WO2022120998A1 - 一种双层微孔芯片、双层微孔芯片制备方法及生物装置 - Google Patents

一种双层微孔芯片、双层微孔芯片制备方法及生物装置 Download PDF

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WO2022120998A1
WO2022120998A1 PCT/CN2020/139641 CN2020139641W WO2022120998A1 WO 2022120998 A1 WO2022120998 A1 WO 2022120998A1 CN 2020139641 W CN2020139641 W CN 2020139641W WO 2022120998 A1 WO2022120998 A1 WO 2022120998A1
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layer
microporous
micropores
double
photoresist
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English (en)
French (fr)
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杨慧
陈希
张翊
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深圳先进技术研究院
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation

Definitions

  • the invention relates to the technical field of medical devices, in particular to a double-layer microporous chip, a method for preparing the double-layered microporous chip, and a biological device.
  • pluripotent stem cells can be used in the fields of disease treatment and regenerative medicine, due to the heterogeneity of their cell functions, Pluripotent stem cells have been difficult to stably differentiate.
  • cancer stem cells a small group of cells called "cancer stem cells" within a tumor group is thought to be the main cause of tumor resistance to chemotherapy drugs. If the heterogeneity between each type of cells can be accurately analyzed, it will help to simplify complex biological phenomena and provide new directions for disease treatment.
  • FACS flow cytometry
  • microfluidic chip devices for single cell screening and analysis using different principles, such as microdroplets, dielectrophoresis, hydrodynamics, and microwell arrays. array) and so on.
  • the microdroplet cell culture method can quickly provide a single-cell culture space. This method is to separate the oily medium and wrap the single cells in the cell culture droplets for culture.
  • the cell culture medium in the droplet cannot be replaced after the droplet is formed. This method is not suitable for cell experiments where the medium needs to be replaced during the process, and the droplet also lacks a matrix that allows cells to attach, so it cannot be applied. in the culture of adherent cells.
  • microwell-designed chips can be applied to the screening and culture of adherent or suspended cells.
  • This design only needs to be in a hole-like structure, and the operation only requires the use of hydrodynamics or gravity to place cells into the micropores.
  • the design size of the microwells must usually be much larger than the size of a single cell, resulting in a general drop of 10-30% in single-cell screening efficiency.
  • the reduction in single-cell capture efficiency caused by the single microwells used by current techniques and limiting dilution methods are both due to Poisson distribution limitations.
  • the design size of the microwell needs to be much larger than the size of a single cell, which greatly reduces the screening efficiency of a single cell.
  • the single microwell used for single cell capture technology cannot accurately control the flow rate of the chip or system, which will lead to single cell capture. Loss of target cells during screening results in reduced screening efficiency. In view of this, it is necessary to provide a double-layer microwell chip with high screening efficiency for single cells.
  • the present invention provides a double-layer microporous chip, comprising: a first microporous structure layer and a second microporous structure layer connected to the first microporous structure layer, the first microporous structure
  • the layer includes several first layer micropores
  • the second microporous structure layer includes several second layer micropores
  • the pore diameters of the first layer micropores and the second layer micropores are the same
  • the pore diameter of the first layer of micropores and the pore diameter of the second layer of micropores are from small to large, and any one of the second layer of micropores has a shape and size that can accommodate only one sample.
  • the first microporous structure layer and the second microporous structure layer are integrally formed, the thickness of the first microporous structure layer is 0.5-20um, and the second microporous structure layer The thickness is 20-60um.
  • the micropores of the first layer are distributed on the first microporous structure layer at uniform intervals, and the micropores of the second layer are distributed at uniform intervals on the second microporous structure layer.
  • the porosity of the pores in the first microporous structure layer and the second microporous structure layer is 3-10%.
  • the vertical axes where the pore diameters of the first layer of micropores and the pore diameters of the second layer of micropores are located are on the same horizontal line and the transverse axes are parallel.
  • the first layer of micropores and the second layer of micropores are circular, triangular, or rhombus-shaped.
  • the first layer of micropores and the second layer of micropores are circular, the diameter of the first layer of micropores is 0.2-20um, and the diameter of the second layer of micropores is 20-60um.
  • the first microporous structure layer and the second microporous structure layer are made of photosensitive transparent polymers or thermosetting transparent polymers.
  • the sample is a biological cell or a fluorescent microsphere.
  • the diameter of the largest circle inscribed in the planar shape of the second layer of microwells is in the range of 1-3 times that contained in the sample, or preferably in the range of 1-2.4 or more preferably 1-1.4 times the range.
  • the depth of the second layer of microwells is to be accommodated in the range of 1-3 times the diameter of the sample, or preferably in the range of 1-2.4 times or more preferably in the range of 1-1.6 times.
  • the captured sample is a fluorescent microsphere
  • the diameter of the fluorescent microsphere is 30um
  • the diameter of the second layer of micropores is 30-35um
  • the depth is 30-55um
  • the present invention provides a method for preparing the double-layer microporous chip, comprising the following steps:
  • a photoresist is coated on the surface of the metal seed layer, and a first photoresist microporous layer is fabricated on the photoresist to obtain the first microporous structure layer. Including several first-layer micropores;
  • a photoresist is coated on the first photoresist microporous layer, and a second photoresist microporous layer is fabricated on the photoresist to obtain the second microporous structure layer.
