WO2022236863A1 - Microsphere having functionalized spot on surface, and preparation method and application of microsphere - Google Patents

Microsphere having functionalized spot on surface, and preparation method and application of microsphere Download PDF

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WO2022236863A1
WO2022236863A1 PCT/CN2021/095043 CN2021095043W WO2022236863A1 WO 2022236863 A1 WO2022236863 A1 WO 2022236863A1 CN 2021095043 W CN2021095043 W CN 2021095043W WO 2022236863 A1 WO2022236863 A1 WO 2022236863A1
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microsphere
functionalized
functionalized spots
spots
surface according
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French (fr)
Chinese (zh)
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徐方成
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厦门依加成科技有限公司
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    • B01J20/28016—Particle form
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    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • B01J20/28078—Pore diameter
    • B01J20/28085—Pore diameter being more than 50 nm, i.e. macropores
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    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
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    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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Definitions

  • the invention relates to the technical field of biomaterials, in particular to a microsphere with functionalized spots on the surface and a preparation method and application thereof.
  • Cells, bacteria, and viruses are the three major categories of materials in the field of biology. They all have the properties of particles. For example, viruses are usually 15-200nm, bacteria are 0.2-8 ⁇ m, and human mammalian cells are usually 5-30 ⁇ m.
  • Existing biological research methods whether it is physiological and biochemical research, or gene and genome research, usually use many of the same cells. For example, in conventional bacterial genome sequencing, hundreds of thousands or even millions of bacterial individuals are usually used to crush together and extract genes as templates, and then perform gene amplification and sequencing.
  • modern science and technology have proved that there are differences between individuals of bacteria, viruses, and cells.
  • the individual viruses are extremely small and belong to nanoscale biological particles, they can usually only be seen under an electron microscope. It is still difficult to separate a large number of individual virus particles with high throughput. Therefore, it is necessary to find new materials or methods that can be used for the isolation of virus single particles, so as to be applied to the isolation and purification of viruses.
  • the existing technology usually uses methods such as flow cytometry and micro-manipulation under an optical microscope to divide bacteria or cells into several categories.
  • the single-cell instrument of 10xGenomics uses microfluidics. Isolate single cells, but usually only cells larger than 5 ⁇ m can be separated, and the highest yield is only about 40%, usually only a dozen percent. Therefore, it is also necessary to develop single-cell isolation methods and instruments based on new principles.
  • the present invention provides a microsphere with functionalized micro or nano spots on the surface, its preparation method and application.
  • the present invention adopts following technical scheme:
  • a microsphere with functionalized spots on the surface comprising a microsphere body and a functionalized spot inlaid on the top of the microsphere body, a gravity ball is embedded in the bottom of the microsphere body, and the gravity ball is used to adjust the The center of gravity of the microsphere body keeps the functionalized spots upward.
  • the diameter of the microsphere body is 2-60 ⁇ m
  • the surface does not have adsorption properties
  • the surface is modified with fluorescence or quantum dots
  • the inside is magnetically modified.
  • microsphere main body adopts polystyrene magnetic microspheres or silica magnetic microspheres.
  • the diameter of the gravity ball is 30%-50% of the main body of the microsphere, and the specific gravity is 2-10.
  • the functionalized spots have adsorption properties and have a diameter of 15nm-30 ⁇ m.
  • the functionalized spots contain substances that can adsorb bioactive particles
  • the substances that can adsorb bioactive particles include adsorbed molecules, adsorbed nanoparticles or modified micro-nanospheres.
  • the functionalized spot is embedded on the top of the microsphere body through gel fixation.
  • the gel is gelatin, agar or polyethylene glycol.
  • the adsorption molecules include streptavidin, protein A or protein G;
  • the adsorption nanoparticles include gold nanoparticles and fullerene nanoparticles with a diameter of 1-20nm;
  • the modified micro-nanospheres It is a micro-nanosphere modified by amino group, carboxyl group, hydroxyl group or streptavidin, with a diameter of 2nm-30 ⁇ m.
  • a method for preparing microspheres with functionalized spots on the surface comprising the steps of:
  • the diameter of the micron hole in step S1 is 2-60 ⁇ m, and the depth is 1-30 ⁇ m.
  • the thickness of the nano-film in step S4 is 3-50 nm; the diameter of the micro-nanopore is 15 nm-30 ⁇ m, and the depth is 15 nm-30 ⁇ m.
  • microspheres with functionalized spots on the surface for the adsorption and separation of biologically active particles.
  • the biologically active particles include virus single particles, protein single particles, nucleic acid single particles, single microorganisms or single cells.
  • the adsorption and separation of the biologically active particles specifically includes the following steps:
  • the present invention has the following advantages compared with the background technology:
  • the bottom of the microsphere of the present invention is embedded with a gravity ball, and the center of gravity of the microsphere body is adjusted through the gravity ball, so that the microsphere body is in a tumbler state in the solution, so that the functionalized spot on the top of the microsphere body can always keep upward , which is conducive to the adsorption, detection and separation of bioactive particles;
  • the functionalized spots on the top of the microspheres contain active substances that adsorb biologically active particles, so they have a good ability to adsorb biologically active particles. According to the size of the biologically active particles, functionalized spots with nanometer or micron size similar to the size of the active particles can be selected.
