WO2022000641A1 - 超疏水微坑阵列芯片及其制备方法和装置 - Google Patents

超疏水微坑阵列芯片及其制备方法和装置 Download PDF

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WO2022000641A1
WO2022000641A1 PCT/CN2020/104470 CN2020104470W WO2022000641A1 WO 2022000641 A1 WO2022000641 A1 WO 2022000641A1 CN 2020104470 W CN2020104470 W CN 2020104470W WO 2022000641 A1 WO2022000641 A1 WO 2022000641A1
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micro
super
chip
hydrophobic
superhydrophobic
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French (fr)
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刘鹏
胡亚伟
陈忠尧
刘畅
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清华大学
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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
    • B01L3/502707—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 the manufacture of the container or its components
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D4/00—Coating compositions, e.g. paints, varnishes or lacquers, based on organic non-macromolecular compounds having at least one polymerisable carbon-to-carbon unsaturated bond ; Coating compositions, based on monomers of macromolecular compounds of groups C09D183/00 - C09D183/16
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D7/00—Features of coating compositions, not provided for in group C09D5/00; Processes for incorporating ingredients in coating compositions
    • C09D7/40—Additives
    • C09D7/60—Additives non-macromolecular
    • C09D7/61—Additives non-macromolecular inorganic

Definitions

  • the invention relates to the technical field of cytology, in particular to a superhydrophobic micropit array chip and a preparation method and device thereof.
  • microfluidic chips are microchannels and micropit arrays.
  • the microfluidic chip of micropit array is widely used in high-throughput drug screening, gene function analysis, cell transfection due to its advantages of high throughput, high integration of experimental operations, and saving a lot of experimental reagent consumption and labor consumption. and stem cell culture microenvironment.
  • the main problem in using the micro-pit array chip is how to avoid cross-talk between the individual micro-pits, thereby resulting in cross-contamination.
  • the researchers made a layer of super-hydrophobic material between each micro-pit and the interior of the micro-pit is hydrophilic, thereby avoiding cross-contamination between different micro-pits.
  • the superhydrophobic layer on the current superhydrophobic microarray chips (SMAR-chips) based on PDMS and glass substrates is formed by UV crosslinking of pre-prepared superhydrophobic prepolymers.
  • the processing process of the PDMS-based SMAR-chip is to inject PDMS material into the micro-pit mold to form a micro-array chip, and then use 3140 glue to graft the prepared superhydrophobic layer attached to the glass slide to the surface of the PDMS micro-pit chip and let it stand.
  • the processing of the chip is completed after 6-8h is uncovered.
  • Glass-based SMAR-chips require auxiliary equipment such as fixtures, PMMA micro-pit molds, and silanized-modified glass slides.
  • the processing process of the two substrates is rather cumbersome and each use needs to be combined with a suitable size petri dish, that is, the use of 3140 glue
  • the SMAR-chip is glued to the bottom of the petri dish, and the chip and petri dish processed under non-sterile conditions need to be sterilized before use.
  • the material of the common petri dish is made of polystyrene, which becomes soft when the temperature is above 80°C, so it cannot be autoclaved. You can choose to sterilize it with gamma rays or immerse it in 75% ethanol after bonding with the chip. Sterilization, which not only increases the risk of contamination, but also increases the tediousness of preparing the chip before use.
  • the present invention provides a super-hydrophobic micro-pit array chip, which is prepared by the following method, and the method includes the following steps: Step 1: forming a micro-pit array and a super-hydrophobic coating by integral injection molding A chip with a layer pool, the diameter of the micropits of the micropit array is 300-1500 ⁇ m, the depth of the micropits is less than 500 ⁇ m and the distance between the micropits is 1500-3000 ⁇ m; and the depth of the superhydrophobic coating pool is greater than 50 ⁇ m, the The wall thickness of the micro-pit between the coating pool and the micro-pit is 50-300 ⁇ m; and, step 2: adding the super-hydrophobic coating into the super-hydrophobic coating pool, and the super-hydrophobic coating solution is volatilized in the micro-pit.
  • a superhydrophobic layer is formed between the micropits of the pit array.
  • the superhydrophobic layer refers to a hydrophobic layer whose surface has a contact angle with water or an aqueous solution greater than 150° and a rolling angle of less than 10°.
  • the superhydrophobic layer makes each micropit Effective physical isolation between medium and aqueous solutions.
  • the superhydrophobic coating solution is a suspension comprising 1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane, titanium oxide nanoparticles and P25 titanium oxide mixed uniformly.
