WO2022062897A1 - 一种用于微粒筛选分离的微流控芯片 - Google Patents

一种用于微粒筛选分离的微流控芯片 Download PDF

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WO2022062897A1
WO2022062897A1 PCT/CN2021/117046 CN2021117046W WO2022062897A1 WO 2022062897 A1 WO2022062897 A1 WO 2022062897A1 CN 2021117046 W CN2021117046 W CN 2021117046W WO 2022062897 A1 WO2022062897 A1 WO 2022062897A1
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microchannel
microfluidic chip
microfluidic
sample storage
sample
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PCT/CN2021/117046
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English (en)
French (fr)
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徐腾
马波
徐健
籍月彤
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中国科学院青岛生物能源与过程研究所
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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
    • 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

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  • the invention relates to the technical field of microfluidics, in particular to a technology for forming and exporting a single particle-encapsulated droplet by using a microfluidic chip, which can be used for single cell screening, single cell separation, single cell sequencing, single cell morphological analysis, single cell morphological analysis, and single cell morphological analysis. culture, drug screening, etc.
  • microorganisms play a pivotal role in the ecosystem and are an indispensable part of biomass synthesis, degradation and recycling.
  • microbes are also closely related to human health - the number of microbes in the human body is equivalent to ten times the number of the body's own cells.
  • the phenotype identification, sorting and genotype analysis of single living cells ie "single cell technology" can avoid the lengthy incubation process of microorganisms and analyze the "deep" heterogeneity and operation mechanism of the living system.
  • single-cell technology for microorganisms has faced this technical problem: how to isolate single microbial cells non-destructively and accurately.
  • FACS fluorescence flow cytometry
  • eppendorf micromanipulator
  • FACS fluorescence flow cytometry
  • eppendorf micromanipulator
  • FACS requires fluorescent labeling of cells, which usually encounters difficulties in labeling or affecting cell viability after labeling.
  • FACS for microbial isolation is expensive.
  • Micro-manipulation technology is complex in a single operation (need to precisely control the position of the capillary tip, including needle entry and withdrawal), and the throughput is low.
  • the purpose of the present invention is to provide a technology for separating and exporting a single target particle.
  • the present invention realizes the detection and capture of a single target particle in a microfluidic chip, and the transfer.
  • particles refer to particles that can be suspended in a non-organic phase solution (such as an aqueous phase) and pass through the microfluidic chip of the present invention, including particles derived from organisms and particles derived from non-organisms, For example, eukaryotic cells, prokaryotic cells, unicellular organisms, virus particles, organelles, particles formed by biological macromolecules, drug particles, drug carrier particles, liposomes, polymer particles and the like.
  • a first aspect of the present invention provides a microfluidic chip, the microfluidic chip sample storage tank, a main microchannel, and a secondary microchannel, the sample storage tank is a closed hollow three-dimensional structure, and the sample storage tank passes through The secondary microchannel communicates with the primary microchannel.
  • the sample storage tank includes an oil storage tank, a sample storage tank, a microchannel, and a sample inlet.
  • the two ends of the microchannel are respectively connected with the oil storage tank and the sample inlet.
  • the sample storage tank is a closed hollow three-dimensional structure.
  • the outlet of the sample storage tank It communicates with the microchannel through the microchannel branch.
  • the oil storage tank is a hollow three-dimensional structure.
  • the surface of the oil storage tank is a hydrophobic and oleophilic surface.
  • the top of the oil storage tank is an opening structure.
  • the oil storage tank has a diameter of 4 to 14 mm and a depth of 0.5 to 2 mm; preferably, the oil storage tank has a diameter of 6 to 10 mm and a depth of 0.5 to 1.5 mm; more preferably, the The diameter of the oil storage tank is 6-10mm, and the depth is 1-1.5mm.
  • the microchannel is a columnar structure.
  • the microchannel is a columnar structure.
  • the width of the microchannel is 10-100um, and the depth is 10-100um.
  • the width of the microchannel is 10-30 ⁇ m, and the depth is 10-30 ⁇ m.
  • the width of the microchannel is 30-50 ⁇ m, and the depth is 30-50 ⁇ m.
  • the width of the microchannel is 50-100 ⁇ m, and the depth is 50-100 ⁇ m.
  • the upper and lower channel walls of the microchannel are transparent optical mirror surfaces.
  • the depth of the microchannel branch is the same as the depth of the microchannel.
  • the sample storage tank is a closed cylinder cavity.
  • the diameter of the sample storage tank is 30-1000 ⁇ m; preferably, the diameter of the sample storage tank is 30-100 ⁇ m.
  • the depth of the sample storage tank is 300-500 ⁇ m.
  • the material of the microfluidic chip is selected from but not limited to quartz, PDMS (polydimethylsiloxane), PMMA (polymethylmethacrylate), borosilicate glass, and calcium fluoride.