  • nano-functionalized spots similar in size to virus particles can be designed, and single virus particles are adsorbed on the functionalized spots, while the main surface of the microsphere does not adsorb Biological particles, the surface of which is modified by fluorescence or quantum dots, and the interior is magnetically modified, and the separation and detection of microspheres can be realized by magnetic adsorption method;
  • the preparation method of the microspheres of the present invention has the advantages of low cost, high efficiency and rapidity
  • microspheres of the present invention have the functions of adsorption and separation of bioactive particles.
  • Fig. 1 is the schematic diagram of microsphere structure and application principle of the present invention
  • Fig. 2 is a schematic diagram of functionalized spots prepared by imprinting in the present invention.
  • a microsphere with functionalized spots on the surface includes a microsphere main body 1 and a functionalized spot 2 embedded on the top of the microsphere main body 1, and the bottom of the microsphere main body 1 is embedded with gravity Ball 3, the gravity ball 3 is used to adjust the center of gravity of the microsphere body 1, so that the functionalized spot 2 keeps upward.
  • the functionalized spots 2 are used to adsorb individual bioactive particles 4 .
  • the microsphere main body 1 adopts commercially available polystyrene magnetic microspheres with a diameter of 5 ⁇ m.
  • the gravity ball 3 has a diameter of 2.5 ⁇ m and a specific gravity of 7.6.
  • the functionalized spot 2 is solidified from a mixed gel containing streptavidin, has adsorption properties, and has a diameter of 100 nm.
  • the mixed gel is composed of 10% gelatin and 0.15% streptavidin.
  • gelatin with a concentration of 8-12% and a mass concentration of 0.05%-0.3% streptavidin can be selected for the gel.
  • a kind of surface has the preparation method of the microsphere of functionalized spot, comprises the steps:
  • the diameter of the cylindrical hole can be selected to be 50-400nm larger than the diameter of the microsphere, the hole depth is 30-750nm smaller than the diameter of the microsphere, the distance between the walls of two adjacent holes is 3-25 ⁇ m, and the thickness of the porous ceramic chip
  • the chip 5 can also choose a single crystal silicon plate chip with a thickness of 2-20 ⁇ m, and the holes of the micron hole array can also be made into tapered holes.
  • the microsphere body can be controlled to be 30-750nm higher than the surface of the chip 5; in addition, the above steps can also adopt the method of filtering to array the microsphere body 1 on the chip 5.
  • the thickness of the optional nano film 7 is 5-50nm
  • the gel of this embodiment uses The commercially available gelatin has a concentration of 10%, and the adsorbent is commercially available surface aminated silica nanospheres with a diameter of 10 nm.
  • the gel can also be made of polymer materials such as agar and polyethylene glycol.
  • microspheres of Example 1 are used for adsorption and separation of virus single particles, which specifically includes the following steps:
  • A2 adding a solution containing virus particles to the surface of the first chip, and the virus particles are adsorbed on the functionalized spots of the microspheres;
  • microspheres with functionalized spots of corresponding size are designed, so that each functionalized spot can only adsorb a single virus particle, thereby realizing the separation of single virus particles.
  • corresponding functionalized spots can be designed according to the size of the bacteria and cells that need to be adsorbed and separated, so as to realize the single particle separation of bioactive particles of different sizes.

Abstract

A microsphere having a functionalized spot on the surface, comprising a microsphere main body and a functionalized spot embedded in the top of the microsphere main body. A gravity ball is embedded in the bottom of the microsphere main body, and the gravity ball is used for adjusting the center of gravity of the microsphere main body to keep the functionalized spot upward. A preparation method for and an application of the microsphere are further disclosed. The microsphere having the functionalized spot can be used for adsorption and separation of bioactive particles.

Description

一种表面具有功能化斑点的微球及其制备方法和应用A microsphere with functionalized spots on the surface and its preparation method and application 技术领域technical field
本发明涉及生物材料技术领域，特别涉及一种表面具有功能化斑点的微球及其制备方法和应用。The invention relates to the technical field of biomaterials, in particular to a microsphere with functionalized spots on the surface and a preparation method and application thereof.