  • the superhydrophobic coating solution is prepared by mixing 1H,1H,2H,2H-perfluorooctyltriethoxysilane with absolute ethanol under mechanical stirring, and then adding Titanium oxide nanoparticles and P25 titanium oxide are mixed and dispersed by ultrasonic to prepare a suspension.
  • the present invention provides an integrated super-hydrophobic micro-pit array chip device, the device comprises the above-mentioned super-hydrophobic micro-pit array chip, a substrate and a water tank, and the super-hydrophobic micro-pit array chip and the water tank are arranged on the Above the substrate, the water tank surrounds the periphery of the chip.
  • the device further includes an outer wall and a rail extending from the base and forming the sink therebetween.
  • the device further comprises a substrate base of a superhydrophobic micropit array chip, and a plurality of superhydrophobic micropit array chips with outer walls are embedded in the substrate base, so as to be assembled into a base comprising a plurality of superhydrophobic micropits A chip array of array chips.
  • the device further includes a cover, the outer wall is made into a square shape compatible with the micro-pit array chip, and the chamfered shape of the four corners facilitates the cooperation of the outer wall and the cover to form a closed space.
  • the chip is made of PMMA or PC material, preferably PC 2458.
  • the present invention provides a method for preparing a superhydrophobic micropit array chip, the method comprising the following steps: Step 1: forming a chip including a micropit array and a superhydrophobic coating pool by integral injection molding, so that the The micro-pit diameter of the micro-pit array is 300-1500 ⁇ m, the micro-pit depth is less than 500 ⁇ m and the micro-pit spacing is 1500-3000 ⁇ m; and the depth of the super-hydrophobic coating pool is greater than 50 ⁇ m, the coating pool and the The wall thickness of the micro-pits between the micro-pits is 50-300 ⁇ m; and, step 2: adding the super-hydrophobic coating into the super-hydrophobic coating pool, and the super-hydrophobic coating solution is volatilized between the micro-pits of the micro-pit array.
  • a super-hydrophobic layer is formed between the two, and the super-hydrophobic layer refers to a hydrophobic layer whose surface has a contact angle with water or an aqueous solution greater than 150° and a rolling angle of less than 10°. Physical isolation.
  • the invention provides a super-hydrophobic micro-pit array chip and a preparation method thereof.
  • the novel super-hydrophobic coating in the invention can be used to modify the surface of glass, PMMA, PC and fabric materials, and has super-hydrophobic performance.
  • the invention designs and injects an integrated micro-pit array device based on PC material.
  • the device integrates a micro-pit array chip and a petri dish, and injects super-hydrophobic paint into the gaps between the micro-pit arrays so that the micro-pit array is formed between the micro-pits.
  • a superhydrophobic layer with a thickness of about 100 ⁇ m was formed on the PC material substrate in between. After autoclaving, 2D/3D of cells and 3D culture of tumor organoids and physiological and biochemical analysis can be performed on the integrated chip.
  • the super-hydrophobic micro-pit array chip is a PC-based material, comprising a micro-pit array and a micro-cell culture chip with a super-hydrophobic surface layer between the micro-pits.
  • the arrangement, size and depth of the micro-pit arrays in the chip can be adjusted, and complete isolation between the micro-pit arrays can be achieved, thereby avoiding cross-contamination between the micro-pits and maintaining good biocompatibility.
  • a dedicated chip substrate base multiple chips can be assembled on it and used in parallel, and the chips are completely isolated from each other. Therefore, the chip can be used for 2D cell culture and 3D tumor organoid culture, as well as biological analysis such as in vitro high-throughput drug screening and drug sensitivity detection.
  • microfluidic chip materials include: siliceous materials, glass and quartz and other polymer materials (such as polymethyl methacrylate PMMA, polycarbonate PC, polydimethylsiloxane PDMS) and the like. Polymers are the most commonly used materials for microfluidic chips.
  • microfluidic chip materials include: Polymethyl methacrylate (PMMA), Polycarbonate (PC), Polydimethylsiloxane (Polydimethylsiloxane, PDMS) etc.
  • PMMA Polymethyl methacrylate
  • PC Polycarbonate
  • PDMS Polydimethylsiloxane
  • These high molecular polymer materials have good biocompatibility and chemical inertness, good optical transparency and insulation, and are easy to process and shape. The most important thing is that it has low cost and simple and mature processing technology, which is very suitable for laboratory research and industrial production. It is a rare and excellent material for microfluidic chips.
  • the present invention designs and injects a new type of chip that integrates a microarray chip and a petri dish.
  • the chip material is PC2458, which is an amorphous thermoplastic resin, which can be used for including medical instruments and equipment. Among them, the material has good impact resistance, dimensional stability and thermal distortion resistance, with a melt index of 300°C and a heat distortion temperature of 1.80Mpa, which can withstand the high pressure sterilization conditions in biological experiments (103.4Kpa, 121.3°C).