  • Another aspect of the present invention provides a microfluidic chip device for forming a single particle-encapsulated droplet, the device comprising the microfluidic chip provided in the first aspect of the present invention and a liquid sampling device, the liquid feeding The sample device communicates with the sample inlet.
  • the liquid sampling device is selected from, but not limited to, a gravity-driven regulated sampling device, a syringe, a peristaltic pump, and a syringe pump.
  • the gravity-driven adjustable sampling device includes a height-adjustable sample holder, a sample container, and a conduit, the sample container communicates with the sampling port through the conduit, and the sample container can be placed in the The height-adjustable sample holder moves up and down.
  • the way of moving the sample container up and down is manual adjustment.
  • the way of moving the sample container up and down is electric adjustment.
  • the height-adjustable sample rack is designed as a sliding rail, and the sliding rail has an electrically movable slider for fixing the sample container; more preferably, the height-adjustable sample rack further includes a height An adjustment controller, the height adjustment controller controls the slider to move up and down on the slide rail.
  • Another aspect of the present invention provides a microfluidic operating system for forming a single particle-encapsulated droplet, comprising the microfluidic chip or microfluidic chip device provided by the present invention, and a particle capturing device, the The particle capture device is selected from optical tweezers.
  • optical tweezers device adopted in the present invention is the prior art in the field.
  • the capturing in the present invention refers to using a particle capturing device including optical tweezers to fix the target particles, so that when the microfluidic chip of the present invention is moved, the target particles do not move with the chip.
  • the microfluidic operating system further includes a sample detection device, and the sample detection device includes but is not limited to a Raman detection device, an optical microscope, and a fluorescence microscope.
  • the microfluidic operating system further includes a device for exporting a single particle-encapsulated droplet, and the device for exporting a single particle-encapsulated droplet is selected from a capillary tube and a pipette tip.
  • Another aspect of the present invention provides a method for exporting a single particle, the method utilizes the microfluidic chip or the microfluidic chip device or the microfluidic operating system provided by the present invention, and includes the steps of: 1. Connect to one end of the main microchannel, and seal the other end of the main channel; 2. place the microfluidic chip and the connected sample solution in a sealed vacuum chamber, and vacuumize (0.01 to 0.5 atmospheres); 3. restore the vacuum chamber to normal pressure , the particle phase solution fills the sample storage tank; 4 The aqueous phase solution is injected into the main microchannel through the injection port of the microfluidic chip to flush the main microchannel, until there are no residual particles in the entire microchannel part, and all the particles in the chip are located in the main microchannel. In the sample storage tank; 5 Use the particle capture device to capture the target particles and drag them into the main microchannel through the secondary microchannel, and the target cells flow out and collect with the water phase of the main channel to complete the sorting.
  • the methods for regulating the liquid flow in the microchannel include but are not limited to gravity-driven regulation, syringe pump-driven regulation, and peristaltic pump-driven regulation.
  • the particle capturing device captures target particles from the sample storage tank.
  • the oil phase is selected from one or more of mineral oil, silicone oil, fluorocarbon oil, vegetable oil, and petroleum ether.
  • the particulate phase contains particulates and a liquid that is immiscible with the oil phase liquid in the oil storage tank; preferably, the particulate phase is a non-organic phase; more preferably, the particulate The phases are aqueous or non-organic buffers.
  • the method for forming a single particle encapsulated droplet and exporting it further includes a sample detection step, the sample detection step is located before step 5, and the method used in the sample detection step is selected from but not limited to Raman Spectral analysis, fluorescence detection, optical microscope detection, conductivity detection;
  • the method for exporting the droplets wrapped with a single target particle includes a capillary export method and a pipette export method;
  • the method for forming a single particle-encapsulated droplet and exporting it further includes performing further operations on the exported target particles, the operations including single-cell sequencing, single-cell morphological analysis, and single-cell culture.
  • the method for collecting the characteristic signal of a single particle in the sample channel is selected from Raman signal collection, fluorescence detection, and optical microscope detection; more preferably, the location where the characteristic signal of a single particle is collected is the detection cell .
  • the laser wavelength of the optical tweezers is 1064 nm.
  • Another aspect of the present invention provides applications of microfluidic chips, microfluidic chip devices or microfluidic manipulation systems, including single particle screening, formation of single particle-encapsulated droplets, or derivation of single particle-encapsulated droplets.