背景技术Background technique
细胞、细菌和病毒是生物学领域中三大类材料，它们都具有颗粒的属性，如病毒通常在15-200nm，细菌在0.2-8μm，而人体哺乳细胞通常在5-30μm。现有的生物学研究方法，无论是生理生化研究，还是基因和基因组研究，通常采用许多相同的细胞。如常规的细菌基因组测序，通常采用几十万甚至几百万个细菌个体，一起破碎后提取基因作为模板，然后进行基因扩增和测序。但现代科学技术已经证明，细菌、病毒、细胞的个体之间是有差异的。在一定条件下，研究这些差异是很有必要的，如新冠肺炎病毒，通过研究一个样本中单个纳米病毒的基因组或生理，有助于了解病毒的突变路径，从而为治疗药物和治疗方法的开发提供理论基础。Cells, bacteria, and viruses are the three major categories of materials in the field of biology. They all have the properties of particles. For example, viruses are usually 15-200nm, bacteria are 0.2-8μm, and human mammalian cells are usually 5-30μm. Existing biological research methods, whether it is physiological and biochemical research, or gene and genome research, usually use many of the same cells. For example, in conventional bacterial genome sequencing, hundreds of thousands or even millions of bacterial individuals are usually used to crush together and extract genes as templates, and then perform gene amplification and sequencing. However, modern science and technology have proved that there are differences between individuals of bacteria, viruses, and cells. Under certain conditions, it is necessary to study these differences, such as the new coronary pneumonia virus, by studying the genome or physiology of a single nanovirus in a sample, it will help to understand the mutation path of the virus, so as to provide a basis for the development of therapeutic drugs and methods. Provide a theoretical basis.
因为病毒的个体极其微小，属于纳米尺度的生物颗粒，通常只能在电子显微镜下才能看到，高通量地分离一大批单个病毒颗粒，在目前还是难以做到。因此，需要寻找新的可用于病毒单颗粒分离的材料或方法，以应用于病毒的分离和纯化。Because the individual viruses are extremely small and belong to nanoscale biological particles, they can usually only be seen under an electron microscope. It is still difficult to separate a large number of individual virus particles with high throughput. Therefore, it is necessary to find new materials or methods that can be used for the isolation of virus single particles, so as to be applied to the isolation and purification of viruses.
细菌、细胞的个体更加大一些，现有技术通常采用流式细胞仪、光学显微镜下微操纵等方法，将细菌或细胞分成几大类，如10xGenomics的单细胞仪器，采用微流控的方法，分离单个细胞，但通常只能分离5μm以上的细胞，而且得率最高只有40％左右，通常只有百分之十几。因此，也有必要开发基于新原理的单细胞分离方法和仪器。Individuals of bacteria and cells are larger. The existing technology usually uses methods such as flow cytometry and micro-manipulation under an optical microscope to divide bacteria or cells into several categories. For example, the single-cell instrument of 10xGenomics uses microfluidics. Isolate single cells, but usually only cells larger than 5 μm can be separated, and the highest yield is only about 40%, usually only a dozen percent. Therefore, it is also necessary to develop single-cell isolation methods and instruments based on new principles.
发明内容Contents of the invention
为解决上述问题，本发明提供了一种表面具有功能化微米或纳米斑点的微球及其制备方法和应用。In order to solve the above problems, the present invention provides a microsphere with functionalized micro or nano spots on the surface, its preparation method and application.
本发明采用以下技术方案：The present invention adopts following technical scheme:
一种表面具有功能化斑点的微球，包括微球主体和镶嵌在所述微球主体顶部的功能化斑点，所述微球主体的底部内嵌有重力球，所述重力球用于调整所述微球主体的重心，使所述功能化斑点保持向上。A microsphere with functionalized spots on the surface, comprising a microsphere body and a functionalized spot inlaid on the top of the microsphere body, a gravity ball is embedded in the bottom of the microsphere body, and the gravity ball is used to adjust the The center of gravity of the microsphere body keeps the functionalized spots upward.
进一步地，所述微球主体的直径为2-60μm，表面不具有吸附特性，表面经荧光或量子点修饰，内部经磁性修饰。Further, the diameter of the microsphere body is 2-60 μm, the surface does not have adsorption properties, the surface is modified with fluorescence or quantum dots, and the inside is magnetically modified.
进一步地，所述微球主体采用聚苯乙烯磁性微球或二氧化硅磁性微球。Further, the microsphere main body adopts polystyrene magnetic microspheres or silica magnetic microspheres.
进一步地，所述重力球的直径为所述微球主体的30％-50％，比重为2-10。Further, the diameter of the gravity ball is 30%-50% of the main body of the microsphere, and the specific gravity is 2-10.
进一步地，所述功能化斑点具有吸附特性，直径为15nm-30μm。Further, the functionalized spots have adsorption properties and have a diameter of 15nm-30μm.
进一步地，所述功能化斑点含有可吸附生物活性颗粒的物质，所述可吸附生物活性颗粒的物质包括吸附分子、吸附纳米颗粒或经修饰的微纳球。Further, the functionalized spots contain substances that can adsorb bioactive particles, and the substances that can adsorb bioactive particles include adsorbed molecules, adsorbed nanoparticles or modified micro-nanospheres.
进一步地，所述功能化斑点通过凝胶固定镶嵌在所述微球主体的顶部。Further, the functionalized spot is embedded on the top of the microsphere body through gel fixation.