  • the material has good physical properties. According to the test method of DIN 5036-1, its transparency rate is 4mm. It has the advantages of self-extinguishing, flame retardancy and non-toxicity. It can satisfy cell culture in biological experiments and ordinary bright field imaging. and fluorescence imaging.
  • FIG. 1 is a schematic diagram of the contact angle formed by the surface droplets of the novel superhydrophobic material of the present invention
  • FIG. 2 is a schematic diagram of an integrated superhydrophobic micropit array chip device of the present invention
  • Fig. 3 is the top/side view of the dish of the integrated superhydrophobic micropit array chip device of the present invention.
  • FIG. 4 is a top/side view of the cover of the integrated superhydrophobic micropit array chip device of the present invention.
  • Fig. 5 is the processing flow chart of the integrated super-hydrophobic micropit array chip of the present invention.
  • FIG. 6 is a schematic structural diagram of a superhydrophobic microarray chip in the integrated superhydrophobic micropit array chip device of the present invention.
  • FIG. 7 is a schematic cross-sectional view of a superhydrophobic microarray chip in the integrated superhydrophobic micropit array chip device of the present invention.
  • Figure 8 is a top/side view of a superhydrophobic micropit array chip fitted with a substrate base
  • Figure 9 is a top/side view of a substrate base for superhydrophobic micropit array chip embedding assembly
  • Figure 10 is a 3D modeling rendering of 21 superhydrophobic microarray chips assembled on a substrate base
  • Fig. 11 is the result diagram of the microdroplet array automatically formed after the PBS liquid is drawn off on the integrated superhydrophobic micropit array chip of the present invention
  • Fig. 12 is the result diagram of the microdroplet array automatically formed after the culture medium is extracted from the integrated superhydrophobic micropit array chip of the present invention.
  • Figure 13 is a graph of the results of a superhydrophobic microarray chip incorporating Matrigel.
  • Fig. 14 is a graph showing the result of superhydrophobic microarray chip added with culture medium.
  • a similar method was used to adjust the ratio of titanium oxide nanoparticles and titanium oxide in the new superhydrophobic material solution to obtain superhydrophobic layers with a contact angle of 150°C, a rolling angle of 10°, 175°, and a rolling angle of 6°.
  • Figure 2 is a schematic diagram of the integrated superhydrophobic micropit array chip device.
  • the fence of the device is made into a square shape that is compatible with the micropit array chip, and the shape of the four corners is chamfered to facilitate the combination of the device cover and the dish.
  • Figure 3 shows the top view and side view of the dish in the device, and the dish size is 37x52x10, in mm.
  • Figure 4 shows the top and side views of the cover in the device, the cover size is 40x55x7 in mm.
  • FIG 6 is a schematic diagram of the structure of the superhydrophobic chip in the integrated superhydrophobic micropit array chip device.
  • the superhydrophobic layer in the figure is the coating formed after the paint in the superhydrophobic coating pool 4 is evaporated, and has high hydrophobicity. , which can effectively isolate the micro-pits.
  • the inner diameter of the micropits is 1.37mm
  • the spacing between the micropits is 2.25mm, which is consistent with the well spacing of the 1536-well plate, and is compatible with detection instruments such as commercial fluorescence scanners.
  • Figure 7 is a schematic diagram of the cross-sectional structure of the superhydrophobic chip in the mold. The yellow color represents the superhydrophobic layer, the depth of the micropit is 300 ⁇ m, the depth of the superhydrophobic coating pool is 100 ⁇ m, and the wall thickness of the micropit between the coating pool and the micropit is 0.19mm.
  • the petri dish and the micro-pit array chip are integrated to form an integrated micro-pit array chip device, and the novel super-hydrophobic coating is organically combined with the device to develop an integrated new type of super-hydrophobic micro-array chip.
  • IN-SMAR-chip The components of IN-SMAR-chip are: PC material micropit array device and new super-hydrophobic coating.
  • the device is divided into a cover and a dish, the length x width x height of the cover are 55mm x 40mm x 7mm, the top wall thickness of the cover is 1mm, and the thickness of the side wall of the cover is 0.79mm.
  • the length x width x height of the dish is 52mm x 37mm x 10mm, the thickness of the bottom of the dish is 1mm, and the thickness of the side wall of the dish is 0.84mm.
  • the number of micro-pits on the chip is adjustable.