  • the second microporous structure layer includes several second layer micropores;
  • the pore diameter of the first layer of micropores and the pore diameter of the second layer of micropores are kept the same, the pore diameter of the first layer of micropores and the pore diameter of the second layer of micropores are from small to large, and any one
  • the second layer of microwells has a shape and size that can accommodate only one sample.
  • the substrate is silicon or silicon nitride or silicon oxide.
  • the step of forming a metal seed layer on the substrate includes:
  • a layer of metal is sputtered on the surface of the substrate by magnetron sputtering, and the metal is chromium, nickel or aluminum.
  • a photoresist is coated on the surface of the metal seed layer, and a first photoresist microporous layer is fabricated on the photoresist to obtain the first microporous structure layer , specifically:
  • a photoresist is spin-coated on the metal seed layer by a soft lithography process, and a first photoresist microporous layer is fabricated on the photoresist to obtain the first microporous structure layer.
  • the step of spin-coating photoresist on the metal seed layer using a soft lithography process specifically includes:
  • the photoresist includes SU8-3005 or SU8-3025.
  • the step of peeling off the metal seed layer to obtain the double-layer microporous chip is specifically:
  • the metal seed layer is peeled off with a metal cleaning solution.
  • the present invention also provides a biological device, comprising an upper casing, the double-layer microporous chip and a lower casing, the upper casing and the lower casing are fixedly installed, and the double-layered The microporous chip is fixed in the space formed by the upper casing and the lower casing.
  • the double-layer microporous chip provided by the present invention includes a first microporous structure layer and a second microporous structure layer connected to the first microporous structure layer, and the first microporous structure layer includes several a first layer of micropores, the second microporous structure layer includes several second layer of micropores, the pore diameter of the first layer of micropores and the pore diameter of the second layer of micropores are consistent, and the first layer of micropores
  • the pore size of the first layer of micropores and the pore size of the second layer of micropores are from small to large, and any one of the second layer of micropores has a shape and size that can only accommodate one sample
  • the double-layered micropores provided by the present invention are Chip, the diameter of the first layer of micropores and the diameter of the second layer of micropores are designed from small to large to reduce the pressure difference between layers, prevent the blockage of the microporous chip, and ensure high throughput And through the interception and screening of the
  • the preparation method of the double-layer microporous chip provided by the present invention combines the soft lithography replication technology and the film transfer technology.
  • the method is simple and fast, and is suitable for the preparation of micropores of different materials.
  • the present invention provides a biological device including the above-mentioned double-layer microporous chip, which can be used for single-cell capture, analysis, and intercellular material transfer in in vitro diagnosis, thereby improving the accuracy of single-cell analysis, thereby improving the accuracy of single-cell analysis. It provides a new method for real-time monitoring of single cells.
  • FIG. 1 is a schematic structural diagram of a double-layer microporous chip provided in Embodiment 1 of the present invention.
  • FIG. 2 is a schematic explanatory diagram of sample capture in the double-layer microporous chip provided in Example 1 of the present invention.
  • Example 3 is a flow chart of the steps of a method for preparing a double-layer microporous chip provided in Example 2 of the present invention
  • Example 4 is a schematic diagram of the principle of a method for preparing a double-layer microporous chip provided in Example 2 of the present invention
  • FIG. 5 is a schematic structural diagram of a biological device provided in Embodiment 3 of the present invention.
  • first and second are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature defined as “first” or “second” may expressly or implicitly include one or more of that feature.
  • “plurality” means two or more, unless otherwise expressly and specifically defined.
  • FIG. 1 is a schematic structural diagram of a double-layer microporous chip 100 according to an embodiment of the present invention, including a first microporous structure layer 110 and a second microporous structure layer connected to the first microporous structure layer 110 120.
  • the connection relationship between the various components will be described in detail below.
  • the first microporous structure layer 110 includes a plurality of first-layer micropores 111
  • the second microporous structure layer 120 includes a plurality of second-layer micropores 121
  • the diameter of the first layer of micropores 111 and the The pore diameters of the second layer of micropores 121 are kept the same, the pore diameter of the first layer of micropores 111 and the pore diameter of the second layer of micropores 121 are from small to large, and any one of the second layer of micropores 121 Available in shapes and sizes to accommodate only one sample.
  • the diameter of the first layer of micropores 111 and the diameter of the second layer of micropores 121 are designed from small to large, the pressure difference between the layers is reduced, the blockage of the microporous chip is prevented, and the guarantee of High-throughput screening through the first layer of micropores 111 is achieved, which improves the signal-to-noise ratio of screening capture and screening capability.
  • the first microporous structure layer 110 and the second microporous structure layer 120 are integrally formed, the thickness of the first microporous structure layer 110 is 0.5-20um, and the second microporous structure layer 110 has a thickness of 0.5-20um.