进一步地，所述凝胶为明胶、琼脂或聚乙二醇。Further, the gel is gelatin, agar or polyethylene glycol.
进一步地，所述吸附分子包括链霉亲合素、蛋白A或蛋白G；所述吸附纳米颗粒包括纳米金颗粒、富勒烯纳米颗粒，直径为1-20nm；所述经修饰的微纳球为经氨基、羧基、羟基或链霉亲和素修饰的微纳球，直径2nm-30μm。Further, the adsorption molecules include streptavidin, protein A or protein G; the adsorption nanoparticles include gold nanoparticles and fullerene nanoparticles with a diameter of 1-20nm; the modified micro-nanospheres It is a micro-nanosphere modified by amino group, carboxyl group, hydroxyl group or streptavidin, with a diameter of 2nm-30μm.
一种表面具有功能化斑点的微球的制备方法，包括如下步骤：A method for preparing microspheres with functionalized spots on the surface, comprising the steps of:
S1、制备含有微米孔阵列的芯片，并将微米球阵列于所述芯片的微米孔中，每个孔洞只含有一个微米球；S1. Prepare a chip containing a microwell array, and place microsphere arrays in the microwells of the chip, each hole containing only one microsphere;
S2、在所述微球上刻蚀一个微米孔，并将重力球压入所述微米孔中，得到内嵌有重力球的微球主体；S2. Etching a micron hole on the microsphere, and pressing the gravity ball into the micron hole to obtain a microsphere body embedded with the gravity ball;
S3、将所述微球主体悬浮于液体中，采用沉降或过滤的方法将所述微球主体阵列在所述芯片上；S3, suspending the microsphere body in the liquid, and arraying the microsphere body on the chip by sedimentation or filtration;
S4、在所述微球主体上方贴一层纳米薄膜，在所述微球主体顶部刻蚀一个微米或纳米大小的微纳孔；S4. Paste a layer of nano film on the top of the microsphere body, and etch a micro-nano hole of micron or nanometer size on the top of the microsphere body;
S5、在所述纳米薄膜上铺一层含有所述可吸附生物活性颗粒的物质的所述混合凝胶，用压印法将所述混合凝胶灌满所述微纳孔；S5. Lay a layer of the mixed gel containing the substance capable of absorbing bioactive particles on the nano-film, and fill the micro-nanopores with the mixed gel by embossing;
S6、去除覆盖在芯片表面的纳米薄膜，未压入微纳孔的混合凝胶被清除，待混合凝胶凝固后，得到具有功能化斑点的微球。S6. Remove the nano film covering the surface of the chip, remove the mixed gel that has not been pressed into the micro-nano pores, and obtain microspheres with functionalized spots after the mixed gel is solidified.
进一步地，步骤S1中所述微米孔的直径为2-60μm，深度为1-30μm。Further, the diameter of the micron hole in step S1 is 2-60 μm, and the depth is 1-30 μm.
进一步地，步骤S4中所述纳米薄膜厚度为3-50nm；所述微纳孔直径为15nm-30μm，深度为15nm-30μm。Further, the thickness of the nano-film in step S4 is 3-50 nm; the diameter of the micro-nanopore is 15 nm-30 μm, and the depth is 15 nm-30 μm.
一种表面具有功能化斑点的微球的应用，所述微球用于生物活性颗粒的吸附和分离。A use of microspheres with functionalized spots on the surface for the adsorption and separation of biologically active particles.
进一步地，所述生物活性颗粒包括病毒单颗粒、蛋白单颗粒、核酸单颗粒、单个微生物或单个细胞。Further, the biologically active particles include virus single particles, protein single particles, nucleic acid single particles, single microorganisms or single cells.
进一步地，所述生物活性颗粒的吸附和分离具体包括如下步骤：Further, the adsorption and separation of the biologically active particles specifically includes the following steps:
A1、将表面具有功能化斑点的所述微球阵列在含有微米孔阵列的第一块芯片上；A1. Arraying the microspheres with functionalized spots on the surface on the first chip containing the microwell array;
A2、将含有生物活性颗粒的溶液添加到所述第一块芯片的表面，生物活性颗粒被吸附在所述微球的功能化斑点上；A2. Add a solution containing bioactive particles to the surface of the first chip, and the bioactive particles are adsorbed on the functionalized spots of the microspheres;
A3、洗去多余的生物活性颗粒，盖上第二块芯片，利用磁性吸附的方法将吸附了生物活性颗粒的微球转移到第二块芯片上。A3. Wash off excess bioactive particles, cover the second chip, and transfer the microspheres adsorbed with bioactive particles to the second chip by magnetic adsorption.