  • the injection-molded chip has 108 holes, the chip size is 23.11mm x 29.61mm, and the hole diameter, depth and hole spacing are respectively 1.37 mm (0.3-1.5mm), 300 ⁇ m (0-500) and 2.25mm (1.5-3mm).
  • the base of the substrate in the device is shown in Figure 9, its length x width x height are 127.63mm x 85.11mm x 14.30mm, and the thickness of the side wall is 0.8mm. compatible.
  • the number of chips that can be accommodated in the substrate base is adjustable.
  • the substrate base has 21 chip slots, and the length x width x height of each slot is 27.04mm x 17.09mm x 2mm, respectively.
  • the wall thickness in between is 1mm.
  • Figure 10 is a 3D modeling rendering of 21 superhydrophobic microarray chips assembled on a substrate base.
  • the embedded superhydrophobic microarray chip in the device is shown in Figure 8, its length x width x height are 25mm x 17.09mm x 8mm, and the sidewall thicknesses are 1mm and 1.3mm, respectively.
  • the number and size of the micro-pits on the chip are adjustable.
  • the chip has 96 micro-pits, the diameter of the micro-pits is 0.8 mm, and the distance between the micro-pits is 1.5 mm.
  • the depth of the superhydrophobic coating pool is 0.1 mm, and the wall thickness of the micro-pit between the coating pool and the micro-pit is 0.19 mm.
  • FIG. 11 is the PBS microdroplet array formed on the chip after extraction
  • Figure 12 is the medium microdroplet array formed on the chip after extraction, which proves that the superhydrophobic and hydrophobic micropit array chip has good superhydrophobicity performance.
  • the 3D culture on the chip is to wrap the cells or tumor organoids in Matrigel, and inoculate them into the micro-wells of the chip through an electric pipette. 600nL Matrigel was added, and after 30min solidification, a relatively uniform glue droplet array was formed in the micro-pit of the chip.