  • the microparticles are cells, including but not limited to bacteria, fungi, and mammalian cells.
  • the sample storage pool in the chip can be maintained as a static flow field during the sorting process, which ensures the stability and accuracy of the sorting, and there is no exposure time limit for imaging in the static flow field, which improves the imaging quality.
  • the single particle screening and removal speed is fast, and the operation time of a single particle from the chip to the test tube is about 15 seconds.
  • Figure 1 is a schematic diagram of the design of a microfluidic chip
  • FIG. 1 Schematic diagram of single cell sorting by microfluidic chip.
  • All the cell suspensions to be sorted are located in the sample storage tank (2).
  • the target cells are moved into the microchannel (3) by the optical tweezers.
  • the target cells enter the oil storage tank (1) with the aqueous phase to form single-cell droplets.
  • the design of the microfluidic chip is shown in Figure 1, and its channel design mainly includes an oil storage pool (1), a sample storage pool (2), a microchannel (3), a sample inlet (4), a cavity (5), Microchannel branch (6).
  • the upper layer of the oil storage tank (1) has no cover sheet and is a semi-open structure.
  • the microchannel (3) and the sample storage pool (2) are closed structures. There are several closed cavities (5) around the sample storage pool (2), and the cavities (5) are independent and not communicated with the sample storage pool.
  • the entire chip structure is prepared by the traditional microfluidic field photolithography method, and is formed by bonding a layer of PDMS with a channel structure and a double-layer mirror glass.
  • the sampling device is a sample holder with adjustable height up and down, and the pure water is passed into the microchannel of the chip through the liquid level difference.
  • the optical tweezers device uses a 1064 nm laser.
  • the oil storage tank (1) has a diameter of 6 to 10 mm and a depth of about 1 mm; the right side is connected with a microchannel.
  • the size of the microchannel (3) is about 10 to 100 ⁇ m wide and 10 to 100 ⁇ m deep (the size of the channel is determined according to the sorted cells. Small-sized cells such as bacteria use 10-30 ⁇ m channels, medium-sized cells such as yeast use 30-50 ⁇ m channels, and mammalian cells generally use 50-100 ⁇ m channels); the connection on the microchannel branch (6) is the sample storage pool (2 ), the volume of the sample storage pool is determined according to the number to be sorted, and is generally designed to be 100 to 1000 ⁇ m. If the sample storage pool is used for single cell culture, it is about 30 to 100 ⁇ m in diameter.
  • the sample storage pool (2) is distributed around the There is a sealed cavity (5); the other end of the microchannel (3) is communicated with the injection port (4).
  • the chip preparation method refers to the traditional PDMS microfluidic chip preparation method: use CAD software to make the chip structure diagram and print out the mask.
  • the SU-8 chip mold was prepared according to the method of soft lithography.
  • the PDMS was poured into the mold, and the PDMS was cured at 80 degrees Celsius for one hour.
  • the cured PDMS is removed from the mold, and the designed channel structure is left on the PDMS. Then, the PDMS layer and the glass are bonded by oxygen plasma bombardment to complete the chip preparation.
  • the fluorescent E. coli suspension needs to be introduced into the sample storage tank (2) of the chip.
  • the chip is first placed in a desiccator connected to a vacuum chamber and a vacuum pump to be evacuated. After a certain period of time, the chip is taken out, and the cell suspension is dispensed with a pipette on the micropipette in the oil storage tank (1). At the outlet of the channel, depending on the residual negative pressure of PDMS, especially the negative pressure provided by the cavity (5), the cell suspension will slowly fill the sample storage tank (2), as shown in Figure 2A. Then, pure water is connected to the chip injection port (4), and the microchannel (3) is continuously flushed. Eventually, the entire microchannel (3) section will be free of residual cells, and all cells in the chip are located in the sample reservoir (2).
  • the fluorescent E. coli is green fluorescent protein, turn on the fluorescent light source, and switch the shutter to blue light excitation. E. coli large cells emitting green fluorescence can be observed in the field of view.
  • the target fluorescent Escherichia coli single cell in the sample storage tank (2) is captured and dragged into the microchannel (3) by using an optical tweezers device, as shown in FIG. 2B .
  • the height of the sampling device was raised, the optical tweezers were released, and the cells flowed into the oil reservoir (1) with the pure water phase, and were encapsulated by the generated water-in-oil droplets, as shown in Figure 2C. All resulting droplets were removed with a capillary to complete single cell isolation.
  • the entire microfluidic chip is calculated by hydrodynamics, and the flow of the pure water phase in the microchannel will not interfere with the sample storage pool connected with it. Therefore, except for the target cells captured and dragged by the optical tweezers, no other cells are introduced to be filled with pure water.
  • the microchannel in the water phase ensures the success rate of single cell separation in the water.