采用上述技术方案后，本发明与背景技术相比，具有如下优点：After adopting the technical solution, the present invention has the following advantages compared with the background technology:
1、本发明的微球底部内嵌有重力球，通过重力球调节微球主体的重心，使微球主体在溶液中处于不倒翁的状态，从而使微球主体顶部的功能化斑点能够一直保持向上，利于生物活性颗粒的吸附、检测和分离；1. The bottom of the microsphere of the present invention is embedded with a gravity ball, and the center of gravity of the microsphere body is adjusted through the gravity ball, so that the microsphere body is in a tumbler state in the solution, so that the functionalized spot on the top of the microsphere body can always keep upward , which is conducive to the adsorption, detection and separation of bioactive particles;
2、微球顶部的功能化斑点含有吸附生物活性颗粒的活性物质，因此具有良好吸附生物颗粒的能力，根据生物活性颗粒的大小，可选择与活性颗粒尺寸相近的纳米或者微米大小的功能化斑点，从而实现生物活性颗粒的单颗粒吸附和分离；如吸附病毒颗粒，可设计与病毒颗粒尺寸相近的纳米功能化斑点，单个病毒颗粒被吸附在该功能化斑点上，而微球主体表面不吸附生物颗 粒，且表面经荧光或量子点修饰，内部经磁性修饰，可通过磁吸附法实现微球的分离和检测；2. The functionalized spots on the top of the microspheres contain active substances that adsorb biologically active particles, so they have a good ability to adsorb biologically active particles. According to the size of the biologically active particles, functionalized spots with nanometer or micron size similar to the size of the active particles can be selected. , so as to realize single-particle adsorption and separation of biologically active particles; for example, to adsorb virus particles, nano-functionalized spots similar in size to virus particles can be designed, and single virus particles are adsorbed on the functionalized spots, while the main surface of the microsphere does not adsorb Biological particles, the surface of which is modified by fluorescence or quantum dots, and the interior is magnetically modified, and the separation and detection of microspheres can be realized by magnetic adsorption method;
3、本发明的微球的制备方法具有低成本、高效快速的优点；3. The preparation method of the microspheres of the present invention has the advantages of low cost, high efficiency and rapidity;
4、本发明的微球具有生物活性颗粒的吸附和分离功能。4. The microspheres of the present invention have the functions of adsorption and separation of bioactive particles.
附图说明Description of drawings
图1为本发明的微球结构和应用原理示意图；Fig. 1 is the schematic diagram of microsphere structure and application principle of the present invention;
图2为本发明的压印制备功能化斑点的示意图。Fig. 2 is a schematic diagram of functionalized spots prepared by imprinting in the present invention.
具体实施方式Detailed ways
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.
实施例一Embodiment one
如图1所示，一种表面具有功能化斑点的微球，包括微球主体1和镶嵌在所述微球主体1顶部的功能化斑点2，所述微球主体1的底部内嵌有重力球3，所述重力球3用于调整所述微球主体1的重心，使所述功能化斑点2保持向上。所述功能化斑点2用于吸附单个生物活性颗粒4。As shown in Figure 1, a microsphere with functionalized spots on the surface includes a microsphere main body 1 and a functionalized spot 2 embedded on the top of the microsphere main body 1, and the bottom of the microsphere main body 1 is embedded with gravity Ball 3, the gravity ball 3 is used to adjust the center of gravity of the microsphere body 1, so that the functionalized spot 2 keeps upward. The functionalized spots 2 are used to adsorb individual bioactive particles 4 .
所述微球主体1采用市售的直径为5μm的聚苯乙烯磁性微球。The microsphere main body 1 adopts commercially available polystyrene magnetic microspheres with a diameter of 5 μm.
所述重力球3的直径为2.5μm，比重为7.6。The gravity ball 3 has a diameter of 2.5 μm and a specific gravity of 7.6.
所述功能化斑点2由含有链霉亲合素的混合凝胶凝固而成，具有吸附特性，直径为100nm。所述混合凝胶由浓度10％的明胶和质量浓度为0.15％链霉亲合素组成。The functionalized spot 2 is solidified from a mixed gel containing streptavidin, has adsorption properties, and has a diameter of 100 nm. The mixed gel is composed of 10% gelatin and 0.15% streptavidin.
这里凝胶起到固定的作用，根据实际制备过程，凝胶可选择浓度为8-12％的明胶；质量浓度为0.05％-0.3％链霉亲合素。Here the gel plays a role of fixation. According to the actual preparation process, gelatin with a concentration of 8-12% and a mass concentration of 0.05%-0.3% streptavidin can be selected for the gel.