  • the thickness of the hole structure layer 120 is 20-60um.
  • the thickness of the first microporous structure 110 is 10 ⁇ m and the thickness of the second microporous structure layer is 30 ⁇ m, when the thickness of the first microporous structure layer and the second microporous structure layer is 1:3, the single The capture efficiency of cells or magnetic beads is better.
  • the first microporous structure layer 110 is provided with perforations on the second microporous structure layer 120
  • the second microporous structure layer 120 is a perforation provided on the first microporous structure layer 110 .
  • the first layer of micropores 111 is evenly spaced on the first microporous structure layer 110
  • the second layer of micropores 121 is evenly spaced and distributed on the second microporous structure layer 120 on.
  • the porosity of the pores in the first microporous structure layer 110 and the second microporous structure layer 120 is 3-10%.
  • the vertical axes where the diameters of the first-layer micro-holes 111 and the second-layer micro-holes 121 are located are on the same horizontal line and the horizontal axes are parallel.
  • the first layer of micropores 111 and the second layer of micropores 121 are circular, triangular, or rhombus-shaped.
  • the diameter of the first layer of microholes 111 and the second layer of microholes 121 is 0.2-20um, and the second layer of microholes 121 The diameter is 20-60um.
  • the diameter of the first microporous structure 110 is 10um
  • the diameter of the second microporous structure layer is 30um
  • the thickness of the first microporous structure layer and the second microporous structure layer is 1:3
  • the single The capture efficiency of cells or magnetic beads is better.
  • the first microporous structure layer 110 and the second microporous structure layer 120 are made of photosensitive transparent polymers or thermosetting transparent polymers.
  • the sample is a biological cell or a fluorescent microsphere.
  • the diameter of the largest circle inscribed in the plane shape of the second layer of micropores 121 is in the range of 1-3 times that of the sample, or preferably in the range of 1-2.4, or more preferably in the range of 1- 1.4 times the range.
  • the depth of the second layer of micropores 121 is to be accommodated in a range of 1-3 times or preferably 1-2.4 times or more preferably 1-1.6 times the diameter of the sample.
  • the captured sample is a fluorescent microsphere
  • the diameter of the fluorescent microsphere is 30um
  • the diameter of the second layer of micropores 121 is 30-35um
  • the depth is 30-55um
  • the diameter of the first microporous structure 110 is 10um
  • the diameter of the second microporous structure layer is 30um
  • the thickness of the first microporous structure layer and the second microporous structure layer is 1:3
  • the single The capture efficiency of cells or magnetic beads is better.
  • FIG. 2 is a schematic explanatory diagram of sample capture in the double-layer microporous chip provided in Embodiment 1 of the present invention, wherein the sphere represents the sample.
  • the size of the micropore should be appropriately determined in consideration of an appropriate ratio of the diameter of the biological sample to be accommodated in the micropore to the size of the micropore.
  • the double-layer microporous chip provided in the above-mentioned embodiment 1 of the present invention includes a first microporous structure layer and a second microporous structure layer connected to the first microporous structure layer, and the first microporous structure layer includes several a first layer of micropores, the second microporous structure layer includes several second layer of micropores, the pore diameter of the first layer of micropores and the pore diameter of the second layer of micropores are consistent, and the first layer of micropores
  • the pore size of the first layer of micropores and the pore size of the second layer of micropores are from small to large, and any one of the second layer of micropores has a shape and size that can only accommodate one sample, the double-layered micropores provided by the present invention are Chip, the diameter of the first layer of micropores and the diameter of the second layer of micropores are designed from small to large to reduce the pressure difference between layers, prevent the blockage of the microporous chip, and ensure high throughput And through
  • the present invention provides a flow chart of steps of a method for preparing a double-layer microporous chip, including the following steps:
  • Step S110 forming a metal seed layer on the substrate.
  • the substrate is silicon or silicon nitride or silicon oxide.
  • the step of forming a metal seed layer on the substrate specifically: using magnetron sputtering to sputter a layer of metal on the surface of the substrate, where the metal is chromium, nickel or aluminum.
  • Step S120 Coating photoresist on the surface of the metal seed layer, and fabricating a first photoresist microporous layer on the photoresist to obtain the first microporous structure layer, the first microporous structure layer.
  • the pore structure layer includes several first layer micropores.
  • a photoresist is coated on the surface of the metal seed layer, and a first photoresist microporous layer is fabricated on the photoresist to obtain the first microporous structure layer , specifically: using a soft lithography process to spin-coat photoresist on the metal seed layer, and making a first photoresist microporous layer on the photoresist to obtain the first microporous structure Floor.
  • the step of spin-coating the photoresist on the metal seed layer by using a soft lithography process specifically includes: firstly rotating the photoresist at a low speed to slowly unscrew the photoresist to the silicon The edge of the sheet; and then spin-coating the photoresist to the edge of the metal seed layer by means of high rotation, the low-speed rotation is 500rpm for 10s, and the high-speed rotation is 2500 Spin glue for 30s.
