖和壳聚糖中的一种或两种的混合物、降解液(透明质酸酶和/或纤维素酶)、支化聚合物(聚丙烯酰胺、丙烯酰胺、聚醚胺)在pH为8的PBS-EDTA溶液中搅拌溶解，配置成共聚物溶液A，各组分的质量百分比为3-10％、8-15％、0.1-6％、4-12％、4-18％、0.5-5％、10-20％、0.1-6％、0.1-20％；
步骤3-2、采用pH为8的PBS-EDTA溶液配制0.8mM的DTT(二硫苏糖醇)或者TECP(三(2-羧乙基)膦)溶液B；
步骤3-3、采用溶液B配制1mM的生物素PEG溶液C，将溶液A和溶液C按照1:1混合得到溶液D，溶液D室温孵育12h；
步骤3-4、向溶液D中加入链霉亲和素，室温孵育1h，得到溶液E；
步骤3-5、将清洗后的由步骤2制得的捕获筛半成品放置于溶液E中，摇床孵育3h；
步骤3-6、采用PBS-EDTA溶液清洗捕获筛，获得生物大分子层及支化包裹的捕获筛。
本发明提供的捕获筛，通过在金属保护层表面覆着上可降解高分子聚合物涂层，采用聚乙二醇、聚乙烯醇、透明脂、透明质酸、海藻酸钠、羟乙基纤维素、聚乳酸、聚酰胺、琼脂糖或葡聚糖或壳聚糖等进行聚合，活化材料表面，使得高分子材料膜具有很好的亲水性。本发明在共聚物中加入降解液，在洗脱目标分子阶段能够实现“无损”洗脱或再培养。一方面提高了目标分子的捕获效率，另一方面在高分子材料中添加降解液，提高了目标捕获物的洗脱效率。
此外，本发明在高分子层的表面覆着上支化聚合物涂层，或在高分子层中添加支化聚合物，支化聚合物表面官能团随相对分子质量和分子尺寸递增，提高纳米粒子在聚合物中的分散能力，增加纳米粒子与聚合物的界面结合力，降低了粒子的表面能，消除粒子的表面电荷，提高粒子与有机相的亲和力，减弱粒子的表面极性，并且引入支化聚合物后显示出具有比改性的有机硅酸盐更好的韧性。支化聚合物的“核壳”结构的内核或外壳作为小分子反应的场所，以此来改善捕获筛的性能。通过引入支化聚合物包括聚丙烯酰胺、丙烯酰胺、聚醚胺等 材料，降低了材料的非特异性吸附和毒性，提高了材料的亲水性和修饰性，同时材料和液体易于分离，无需离心操作，可高效捕获细胞、细菌及目标生物分子，并且捕获后的细胞活性无明显损伤。
本发明的捕获筛具有如下特点：(1)本发明的捕获筛，是将目标分子(生物小分子，DNA，RNA，抗体蛋白等)通过光交联共价连接在在高分子聚合物捕获筛载体上，与常规的捕获筛相比，其抗非特异性能力有大幅提高，大大提高了药物靶标的捕获成功率，达到93％。(2)本发明采用的高分子共聚物和支化聚合物能够增加捕获物的种类，如抗体、抗原、适配体、蛋白质或其组合。(3)整个杂交过程非特异性吸附小，无明显的相互交叉干扰。(4)高分子材料制成的捕获芯片材料价格低、易加工，适于工业大量生产，样品试剂消耗少，可以根据生化分析的需要进行各种生物化学改性，满足生化分析的需要，成本低、批量化制造，高分子材料的组成配比非单一材料，能够根据配比情况能够满足微流控芯片的工艺需求。(5)本发明捕获筛采用的添加有降解液的高分子层能够进行快速降解，实现捕获物的“无损”脱附及再培养，洗脱效率达85％以上。
上述实施例只为说明本发明的技术构思及特点，是一种优选的实施例，其目的在于熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限定本发明的保护范围。凡根据本发明的原理所作的等效变换或修饰，都应涵盖在本发明的保护范围之内。