实施例二Embodiment two
如图2所示，一种表面具有功能化斑点的微球的制备方法，包括如下步 骤：As shown in Figure 2, a kind of surface has the preparation method of the microsphere of functionalized spot, comprises the steps:
S1、制备含有微米孔阵列的芯片5，并将微米球阵列于所述芯片5的微米孔中，每个孔洞只含有一个微米球；本实施例的芯片采用多孔陶瓷芯片，微米孔阵列的孔洞为圆柱形孔6，圆柱形孔的直径比微米球大200nm，孔深比微米球直径小300nm，相邻两孔壁间距为5μm；S1. Prepare a chip 5 containing a micro-hole array, and put the micro-ball array in the micro-hole of the chip 5, each hole only contains one micro-ball; the chip of this embodiment adopts a porous ceramic chip, and the holes of the micro-hole array It is a cylindrical hole 6, the diameter of the cylindrical hole is 200nm larger than that of the microsphere, the hole depth is 300nm smaller than the diameter of the microsphere, and the distance between the walls of two adjacent holes is 5 μm;
根据实际的微米球尺寸，可以选择圆柱形孔的直径比微米球大50-400nm，孔深比微米球的直径小30-750nm，相邻两孔壁间距为3-25μm，多孔陶瓷芯片的厚度为0.5-3mm，此外，芯片5还可选择厚度为2-20μm的单晶硅板芯片，微米孔阵列的孔洞还可制作成锥形孔。According to the actual microsphere size, the diameter of the cylindrical hole can be selected to be 50-400nm larger than the diameter of the microsphere, the hole depth is 30-750nm smaller than the diameter of the microsphere, the distance between the walls of two adjacent holes is 3-25μm, and the thickness of the porous ceramic chip In addition, the chip 5 can also choose a single crystal silicon plate chip with a thickness of 2-20 μm, and the holes of the micron hole array can also be made into tapered holes.
S2、采用通孔掩膜光刻技术在所述30μm微米球3上刻蚀一个直径为15μm，深度为15μm的微米孔，并将重力球3压入所述微米孔中，得到内嵌有重力球3的微球主体1；S2. Etching a micron hole with a diameter of 15 μm and a depth of 15 μm on the 30 μm microsphere 3 by using the through-hole mask photolithography technology, and pressing the gravity ball 3 into the micron hole to obtain the embedded gravity Microsphere body 1 of sphere 3;
S3、将所述微球主体1悬浮于液体中，采用重力沉降的方法将所述微球主体1阵列在所述芯片5上，所述微球主体高处芯片5表面500nm；S3, suspending the microsphere body 1 in the liquid, arraying the microsphere body 1 on the chip 5 by gravity settling, the height of the microsphere body is 500 nm above the surface of the chip 5;
根据实际的微米球尺寸，可控制微球主体高出芯片5表面30-750nm；此外，上述步骤还可以采用过滤的方法将所述微球主体1阵列在所述芯片5上。According to the actual microsphere size, the microsphere body can be controlled to be 30-750nm higher than the surface of the chip 5; in addition, the above steps can also adopt the method of filtering to array the microsphere body 1 on the chip 5.
S4、在所述微球主体上方贴一层纳米薄膜7，采用通孔掩膜技术在所述微球主体1顶部刻蚀一个微纳孔，所述纳米薄膜7厚度为20nm，所述微纳孔直径为15μm，深度为10μm；S4. Paste a layer of nano film 7 above the microsphere body, etch a micro-nano hole on the top of the microsphere body 1 using through-hole mask technology, the thickness of the nano-film 7 is 20nm, and the micro-nano The hole diameter is 15 μm and the depth is 10 μm;
根据实际需要，可选择的纳米薄膜7的厚度为5-50nm；According to actual needs, the thickness of the optional nano film 7 is 5-50nm;
S5、在所述纳米薄膜7上铺一层含有链霉亲合素的混合凝胶溶液8，用压印法将所述混合凝胶溶液8灌满微纳孔；本实施例的凝胶采用市售的浓度为10％的明胶，吸附物质为直径为10nm市售的表面氨基化二氧化硅纳米球，此外，凝胶还可以选择琼脂、聚乙二醇等高分子材料。S5. Spread a layer of mixed gel solution 8 containing streptavidin on the nano-film 7, and fill the micro-nanopores with the mixed gel solution 8 by embossing; the gel of this embodiment uses The commercially available gelatin has a concentration of 10%, and the adsorbent is commercially available surface aminated silica nanospheres with a diameter of 10 nm. In addition, the gel can also be made of polymer materials such as agar and polyethylene glycol.
S6、去除覆盖在芯片5表面的纳米薄膜7，未压入微纳孔的混合凝胶溶液被清除，待混合凝胶凝固后，得到具有功能化斑点的微球。S6. The nano film 7 covering the surface of the chip 5 is removed, the mixed gel solution that has not been pressed into the micro-nano pores is removed, and after the mixed gel is solidified, microspheres with functionalized spots are obtained.
实施例三Embodiment Three
一种表面具有功能化斑点的微球的应用，本实施例采用实施例一的微球进行病毒单颗粒的吸附和分离，具体包括如下步骤：An application of microspheres with functionalized spots on the surface. In this example, the microspheres of Example 1 are used for adsorption and separation of virus single particles, which specifically includes the following steps:
A1、将表面具有功能化斑点的所述微球阵列在含有微米孔阵列的第一块芯片上；A1. Arraying the microspheres with functionalized spots on the surface on the first chip containing the microwell array;
A2、将含病毒颗粒的溶液添加到所述第一块芯片的表面，病毒颗粒被吸附在所述微球的功能化斑点上；A2, adding a solution containing virus particles to the surface of the first chip, and the virus particles are adsorbed on the functionalized spots of the microspheres;
A3、洗去多余的病毒颗粒，盖上第二块芯片，利用磁性吸附的方法将吸附了病毒颗粒的微球转移到第二块芯片上。A3. Wash away excess virus particles, cover the second chip, and transfer the microspheres with adsorbed virus particles to the second chip by magnetic adsorption.