  • adding 2 ⁇ L of medium to each microwell can perform droplet 3D culture of cells or tumor organoids in the chip. By adding an equal volume of medium, a uniform droplet array can be formed. In-pit conditions can be independently controlled, so samples in each micro-pit can be analyzed differently without crosstalk. To avoid evaporation of the liquid in the microwell array, a certain volume of sterile water can be added to the sterile water tank.

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Abstract

一种超疏水微坑阵列芯片，所述芯片通过以下方法制备：通过一体注塑形成包括微坑阵列（5）和超疏水涂层池（4）的芯片，将超疏水涂料加入到超疏水涂层池中，所述超疏水涂料溶液挥发后在所述微坑阵列的微坑之间形成超疏水层，所述超疏水层是指其表面与水或水溶液的接触角大于150°、滚动角小于10°的疏水层，所述超疏水层使得各个微坑中水溶液之间有效的物理隔绝。

Description

超疏水微坑阵列芯片及其制备方法和装置 技术领域
本发明涉及细胞学技术领域，特别涉及超疏水微坑阵列芯片及其制备方法和装置。
背景技术
目前，微流控芯片的主要形式有微管道和微坑阵列。其中微坑阵列的微流控芯片以其通量高、实验操作集成化高以及能够节省大量的实验试剂消耗和人力消耗等优势被广泛应用于高通量药物筛选、基因功能分析、细胞转染和干细胞培养微环境等研究。
使用微坑阵列芯片存在的主要问题是如何避免各个微坑之间发生相互串扰，进而产生交叉污染。研究者通过对微坑芯片进行表面修饰，使各个微坑之间存在一层超疏水材料而微坑内部亲水，从而避免了不同微坑之间发生交叉污染。
目前基于PDMS和玻璃基底的超疏水微阵列芯片(SMAR-芯片)上的超疏水层是由提前配制好的超疏水预聚物通过紫外交联形成。其中PDMS基底的SMAR-芯片的加工过程是将PDMS材料注入微坑模具已形成微阵列芯片，再使用3140胶将制备好的附在玻片上的超疏水层嫁接到PDMS微坑芯片表面并静置6-8h揭开后完成芯片的加工。玻璃基底的SMAR-芯片的加工过程中需要夹具、PMMA微坑模具和经硅烷化修饰的玻片等辅助设备，使用夹具将玻片与微坑模具加紧，并向空隙中注入超疏水预聚物溶液，若夹具拧的过松会导致超疏水预聚物流到模具微柱与玻片的接触面，导致曝光后会在芯片的微坑底部形成薄薄一层超疏水层，从而影响对微坑中培养物的成像；若夹具拧的太紧，存在玻片被压碎和PMMA模具被压变形的风险。因此使得PDMS基底和玻片基底的SMAR-芯片的加工引入了许多人为因素。
尽管以上两种基底的SMAR-芯片的生物相容性以及2D和3D培养的性能良好，但两者的加工过程较为繁琐且每次使用时需配合合适尺寸的培养皿组 合使用，即使用3140胶将SMAR-芯片粘至培养皿底部，且非无菌条件加工的芯片与培养皿组合后需要进行灭菌后方可使用。普通培养皿的材质是由聚苯乙烯组成，在80℃以上则会变软，因而无法进行高压灭菌，可选择用伽马射线进行灭菌或与芯片粘合后共同浸没在75％乙醇中灭菌，这样既增加了污染的风险，也使芯片使用前的准备工作繁琐度增加。
发明内容
为了解决上述技术问题，本发明提供一种一种超疏水微坑阵列芯片，所述芯片通过以下方法制备，所述方法包括以下步骤：步骤1：通过一体注塑形成包括微坑阵列和超疏水涂层池的芯片，所述微坑阵列的微坑直径在300-1500μm，微坑深度为小于500μm和微坑间距为1500-3000μm；和所述超疏水涂层池的深度为大于50μm，所述涂层池与所述微坑之间的微坑壁厚为50-300μm；和，步骤2：将超疏水涂料加入到超疏水涂层池中，所述超疏水涂料溶液挥发后在所述微坑阵列的微坑之间形成超疏水层，所述超疏水层是指其表面与水或水溶液的接触角大于150°、滚动角小于10°的疏水层，所述超疏水层使得各个微坑中水溶液之间有效的物理隔绝。
在一种实施方式中，所述超疏水涂料溶液是括1H,1H,2H,2H-全氟辛基三乙氧基硅烷、氧化钛纳米粒子和P25氧化钛混匀的悬浮液。
在一种实施方式中，所述超疏水涂料溶液通过以下方法制备，将1H,1H,2H,2H-全氟辛基三乙氧基硅烷与无水乙醇在机械力的搅拌下混合，再加入氧化钛纳米粒子和P25氧化钛混匀并超声分散后制备成悬浮液。
在一种实施方式中，本发明提供一种集成超疏水微坑阵列芯片装置，所述装置包括上述的超疏水微坑阵列芯片、基底和水槽，所述超疏水微坑阵列芯片和水槽设置在所述基底之上，所述水槽围绕在所述芯片外周。