Claims (13)

  1. 一种用于捕获生物分子、细胞或细菌的捕获筛，包括基体层、形成于所述基体层上的保护层及形成于所述保护层上的高分子层，其特征在于：所述高分子层的原料包括：聚乙烯醇和/或聚乙二醇；透明质酸；羟乙基纤维素；琼脂糖和/或葡聚糖。
  2. 根据权利要求1所述的捕获筛，其特征在于：所述高分子层的原料还包括丙烯醇、海藻酸钠、聚乳酸、聚酰胺、壳聚糖中的至少一种。
  3. 根据权利要求1所述的捕获筛，其特征在于：所述高分子层的原料还包括降解液。
  4. 根据权利要求3所述的捕获筛，其特征在于：所述降解液选自透明质酸酶、纤维素酶中的一种或两种的组合。
  5. 根据权利要求1所述的捕获筛，其特征在于：所述高分子层的原料被配置为共聚物溶液，按质量百分比计，所述共聚物溶液包含聚乙烯醇和聚乙二醇和丙烯醇中一种或两种的混合物3-10％、透明质酸8-15％、海藻酸钠0.1-6％、羟乙基纤维素4-12％、聚乳酸4-18％、聚酰胺0.5-5％、琼脂糖和葡聚糖和壳聚糖中的一种或两种的混合物10-20％、降解液0.1-6％。
  6. 根据权利要求1所述的捕获筛，其特征在于：所述高分子层的原料还包括支化聚合物。
  7. 根据权利要求6所述的捕获筛，其特征在于：所述支化聚合物选自聚丙烯酰胺、丙烯酰胺、聚醚胺、聚酰胺-胺、聚酯酰胺、聚(酰胺-酯)、聚苯醚、聚乙二醇中的一种或多种的组合。
  8. 根据权利要求6所述的捕获筛，其特征在于：按质量百分比计，所述高分子层的原料被配置为共聚物溶液，按质量百分比计，所述共聚物溶液包含聚乙烯醇和聚乙二醇和丙烯醇中一种或两种的混合物3-10％、透明质酸8-15％、海藻酸钠0.1-6％、羟乙基纤维素4-12％、聚乳酸4-18％、聚酰胺0.5-5％、琼脂糖和葡聚糖和壳聚糖中的一种或两种的混合物10-20％、降解液0.1-6％、支化聚合物0.1-20％。
  9. 根据权利要求1所述的捕获筛，其特征在于：所述捕获筛还包括形成于所述高分子层上的支化聚合物层。
  10. 根据权利要求9所述的捕获筛，其特征在于：所述支化聚合物层的原料选自聚丙烯酰胺、丙烯酰胺、聚醚胺、聚酰胺-胺、聚酯酰胺、聚(酰胺-酯)、聚苯醚、聚乙二醇中的一种或多种的组合。
  11. 根据权利要求9所述的捕获筛，其特征在于：所述支化聚合物层的原料被配置成溶液，按质量百分比计，所述溶液包含将聚丙烯酰胺5-20％、丙烯酰胺1-16％、聚醚胺0.1-8％、聚酰胺-胺2-16％、聚酯酰胺2.5-10％、聚(酰胺-酯)3-15％、聚苯醚0.1-3％、聚乙二醇2-12％。
  12. 根据权利要求1-11任一项所述的捕获筛，其特征在于：所述高分子的原料包括聚乙烯醇和聚乙二醇和丙烯醇中的一种或两种的混合物、透明质酸、海藻酸钠、羟乙基纤维素、聚乳酸、聚酰胺、琼脂糖和葡聚糖和壳聚糖中的一种或两种的混合物、降解液，所述降解液选自透明质酸酶、纤维素酶中的一种或两种的组合。
  13. 根据权利要求1所述的捕获筛，其特征在于：所述基体层的材料为不锈钢；所述保护层的材料为贵金属或其合金。
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