  • the photoresist includes SU8-3005 or SU8-3025.
  • the metal seed layer needs to undergo several processes such as pre-baking, exposure, post-baking, and development.
  • Step S130 coating photoresist on the first photoresist microporous layer, and fabricating a second photoresist microporous layer on the photoresist to obtain the second microporous structure layer,
  • the second microporous structure layer includes several second layer micropores.
  • step S120 For the specific technical solution adopted in this embodiment, reference may be made to step S120, which will not be repeated here.
  • Step S140 peeling off the metal seed layer to obtain the double-layer microporous chip, the pore diameter of the first layer of micropores and the pore diameter of the second layer of micropores are consistent, and the first layer of micropores
  • the pore diameter of the pores and the pore diameter of the second layer of micropores are from small to large, and any one of the second layer of micropores has a shape and size that can accommodate only one sample.
  • the step of peeling off the metal seed layer to obtain the double-layer microporous chip is specifically as follows: finally, the metal seed layer is peeled off with a metal cleaning solution.
  • FIG. 4 is a schematic diagram of the fabrication process of the double-layer microporous chip provided in Embodiment 2 of the present invention.
  • the preparation method of the double-layer microporous chip provided by the invention combines the soft lithography replication technology and the film transfer technology, and the method is simple and fast, and is suitable for the preparation of micropores of different materials.
  • FIG. 5 is a schematic structural diagram of a biological device provided in Embodiment 3 of the present invention, including an upper casing 310 , the double-layer microporous chip 100 described in Embodiment 1 above, and a lower casing 320 .
  • the casing 310 and the lower casing 320 are fixedly installed, and the double-layer microporous chip 100 is fixed in the space formed by the upper casing 310 and the lower casing 320 .
  • the upper casing 310 , the double-layer microporous chip 100 and the lower casing 320 are firmly bonded by magnetic sheets at four vertices.