在一种实施方式中，所述装置还包括外壁和围栏，所述装置的外壁和围栏从所述基底伸出并且在它们之间形成所述水槽。
在一种实施方式中，所述装置还包括超疏水微坑阵列芯片的基板底座，将多个带外壁的超疏水微坑阵列芯片嵌入所述基板底座，从而组装成包含多个超疏水微坑阵列芯片的芯片阵列。
在一种实施方式中，所述装置还包括盖，所述外壁制作成能兼容微坑阵列芯片的方形，四角倒角形状利于所述外壁与所述盖的配合，形成封闭空间。
在一种实施方式中，所述芯片是PMMA或PC材质，优选地是PC 2458。
在一种实施方式中，本发明提供一种超疏水微坑阵列芯片的制备方法，所述方法包括以下步骤：步骤1：通过一体注塑形成包括微坑阵列和超疏水涂层池的芯片，所述微坑阵列的微坑直径在300-1500μm，微坑深度为小于500μm和微坑间距为1500-3000μm；和所述超疏水涂层池的深度为大于50μm，所述涂层池与所述微坑之间的微坑壁厚为50-300μm；和，步骤2：将超疏水涂料加入到超疏水涂层池中，所述超疏水涂料溶液挥发后在所述微坑阵列的微坑之间形成超疏水层，所述超疏水层是指其表面与水或水溶液的接触角大于150°、滚动角小于10°的疏水层，所述超疏水层使得各个微坑中水溶液之间有效的物理隔绝。
本发明提出了一种超疏水微坑阵列芯片及其制备方法，本发明中新型超疏水涂料可用于修饰玻璃、PMMA、PC以及布艺材料表面，具有超疏水性能。本发明设计并注塑了一款基于PC材质的集成式微坑阵列装置，该装置是将微坑阵列芯片与培养皿集成化，并将超疏水涂料注入微坑阵列间的空隙中以在微坑之间的PC材料基底上形成厚度约100μm的超疏水层。通过高压灭菌后，可在该集成芯片上细胞的2D/3D以及肿瘤类器官的3D式培养以及生理生化分析。
在本发明一种实施方式中，超疏水微坑阵列芯片是一种基于PC材质，包括微坑阵列以及微坑之间为超疏水表层的微型细胞培养芯片。芯片中微坑阵列的排列方式、微坑尺寸、深度等均可调节，并可以实现微坑阵列间的完全隔离继而能够避免微坑之间的交叉污染并保持很好的生物相容性。此外，通过结合专用的芯片基板底座可以将多块芯片组装于其上并行使用，芯片与芯片之间相互独立完全隔离。因此该芯片可用于2D细胞培养以及3D肿瘤类器官的培养以及体外高通量药物筛选和药敏检测等生物分析。
在选择用于加工微流控芯片材料方面，不仅需要充分考虑材料的导热性、透光性、耐热性、加工工艺的精度和难度、经济成本以及对芯片复杂结构稳定性维持方面的问题，还要考虑到涉及生化反应过程中，不同材料本身的生物相容性和抗化学试剂的腐蚀性等方面的差异。目前常用的微流控芯片材料有：硅质材料、玻璃和石英等高分子聚合材料(如聚甲基丙烯酸甲酯PMMA，聚碳酯PC，聚二甲基硅氧烷PDMS)等。高分子聚合物是目前微流控芯片最常用的材料。常见用于微流控芯片材料的高分子聚合物包括：聚甲基丙稀酸甲酯(Poly methyl methacrylate,PMMA)，聚碳酸酯(Polycarbonate， PC)，聚二甲基硅氧烷(Polydimethylsiloxane，PDMS)等。这些高分子聚合物材料具有良好的生物相容性及化学惰性，光学透明和绝缘性好，便于加工成型。最重要的是，其成本低廉，加工工艺简单成熟，非常适合实验室研究和工业化生产，是做为微流控芯片不可多得的优良材料。
在本发明一种实施方式中，本发明设计并注塑了一款微阵列芯片与培养皿集成化的新型芯片，该芯片材料为PC2458，是一种非晶型热塑性树脂，可用于包括医疗仪器设备中，该材料具有良好的抗冲力、尺寸稳定性和抗热畸变性能，其中熔融指数为300℃，热变形温度压力为1.80Mpa，可以耐受住生物实验中的高压灭菌条件(103.4Kpa，121.3℃)。该材料具有较好的物理性能，根据DIN 5036-1的测试方法，其透明率为4mm，具有自熄、阻燃和无毒等优点，能够满足于生物实验中的细胞培养以及普通明场成像和荧光成像等。
附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例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申请中记载的一些实施例，对于本领域普通技术人员来说，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它的附图。
图1是本发明的新型超疏水材料表面液滴形成的接触角示意图；
图2是本发明集成超疏水微坑阵列芯片装置示意图；
图3是本发明集成超疏水微坑阵列芯片装置的皿的顶/侧视图；
图4是本发明集成超疏水微坑阵列芯片装置的盖的顶/侧视图；
图5是本发明集成超疏水微坑阵列芯片的加工流程图；
图6是本发明集成超疏水微坑阵列芯片装置中超疏水微阵列芯片的结构示意图；