本实施例根据需要吸附和分离的病毒单颗粒的尺寸，设计了具有相应尺寸的功能化斑点的微球，使得每个功能化斑点仅能吸附单个病毒颗粒，从而实现病毒单颗粒的分离。此外，还可以根据需要吸附和分离的细菌、细胞的尺寸设计相对应的功能化斑点，以实现不同尺寸的生物活性颗粒的单颗粒分离。In this example, according to the size of virus single particles to be adsorbed and separated, microspheres with functionalized spots of corresponding size are designed, so that each functionalized spot can only adsorb a single virus particle, thereby realizing the separation of single virus particles. In addition, corresponding functionalized spots can be designed according to the size of the bacteria and cells that need to be adsorbed and separated, so as to realize the single particle separation of bioactive particles of different sizes.
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应该以权利要求的保护范围为准。The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present invention can easily think of changes or Replacement should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (15)

  1. 一种表面具有功能化斑点的微球，其特征在于：包括微球主体和镶嵌在所述微球主体顶部的功能化斑点，所述微球主体的底部内嵌有重力球，所述重力球用于调整所述微球主体的重心，使所述功能化斑点保持向上。A microsphere with functionalized spots on the surface is characterized in that: it includes a microsphere body and a functionalized spot inlaid on the top of the microsphere body, a gravity ball is embedded in the bottom of the microsphere body, and the gravity ball Used to adjust the center of gravity of the microsphere body so that the functionalized spots remain upward.
  2. 如权利要求1所述的一种表面具有功能化斑点的微球，其特征在于：所述微球主体的直径为2-60μm，表面不具有吸附特性，表面经荧光或量子点修饰，内部经磁性修饰。A microsphere with functionalized spots on the surface according to claim 1, characterized in that: the diameter of the microsphere body is 2-60 μm, the surface does not have adsorption properties, the surface is modified by fluorescence or quantum dots, and the interior is Magnetic modification.
  3. 如权利要求2所述的一种表面具有功能化斑点的微球，其特征在于：所述微球主体采用聚苯乙烯磁性微球或二氧化硅磁性微球。A microsphere with functionalized spots on the surface according to claim 2, characterized in that: the main body of the microsphere is made of polystyrene magnetic microspheres or silicon dioxide magnetic microspheres.
  4. 如权利要求2所述的一种表面具有功能化斑点的微球，其特征在于：所述重力球的直径为所述微球主体的30％-50％，比重为2-10。A microsphere with functionalized spots on the surface according to claim 2, characterized in that: the diameter of the gravity ball is 30%-50% of the main body of the microsphere, and the specific gravity is 2-10.
  5. 如权利要求1所述的一种表面具有功能化斑点的微球，其特征在于：所述功能化斑点具有吸附特性，直径为15nm-30μm。A microsphere with functionalized spots on the surface according to claim 1, characterized in that: the functionalized spots have adsorption properties and have a diameter of 15nm-30μm.
  6. 如权利要求5所述的一种表面具有功能化斑点的微球，其特征在于：所述功能化斑点含有可吸附生物活性颗粒的物质，所述可吸附生物活性颗粒的物质包括吸附分子、吸附纳米颗粒或经修饰的微纳球。A microsphere with functionalized spots on the surface according to claim 5, characterized in that: the functionalized spots contain substances that can adsorb bioactive particles, and the substances that can adsorb bioactive particles include adsorption molecules, adsorption Nanoparticles or modified micro-nanospheres.
  7. 如权利要求1所述的一种表面具有功能化斑点的微球，其特征在于：所述功能化斑点通过凝胶固定镶嵌在所述微球主体的顶部。A microsphere with functionalized spots on the surface according to claim 1, characterized in that: the functionalized spots are fixed and embedded on the top of the microsphere body by gel.
  8. 如权利要求7所述的一种表面具有功能化斑点的微球，其特征在于：所述凝胶为明胶、琼脂或聚乙二醇。A microsphere with functionalized spots on the surface according to claim 7, characterized in that: the gel is gelatin, agar or polyethylene glycol.
  9. 如权利要求6所述的一种表面具有功能化斑点的微球，其特征在于：所述吸附分子包括链霉亲合素、蛋白A或蛋白G；所述吸附纳米颗粒包括纳米金颗粒、富勒烯纳米颗粒，直径为1-20nm；所述经修饰的微纳球为经氨基、羧基、羟基或链霉亲和素修饰的微纳球，直径2nm-30μm。A microsphere with functionalized spots on the surface according to claim 6, characterized in that: the adsorption molecules include streptavidin, protein A or protein G; the adsorption nanoparticles include gold nanoparticles, rich The strene nanoparticle has a diameter of 1-20nm; the modified micro-nanosphere is a micro-nanosphere modified by amino group, carboxyl group, hydroxyl group or streptavidin, and has a diameter of 2nm-30μm.