  • the biological device provided in Example 3 of the present invention can be used for single-cell capture, analysis, and intercellular material transfer in in vitro diagnosis, which improves the accuracy of single-cell analysis, thereby providing a new type of single-cell real-time monitoring. means.

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Abstract

本发明提供的双层微孔芯片，包括第一微孔结构层以及与所述第一微孔结构层连接的第二微孔结构层，第一微孔结构层包括若干个第一层微孔，第二微孔结构层包括若干个第二层微孔，第一层微孔的孔径以及所述第二层微孔的孔径均保持一致，第一层微孔的孔径及所述第二层微孔的孔径由小到大，且任意一个所述第二层微孔具有仅可容纳一个样本的形状以及尺寸。本发明提供的双层微孔芯片，第一层微孔的孔径及所述第二层微孔的孔径由小到大设计减少了层与层之间的压力差，防止了微孔芯片的堵塞，保证了高通量且通过第一层微孔的截留筛选，提高了筛选捕获的信噪比以及筛选能力。本发明还提供了上述双层微孔芯片制备方法以及包括上述双层微孔芯片的生物装置。

Description

一种双层微孔芯片、双层微孔芯片制备方法及生物装置 技术领域
本发明涉及医疗器械技术领域，特别涉及一种双层微孔芯片、双层微孔芯片制备方法及生物装置。
 背景技术
细胞异质性研究在人类疾病治疗中一直是一项重要的课题，举例来说，多能性干细胞虽可应用于疾病治疗和再生医学等领域，但由于其细胞功能上的异质性，让多能干细胞一直难以稳定的进行细胞分化。另在癌症研究中，肿瘤组群内一小群被称为【肿瘤干细胞】的细胞被认为是造成肿瘤对化疗药物产生抗药性的主要原因。如果能够精确的分析出每种细胞之间的异质性，将有助于简化复杂的生物现象，并提供疾病治疗新的方向。然而单细胞的分析不同于整个细胞群体，在实验工具上更加具有挑战性。传统的单细胞实验的进行往往依赖于有限稀释法（limiting dilution）或者流式细胞仪（FACS）来完成。
近年来，已经有诸多微流控芯片装置利用不同的原理进行单细胞的筛选以及分析，例如微液滴（microdroplets）、介电泳（dielectrophoresis）、流体动力学(hydrodynamics)以及微孔阵列（microwell array）等方式。对于需要进行单细胞培养的应用而言，微液滴细胞培养法能够快速地提供单细胞培养空间，此方法是投过油性介质的分离，将单细胞包裹在细胞培养液滴内进行培养。然而液滴内的细胞培养液在液滴形成之后就无法被置换了，此方法不适用于过程中需要更换培养液的细胞实验，且液滴中也缺乏能够让细胞附着的基质，故无法应用于贴附型细胞的培养。反而微孔设计的芯片能够应用于贴壁或者悬浮的细胞的筛选以及培养。此设计只需要在有孔洞状的结构中，而操作上只需要使用流体力学或者重力的方式就将细胞置入微孔内。然而为了让细胞拥有足够的生长空间，微孔的设计尺寸通常必须远大于一个单细胞的大小，导致了单细胞筛选效率普遍下降了10-30%。目前技术使用的单一微孔所造成的的单细胞捕获效率的降低与有限稀释法都是由于泊松分布的限制。
 技术问题
微孔的设计尺寸需要远大于一个单细胞的大小，导致单细胞的筛选效率大大降低，另外使用的单一微孔进行单细胞捕获的技术其芯片或者系统的流量无法得到精确控制，会导致单细胞筛选的过程中出现目标细胞的丢失导致筛选效率的降低。鉴于此，有必要提供一种单细胞的筛选效率高的双层微孔芯片。
 技术解决方案
第一方面，本发明提供了一种双层微孔芯片，包括：第一微孔结构层以及与所述第一微孔结构层连接的第二微孔结构层，所述第一微孔结构层包括若干个第一层微孔，所述第二微孔结构层包括若干个第二层微孔，所述第一层微孔的孔径以及所述第二层微孔的孔径均保持一致，所述第一层微孔的孔径及所述第二层微孔的孔径由小到大，且任意一个所述第二层微孔具有仅可容纳一个样本的形状以及尺寸。
在其中一些实施例中，所述第一微孔结构层与所述第二微孔结构层一体成型，所述第一微孔结构层的厚度为0.5-20um，所述第二微孔结构层的厚度为20-60um。
在其中一些实施例中，所述第一层微孔均匀间隔分布在所述第一微孔结构层上，所述第二层微孔均匀间隔分布在所述第二微孔结构层上。
在其中一些实施例中，所述第一微孔结构层以及所述第二微孔结构层中微孔的孔隙率为3-10%。
在其中一些实施例中，所述第一层微孔的孔径以及所述第二层微孔的孔径所在的竖直轴线处于同一水平线且横轴平行。
在其中一些实施例中，所述第一层微孔及所述第二层微孔为圆形或三角形或菱形。
在其中一些实施例中，所述第一层微孔及所述第二层微孔为圆形，所述第一层微孔的直径为0.2-20um，所述第二层微孔的直径为20-60um。
在其中一些实施例中，第一微孔结构层以及所述第二微孔结构层采用感光性透明高分子或者热固性透明高分子制成。
在其中一些实施例中，所述样本为生物体细胞或荧光微球。
在其中一些实施例中，所述第二层微孔的平面形状内切的最大圆的直径为容纳到所述样本的1-3倍的范围或优选1-2.4的范围或更优选1-1.4倍的范围。
在其中一些实施例中，所述第二层微孔的深度为要容纳为所述样本直径的1-3倍或优选1-2.4的范围或更优选1-1.6倍的范围。