图7是本发明集成超疏水微坑阵列芯片装置中超疏水微阵列芯片的剖面示意图；
图8是适配基板底座的超疏水微坑阵列芯片的顶/侧视图；
图9是用于超疏水微坑阵列芯片嵌入组装的基板底座的顶/侧视图；
图10是21个超疏水微阵列芯片组装于基板底座上的3D建模效果图；
图11是本发明集成超疏水微坑阵列芯片上抽走PBS液体后自动形成的微液滴阵列结果图；
图12是本发明集成超疏水微坑阵列芯片上抽走培养基后自动形成的微液滴阵列结果图；
图13是加入了Matrigel的超疏水微阵列芯片结果图；和
图14是加入了培养基的超疏水微阵列芯片结果图。
具体实施方式
为了使本领域技术领域人员更好地理解本申请中的技术方案，下面将结合实施例对本发明作进一步说明，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都应当属于本申请保护的范围。
一.新型超疏水涂料的合成
将1g 1H,1H,2H,2H-全氟辛基三乙氧基硅烷与99g无水乙醇在机械力的搅拌下混合2h,再加入6g氧化钛纳米粒子和6g P25氧化钛混匀并超声分散30min后就制备成约30mL悬浮液，即新型超疏水材料溶液。该溶液可在PMMA、培养皿以及PC等各种材料表面形成涂层，涂层中的酒精仅需约2min就可蒸发完并形成一层超疏水层。经过测试，如图1所示，液滴在涂层上形成的接触角为164.4°；其滚动角为8°。采用类似方法，调整新型超疏水材料溶液中氧化钛纳米粒子和氧化钛的比例，得到接触角150℃、滚动角10°，175°、滚动角6°的超疏水层。
二.集成超疏水微坑阵列芯片装置的结构与功能说明
如图2所示是集成超疏水微坑阵列芯片装置示意图，将装置的围栏制作成能兼容微坑阵列芯片的方形，四角倒角形状利于装置盖与皿的组合。如图3所示是装置中皿的顶视图和侧视图，皿尺寸为37x52x10，单位mm。图中：1、皿壁，2、无菌水槽，3、围栏，4、超疏水涂层池，5、微坑。如图4所示是装置中盖的顶视图和侧视图，盖尺寸为40x55x7，单位mm。
三.超疏疏水微坑阵列芯片的加工与结构说明
使用移液枪吸取新配制的新型超疏水涂料，注入集成超疏水微坑阵列芯片装置上的超疏水涂层池4中，待涂料中的酒精蒸发，就完成了一张IN-SMAR芯片的加工，如图5所示是芯片的加工过程，(左)为集成超疏水微坑阵列芯片装置，(中)正在注入新型超疏水涂料，(右)集成式新型超疏水芯片。
如图6所示是集成超疏水微坑阵列芯片装置中超疏水芯片的结构示意图，图中的超疏水层是超疏水涂层池4中的涂料蒸发之后形成的涂层，具有较高的疏水性，可将微坑之间进行有效的物理隔绝。微坑的内径为1.37mm，微坑间距为2.25mm，与1536孔板的孔间距保持一致，可与商用的荧光扫描仪等检测仪器兼容。如图7所示是模具中超疏水芯片的剖面结构示意图，黄色代表超疏水层，微坑深度为300μm，超疏水涂层池深度为100μm，涂层池与微坑之间的微坑壁厚为0.19mm。
本发明中将培养皿、微坑阵列芯片集成化形成一款集成式微坑阵列芯片装置，再将新型超疏水涂料与装置进行有机的结合，进而开发出一款集成式新型超疏水微阵列芯片。
IN-SMAR-chip的组成分别是：PC材质的微坑阵列装置、新型超疏水涂料。
装置参数说明：装置分为盖和皿，盖的长x宽x高分别为55mm x 40mm x 7mm，盖顶部壁厚1mm，盖侧壁厚0.79mm。皿的长x宽x高分别为52mm x 37mm x 10mm，皿底部厚度1mm，皿侧壁厚度0.84mm。
装置中微坑阵列芯片参数说明：芯片上微坑数量可调，在一种实施方式中，注塑的芯片有108孔，芯片尺寸为23.11mm x 29.61mm，孔直径、深度及孔间距分别为1.37mm(0.3-1.5mm)、300μm(0-500)和2.25mm(1.5-3mm)。
基于基板底座的嵌入式芯片套组的装置参数说明：
装置中基板底座如图9，其长x宽x高分别为127.63mm x 85.11mm x 14.30mm，侧壁厚度0.8mm，其平面尺寸与CORNING 96孔板尺寸一致，可与部分仪器的适配尺寸兼容。基板底座的可容纳芯片数量可调，在图9的实施方式中，基板底座带有21个芯片槽位，每个槽位的长x宽x高分别为27.04mm x 17.09mm x 2mm，槽位之间的壁厚1mm。图10是21个超疏水微阵列芯片组装于基板底座上的3D建模效果图。
装置中的嵌入式超疏水微阵列芯片如图8，其长x宽x高分别为25mm x 17.09mm x 8mm，侧壁厚分别为1mm和1.3mm。芯片上的微坑数量和尺寸可调，在图8的实施方式中，芯片有96个微坑，微坑直径为0.8mm，微坑间距为1.5mm。超疏水涂层池深度为0.1mm，涂层池与微坑之间的微坑壁厚为0.19mm。
四.超疏疏水微坑阵列芯片的超疏水性能验证