  10. 如权利要求1所述的一种表面具有功能化斑点的微球的制备方法，其特征在于：包括如下步骤：A method for preparing microspheres with functionalized spots on the surface according to claim 1, characterized in that: comprising the steps of:
    S1、制备含有微米孔阵列的芯片，并将微米球阵列于所述芯片的微米孔中，每个孔洞只含有一个微米球；S1. Prepare a chip containing a microwell array, and place microsphere arrays in the microwells of the chip, each hole containing only one microsphere;
    S2、在所述微球上刻蚀一个微米孔，并将重力球压入所述微米孔中，得到内嵌有重力球的微球主体；S2. Etching a micron hole on the microsphere, and pressing the gravity ball into the micron hole to obtain a microsphere body embedded with the gravity ball;
    S3、将所述微球主体悬浮于液体中，采用沉降或过滤的方法将所述微球主体阵列在所述芯片上；S3, suspending the microsphere body in the liquid, and arraying the microsphere body on the chip by sedimentation or filtration;
    S4、在所述微球主体上方贴一层纳米薄膜，在所述微球主体顶部刻蚀一个微米或纳米大小的微纳孔；S4. Paste a layer of nano film on the top of the microsphere body, and etch a micro-nano hole of micron or nanometer size on the top of the microsphere body;
    S5、在所述纳米薄膜上铺一层含有所述可吸附生物活性颗粒的物质的混合凝胶，用压印法将所述混合凝胶灌满所述微纳孔；S5. Laying a layer of mixed gel containing the substance capable of absorbing bioactive particles on the nano-film, and filling the mixed gel with the micro-nanopores by embossing;
    S6、去除覆盖在芯片表面的纳米薄膜，未压入微纳孔的混合凝胶被清除，待混合凝胶凝固后，得到具有功能化斑点的微球。S6. Remove the nano film covering the surface of the chip, remove the mixed gel that has not been pressed into the micro-nano pores, and obtain microspheres with functionalized spots after the mixed gel is solidified.
  11. 如权利要求10所述的一种表面具有功能化斑点的微球的制备方法，其特征在于：步骤S1中所述微米孔的直径为2-60μm，深度为1-30μm。The method for preparing microspheres with functionalized spots on the surface according to claim 10, characterized in that: the diameter of the micron hole in step S1 is 2-60 μm, and the depth is 1-30 μm.
  12. 如权利要求11所述的一种表面具有功能化斑点的微球的制备方法，其特征在于：步骤S4中所述纳米薄膜厚度为3-50nm；所述微纳孔直径为15nm-30μm，深度为15nm-30μm。A method for preparing microspheres with functionalized spots on the surface according to claim 11, characterized in that: the thickness of the nano film in step S4 is 3-50nm; the diameter of the micro-nanopore is 15nm-30μm, and the depth 15nm-30μm.
  13. 如权利要求1所述的一种表面具有功能化斑点的微球的应用，其特征在于：所述微球用于生物活性颗粒的吸附和分离。The application of a microsphere with functionalized spots on the surface according to claim 1, characterized in that: the microsphere is used for the adsorption and separation of bioactive particles.
  14. 如权利要求13所述的一种表面具有功能化斑点的微球的应用，其特征在于：所述生物活性颗粒包括病毒单颗粒、蛋白单颗粒、核酸单颗粒、单个微生物或单个细胞。The application of a microsphere with functionalized spots on the surface according to claim 13, characterized in that: the biologically active particles include virus single particles, protein single particles, nucleic acid single particles, single microorganisms or single cells.
  15. 如权利要求14所述的一种表面具有功能化斑点的微球的应用，其特征在于：所述生物活性颗粒的吸附和分离具体包括如下步骤：The application of microspheres with functionalized spots on the surface according to claim 14, characterized in that: the adsorption and separation of the bioactive particles specifically comprises the following steps:
    A1、将表面具有功能化斑点的所述微球阵列在含有微米孔阵列的第一块芯片上；A1. Arraying the microspheres with functionalized spots on the surface on the first chip containing the microwell array;
    A2、将含有生物活性颗粒的溶液添加到所述第一块芯片的表面，生物活性颗粒被吸附在所述微球的功能化斑点上；A2. Add a solution containing bioactive particles to the surface of the first chip, and the bioactive particles are adsorbed on the functionalized spots of the microspheres;
    A3、洗去多余的生物活性颗粒，盖上第二块芯片，利用磁性吸附的方法将吸附了生物活性颗粒的微球转移到第二块芯片上。A3. Wash away excess bioactive particles, cover the second chip, and transfer the microspheres adsorbed with bioactive particles to the second chip by magnetic adsorption.
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