在其中一些实施例中，当该捕获的样本为荧光微球时，并且所述荧光微球的直径为30um时，所述第二层微孔直径为30-35um，深度为30-55um。
第二方面，本发明提供了一种所述的双层微孔芯片的制备方法，包括下述步骤：
在衬底上形成一层金属种子层；
在所述金属种子层表面涂覆光刻胶，并在所述光刻胶上制作第一层光刻胶微孔层，得到所述第一微孔结构层，所述第一微孔结构层包括若干个第一层微孔；
在所述第一层光刻胶微孔层涂覆光刻胶，并在所述光刻胶上制作第二层光刻胶微孔层，得到所述第二微孔结构层，所述第二微孔结构层包括若干个第二层微孔；
将所述金属种子层进行剥离，得到所述双层微孔芯片；
所述第一层微孔的孔径以及所述第二层微孔的孔径均保持一致，所述第一层微孔的孔径及所述第二层微孔的孔径由小到大，且任意一个所述第二层微孔具有仅可容纳一个样本的形状以及尺寸。
在其中一些实施例中，所述的衬底为硅或氮化硅或氧化硅。
在其中一些实施例中，在衬底上形成一层金属种子层的步骤中，具体为：
利用磁控溅射在衬底表面溅射一层金属，所述金属为铬或镍或铝。
在其中一些实施例中，在所述金属种子层表面涂覆光刻胶，并在所述光刻胶上制作第一层光刻胶微孔层，得到所述第一微孔结构层的步骤中，具体为：
利用软光刻的工艺在所述金属种子层旋涂光刻胶，并在所述光刻胶上制作第一层光刻胶微孔层，得到所述第一微孔结构层。
在其中一些实施例中，利用软光刻的工艺在所述金属种子层旋涂光刻胶的步骤中具体包括：
在其中一些实施例中，所述的光刻胶包括SU8-3005或SU8-3025。
在其中一些实施例中，将所述金属种子层进行剥离，得到所述双层微孔芯片的步骤中，具体为：
最后利用金属清洗液对所述金属种子层进行剥离。
第三方面，本发明还提供了一种生物装置，包括上壳体、所述的双层微孔芯片以及下壳体，所述上壳体及所述下壳体固定安装，所述双层微孔芯片固定于所述上壳体及所述下壳体形成的空间内。
 有益效果
第一方面，本发明提供的双层微孔芯片，包括第一微孔结构层以及与所述第一微孔结构层连接的第二微孔结构层，所述第一微孔结构层包括若干个第一层微孔，所述第二微孔结构层包括若干个第二层微孔，所述第一层微孔的孔径以及所述第二层微孔的孔径均保持一致，所述第一层微孔的孔径及所述第二层微孔的孔径由小到大，且任意一个所述第二层微孔具有仅可容纳一个样本的形状以及尺寸，本发明提供的双层微孔芯片，所述第一层微孔的孔径及所述第二层微孔的孔径由小到大设计减少了层与层之间的压力差，防止了微孔芯片的堵塞，保证了高通量且通过第一层微孔的截留筛选，提高了筛选捕获的信噪比以及筛选能力。
第二方面，本发明提供的双层微孔芯片的制备方法，结合了软光刻复制技术以及膜转移技术，此方法简便、快速，适用于不同材质的微孔的制备。
第三方面，本发明提供了一种包括上述双层微孔芯片的生物装置，能够用于体外诊断的单细胞捕获、分析以及胞间物质传输等方面，提高了单细胞分析的准确性，从而为单细胞的实时监测提供了新型的手段。
 附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对本发明实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面所描述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为本发明实施例1提供的双层微孔芯片的结构示意图；
图2为本发明实施例1提供的双层微孔芯片内样本捕获的概略说明图。
图3为本发明实施例2提供的双层微孔芯片的制备方法的步骤流程图；
图4为本发明实施例2提供的双层微孔芯片的制备方法的原理示意图；
图5为本发明实施例3提供的生物装置的结构示意图。
 本发明的实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
在本发明的描述中，需要理解的是，术语“上”、“下”、“水平”、“内”、“外”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本发明的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
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。
实施例 1
请参阅图1，为本发明一实施方式提供的双层微孔芯片100的结构示意图，包括第一微孔结构层110以及与所述第一微孔结构层110连接的第二微孔结构层120。以下详细说明各个部件之间的连接关系。
所述第一微孔结构层110包括若干个第一层微孔111，所述第二微孔结构层120包括若干个第二层微孔121，所述第一层微孔111的孔径以及所述第二层微孔121的孔径均保持一致，所述第一层微孔111的孔径及所述第二层微孔121的孔径由小到大，且任意一个所述第二层微孔121具有仅可容纳一个样本的形状以及尺寸。
可以理解，由于所述第一层微孔111的孔径及所述第二层微孔的121孔径由小到大设计减少了层与层之间的压力差，防止了微孔芯片的堵塞，保证了高通量且通过第一层微孔111的截留筛选，提高了筛选捕获的信噪比以及筛选能力。
在其中一些实施例中，所述第一微孔结构层110与所述第二微孔结构层120一体成型，所述第一微孔结构层110的厚度为0.5-20um，所述第二微孔结构层120的厚度为20-60um。
可以理解，在第一微孔结构110的厚度为10um，第二微孔结构层的厚度为30um，当第一微孔结构层与第二微孔结构层的厚度为1:3的时候，单细胞或者磁珠的捕获效率较优。