向加工好的超疏疏水微坑阵列芯片中分别加入5mL的PBS或培养基浸没芯片，静置5min，将液体抽走后会在芯片表面自动形成体积较均一的微液滴阵列。如图11是抽走后在芯片上形成的PBS微液滴阵列，如图12是抽走后在芯片上形成的培养基微液滴阵列，证明了超疏疏水微坑阵列芯片良好的超疏水性能。
五.超疏疏水微坑阵列芯片用于3D培养体系的验证
芯片上的3D培养是将细胞或肿瘤类器官包裹入基质胶(Matrigel)中，通过电动移液枪接种至芯片微坑中，如图13所示是接种了Matrigel的芯片，每个微坑中加入600nL Matrigel，待30min凝固后即在芯片微坑中形成较均一的胶滴阵列。如图14，向每个微坑中补加2μL培养基可对芯片中的细胞或肿瘤类器官进行液滴式3D培养，通过加入等体积的培养基，可形成均一的液滴阵列，由于微坑中条件可独立控制，因此可以对每个微坑中的样本进行不同的分析而不会发生串扰。为了避免微坑阵列中的液体蒸发，可在无菌水槽中加入一定体积的无菌水。
应该理解到披露的本发明不仅仅限于描述的特定的方法、方案和物质，因为这些均可变化。还应理解这里所用的术语仅仅是为了描述特定的实施方式方案的目的，而不是意欲限制本发明的范围，本发明的范围仅受限于所附的权利要求。
本领域的技术人员还将认识到，或者能够确认使用不超过常规实验，在本文中所述的本发明的具体的实施方案的许多等价物。这些等价物也包含在所附的权利要求中。

Claims (10)

  1. 一种超疏水微坑阵列芯片，其特征在于，所述芯片通过以下方法制备，所述方法包括以下步骤：
    步骤1：通过一体注塑形成包括微坑阵列和超疏水涂层池的芯片，所述微坑阵列的微坑直径在300-1500μm，微坑深度为小于500μm和微坑间距为1500-3000μm；和所述超疏水涂层池的深度为大于50μm，所述涂层池与所述微坑之间的微坑壁厚为50-300μm；和
    步骤2：将超疏水涂料加入到超疏水涂层池中，所述超疏水涂料溶液挥发后在所述微坑阵列的微坑之间形成超疏水层，所述超疏水层是指其表面与水或水溶液的接触角大于150°、滚动角小于10°的疏水层，所述超疏水层使得各个微坑中水溶液之间有效的物理隔绝。
  2. 根据权利要求1所述的芯片，其特征在于，所述超疏水涂料溶液是括1H,1H,2H,2H-全氟辛基三乙氧基硅烷、氧化钛纳米粒子和P25氧化钛混匀的悬浮液；优选地，所述超疏水涂料溶液通过以下方法制备，将1H,1H,2H,2H-全氟辛基三乙氧基硅烷与无水乙醇在机械力的搅拌下混合，再加入氧化钛纳米粒子和P25氧化钛混匀并超声分散后制备成悬浮液。
  3. 一种集成超疏水微坑阵列芯片装置，其特征在于，所述装置包括权利要求1-2任一所述的超疏水微坑阵列芯片、基底和水槽，所述超疏水微坑阵列芯片和水槽设置在所述基底之上，所述水槽围绕在所述芯片外周。
  4. 根据权利要求3所述的装置，其特征在于，所述装置还包括外壁和围栏，所述装置的外壁和围栏从所述基底伸出并且在它们之间形成所述水槽。
  5. 根据权利要求4所述的装置，其特征在于，所述装置还包括超疏水微坑阵列芯片的基板底座，将多个带外壁的超疏水微坑阵列芯片嵌入所述基板底座，从而组装成包含多个超疏水微坑阵列芯片的芯片阵列。
  6. 根据权利要求4所述的装置，其特征在于，所述装置还包括盖，所述外壁制作成能兼容微坑阵列芯片的方形，四角倒角形状利于所述外壁与所述盖的配合，形成封闭空间。
  7. 根据权利要求4-6任一所述的装置，所述芯片是PMMA或PC材质，优选地是PC 2458。
  8. 一种超疏水微坑阵列芯片的制备方法，其特征在于，所述方法包括以下步骤：
    步骤1：通过一体注塑形成包括微坑阵列和超疏水涂层池的芯片，所述微坑阵列的微坑直径在300-1500μm，微坑深度为小于500μm和微坑间距为1500-3000μm；和所述超疏水涂层池的深度为大于50μm，所述涂层池与所述微坑之间的微坑壁厚为50-300μm；和
    步骤2：将超疏水涂料加入到超疏水涂层池中，所述超疏水涂料溶液挥发后在所述微坑阵列的微坑之间形成超疏水层，所述超疏水层是指其表面与水或水溶液的接触角大于150°、滚动角小于10°的疏水层，所述超疏水层使得各个微坑中水溶液之间有效的物理隔绝。
  9. 根据权利要求8所述的方法，其特征在于，所述超疏水涂料溶液是括1H,1H,2H,2H-全氟辛基三乙氧基硅烷、氧化钛纳米粒子和P25氧化钛混匀的悬浮液。
  10. 根据权利要求9所述的方法，其特征在于，所述超疏水涂料溶液通过以下方法制备，将1H,1H,2H,2H-全氟辛基三乙氧基硅烷与无水乙醇在机械力的搅拌下混合，再加入氧化钛纳米粒子和P25氧化钛混匀并超声分散后制备成悬浮液。
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