具体地，所述第一微孔结构层110设置在所述第二微孔结构层120上的穿孔，所述第二微孔结构层120为设置在第一微孔结构层110上的穿孔。
在其中一些实施例中，所述第一层微孔111均匀间隔分布在所述第一微孔结构层110上，所述第二层微孔121均匀间隔分布在所述第二微孔结构层120上。
进一步地，所述第一微孔结构层110以及所述第二微孔结构层120中微孔的孔隙率为3-10%。
可以理解，当第一微孔结构层以及所述第二微孔结构层的孔隙率为5%的时候，单细胞或者磁珠的捕获效率较优。
在其中一些实施例中，所述第一层微孔111的孔径以及所述第二层微孔121的孔径所在的竖直轴线处于同一水平线且横轴平行。
在其中一些实施例中，所述第一层微孔111及所述第二层微孔121为圆形或三角形或菱形。
进一步地，当所述第一层微孔111及所述第二层微孔121为圆形时，所述第一层微孔111的直径为0.2-20um，所述第二层微孔121的直径为20-60um。
可以理解，在第一微孔结构110的直径为10um，第二微孔结构层的直径为30um，当第一微孔结构层与第二微孔结构层的厚度为1:3的时候，单细胞或者磁珠的捕获效率较优。
在其中一些实施例中，第一微孔结构层110以及所述第二微孔结构层120采用感光性透明高分子或者热固性透明高分子制成。
在其中一些实施例中，所述样本为生物体细胞或荧光微球。
在其中一些实施例中，所述第二层微孔121的平面形状内切的最大圆的直径为容纳到所述样本的1-3倍的范围或优选1-2.4的范围或更优选1-1.4倍的范围。
在其中一些实施例中，所述第二层微孔121的深度为要容纳为所述样本直径的1-3倍或优选1-2.4的范围或更优选1-1.6倍的范围。
在其中一些实施例中，当该捕获的样本为荧光微球时，并且所述荧光微球的直径为30um时，所述第二层微孔121直径为30-35um，深度为30-55um。
可以理解，在第一微孔结构110的直径为10um，第二微孔结构层的直径为30um，当第一微孔结构层与第二微孔结构层的厚度为1:3的时候，单细胞或者磁珠的捕获效率较优。
请参阅图2，为本发明实施例1提供的双层微孔芯片内样本捕获的概略说明图，其中圆球表示样本。
可以理解，在实际中微孔的尺寸应考虑要容纳在微孔中的生物样本的直径与微孔尺寸的恰当比来适当决定。
本发明上述实施例1提供的双层微孔芯片，包括第一微孔结构层以及与所述第一微孔结构层连接的第二微孔结构层，所述第一微孔结构层包括若干个第一层微孔，所述第二微孔结构层包括若干个第二层微孔，所述第一层微孔的孔径以及所述第二层微孔的孔径均保持一致，所述第一层微孔的孔径及所述第二层微孔的孔径由小到大，且任意一个所述第二层微孔具有仅可容纳一个样本的形状以及尺寸，本发明提供的双层微孔芯片，所述第一层微孔的孔径及所述第二层微孔的孔径由小到大设计减少了层与层之间的压力差，防止了微孔芯片的堵塞，保证了高通量且通过第一层微孔的截留筛选，提高了筛选捕获的信噪比以及筛选能力。
实施例 2
请参阅图3，为本发明提供了一种所述的双层微孔芯片的制备方法的步骤流程图，包括下述步骤：
步骤S110：在衬底上形成一层金属种子层。
在其中一些实施例中，所述的衬底为硅或氮化硅或氧化硅。
在其中一些实施例中，在衬底上形成一层金属种子层的步骤中，具体为：利用磁控溅射在衬底表面溅射一层金属，所述金属为铬或镍或铝。
步骤S120：在所述金属种子层表面涂覆光刻胶，并在所述光刻胶上制作第一层光刻胶微孔层，得到所述第一微孔结构层，所述第一微孔结构层包括若干个第一层微孔。
在其中一些实施例中，在所述金属种子层表面涂覆光刻胶，并在所述光刻胶上制作第一层光刻胶微孔层，得到所述第一微孔结构层的步骤中，具体为：利用软光刻的工艺在所述金属种子层旋涂光刻胶，并在所述光刻胶上制作第一层光刻胶微孔层，得到所述第一微孔结构层。
具体地，在其中一些实施例中，利用软光刻的工艺在所述金属种子层旋涂光刻胶的步骤中具体包括：先以低速旋转的方式，使光刻胶慢慢旋开至硅片的边缘；再通过高旋转的方式，将光刻胶旋涂至所述金属种子层边缘，所述的低速旋转的方式为转速500rpm旋胶10s，所述的高旋转的方式为以转速2500旋胶30s。
在其中一些实施例中，所述的光刻胶包括SU8-3005或SU8-3025。
可以连接，利用光刻技术在所述金属种子层旋涂光刻胶过程中，需要对所述金属种子层经过前烘、曝光、后烘、显影等几步工艺。
步骤S130：在所述第一层光刻胶微孔层涂覆光刻胶，并在所述光刻胶上制作第二层光刻胶微孔层，得到所述第二微孔结构层，所述第二微孔结构层包括若干个第二层微孔。
在本实施例中采用的具体技术方案可参考步骤S120中，这里不再赘述。
步骤S140：将所述金属种子层进行剥离，得到所述双层微孔芯片，所述第一层微孔的孔径以及所述第二层微孔的孔径均保持一致，所述第一层微孔的孔径及所述第二层微孔的孔径由小到大，且任意一个所述第二层微孔具有仅可容纳一个样本的形状以及尺寸。
在其中一些实施例中，将所述金属种子层进行剥离，得到所述双层微孔芯片的步骤中，具体为：最后利用金属清洗液对所述金属种子层进行剥离。
请再参阅图4，为本发明实施例2提供的双层微孔芯片的制备工艺原理图。
本发明提供的双层微孔芯片的制备方法，结合了软光刻复制技术以及膜转移技术，此方法简便、快速，适用于不同材质的微孔的制备。
实施例 3
请参阅图5，为本发明实施例3提供的一种生物装置的结构示意图，包括上壳体310、上述实施例1中所述的双层微孔芯片100以及下壳体320，所述上壳体310及所述下壳体320固定安装，所述双层微孔芯片100固定于所述上壳体310及所述下壳体320形成的空间内。
在其中一些实施例中，所述上壳体310、双层微孔芯片100以及下壳体320通过四个顶点的磁片进行牢固键合。
本发明实施例3中提供的生物装置，能够用于体外诊断的单细胞捕获、分析以及胞间物质传输等方面，提高了单细胞分析的准确性，从而为单细胞的实时监测提供了新型的手段。
以上仅为本发明的较佳实施例而已，仅具体描述了本发明的技术原理，这些描述只是为了解释本发明的原理，不能以任何方式解释为对本发明保护范围的限制。基于此处解释，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进，及本领域的技术人员不需要付出创造性的劳动即可联想到本发明的其他具体实施方式，均应包含在本发明的保护范围之内。

Claims (18)

  1. 一种双层微孔芯片，其特征在于，包括：第一微孔结构层以及与所述第一微孔结构层连接的第二微孔结构层，所述第一微孔结构层包括若干个第一层微孔，所述第二微孔结构层包括若干个第二层微孔，所述第一层微孔的孔径以及所述第二层微孔的孔径均保持一致，所述第一层微孔的孔径及所述第二层微孔的孔径由小到大，且任意一个所述第二层微孔具有仅可容纳一个样本的形状以及尺寸。
  2. 如权利要求1所述的双层微孔芯片，其特征在于，所述第一微孔结构层与所述第二微孔结构层一体成型，所述第一微孔结构层的厚度为0.5-20um，所述第二微孔结构层的厚度为20-60um。
  3. 如权利要求1所述的双层微孔芯片，其特征在于，所述第一层微孔均匀间隔分布在所述第一微孔结构层上，所述第二层微孔均匀间隔分布在所述第二微孔结构层上。
  4. 如权利要求3所述的双层微孔芯片，其特征在于，所述第一微孔结构层以及所述第二微孔结构层中微孔的孔隙率为3-10%。
  5. 如权利要求4所述的双层微孔芯片，其特征在于，所述第一层微孔的孔径以及所述第二层微孔的孔径所在的竖直轴线处于同一水平线且横轴平行。
  6. 如权利要求1所述的双层微孔芯片，其特征在于，所述第一层微孔及所述第二层微孔为圆形或三角形或菱形。
  7. 如权利要求6所述的双层微孔芯片，其特征在于，所述第一层微孔及所述第二层微孔为圆形，所述第一层微孔的直径为0.2-20um，所述第二层微孔的直径为20-60um。
  8. 如权利要求1所述的双层微孔芯片，其特征在于，第一微孔结构层以及所述第二微孔结构层采用感光性透明高分子或者热固性透明高分子制成。
  9. 如权利要求1所述的双层微孔芯片，其特征在于，所述样本为生物体细胞或荧光微球。
  10. 如权利要求9所述的双层微孔芯片，其特征在于，所述第二层微孔的平面形状内切的最大圆的直径为容纳到所述样本的1-3倍的范围或优选1-2.4的范围或更优选1-1.4倍的范围。
  11. 如权利要求9所述的双层微孔芯片，其特征在于，所述第二层微孔的深度为要容纳为所述样本直径的1-3倍或优选1-2.4的范围或更优选1-1.6倍的范围。
  12. 如权利要求9所述的双层微孔芯片，其特征在于，当该捕获的样本为荧光微球时，并且所述荧光微球的直径为30um时，所述第二层微孔直径为30-35um，深度为30-55um。
  13. 一种如权利要求1至12任一项所述的双层微孔芯片的制备方法，其特征在于，包括下述步骤：
    在衬底上形成一层金属种子层；
    在所述金属种子层表面涂覆光刻胶，并在所述光刻胶上制作第一层光刻胶微孔层，得到所述第一微孔结构层，所述第一微孔结构层包括若干个第一层微孔；
    在所述第一层光刻胶微孔层涂覆光刻胶，并在所述光刻胶上制作第二层光刻胶微孔层，得到所述第二微孔结构层，所述第二微孔结构层包括若干个第二层微孔；
    将所述金属种子层进行剥离，得到所述双层微孔芯片；
    所述第一层微孔的孔径以及所述第二层微孔的孔径均保持一致，所述第一层微孔的孔径及所述第二层微孔的孔径由小到大，且任意一个所述第二层微孔具有仅可容纳一个样本的形状以及尺寸。
  14. 如权利要求13所述的双层微孔芯片的制备方法，其特征在于，所述的衬底为硅或氮化硅或氧化硅。
  15. 如权利要求13所述的双层微孔芯片的制备方法，其特征在于，在衬底上形成一层金属种子层的步骤中，具体为：
    利用磁控溅射在衬底表面溅射一层金属，所述金属为铬或镍或铝。
  16. 如权利要求13所述的双层微孔芯片的制备方法，其特征在于，在所述金属种子层表面涂覆光刻胶，并在所述光刻胶上制作第一层光刻胶微孔层，得到所述第一微孔结构层的步骤中，具体为：
    利用软光刻的工艺在所述金属种子层旋涂光刻胶，并在所述光刻胶上制作第一层光刻胶微孔层，得到所述第一微孔结构层。
  17. 如权利要求16所述的双层微孔芯片的制备方法，其特征在于，所述的光刻胶包括SU8-3005或SU8-3025。
    18如权利要求13所述的双层微孔芯片的制备方法，其特征在于，将所述金属种子层进行剥离，得到所述双层微孔芯片的步骤中，具体为：
    最后利用金属清洗液对所述金属种子层进行剥离。
  18. 一种生物装置，其特征在于，包括上壳体、权利要求1至13任一项所述的双层微孔芯片以及下壳体，所述上壳体及所述下壳体固定安装，所述双层微孔芯片固定于所述上壳体及所述下壳体形成的空间内。
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