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Abstract

本发明提供一种可用于单个微粒筛选并形成液滴包裹导出的微流控芯片，该微流控芯片包括样品储存池，主微通道、以及副微通道。该微流控芯片借助真空负压装置完成样品填充，之后与液体进样装置、显微光镊相连，可组成一种用于形成单个微粒分选的微流控芯片装置。

Description

一种用于微粒筛选分离的微流控芯片 技术领域
本发明涉及微流控技术领域，具体涉及一种利用微流控芯片形成单个微粒包裹液滴并导出的技术，可用于单细胞筛选、单细胞分离、单细胞测序、单细胞形态分析、单细胞培养、药物筛选等领域。
背景技术
微生物作为地球生物种最丰富的物种，在生态系统中起到举足轻重的作用，是生物质合成、降解、循环中不可缺少的一环。同时，微生物也与人类健康息息相关-人体中的微生物数量相当于人体自身细胞的十倍。然而，至今为止，超过百分之九十的微生物无法在实验室条件下培养。对单个活体细胞的表型识别、分选及基因型分析(即“单细胞技术”)，能够避免微生物冗长的孵育过程，解析生命体系最“深”层次的异质性和运作机制。一直以来，针对微生物的单细胞技术都面临着这一技术难题：如何无损地、精准地分离出单个微生物细胞。
目前能够实现微生物单细胞分离的技术主要有荧光流式技术(FACS)、微操纵技术(micromanipulator,eppendorf)。但是都存在各自的技术局限，如FACS需要对细胞进行荧光标记，通常会遇到标记困难或标记后影响细胞活性的问题，另外满足微生物分离的FACS价格高昂。微操纵技术单次操作复杂(需要精准调控毛细管尖位置，包括进针、退针)，并且通量较低。
国内外研究组报道过通过微流控方法或激光弹射策略分离得到微生物单细胞的方法。如F.Teng,et.al.,Nondestructive Identification and Accurate Isolation of Single Cells through a Chip with Raman Optical Tweezers,Anal.Chem.这篇文章，运用了光镊把细胞从细胞池拖拽到分选池，再用移液枪分选取出单细胞。但是单个细胞拖拽距离有数个毫米，需要较长的操纵时间，另外采用的分离通道结构较宽，无法在视野中将整个通道成像，容易忽视非目标细胞的存在。
Y.Wang,et.al.,Raman Activated Cell Ejection for Isolation of Single Cells,Anal.Chem.这篇文章提供了一种脉冲激光弹射的方法。但是弹射前需将细胞自然干燥在弹 射基片上，加上脉冲激光在干燥基片上会产生强烈的光热现象，将严重影响分离后细胞的生理活性。
发明内容
有鉴于此，本发明的目的是提供一种将目标单个微粒分离并导出的技术，本发明实现了单个目标微粒在微流控芯片内的检测和捕获，以及从微流控芯片到外试管的转移。
本发明所称的“微粒”，是指能够悬浮在非有机相溶液(例如水相)、并在本发明微流控芯片内通过的颗粒，包括生物体来源的和非生物体来源的颗粒，例如真核细胞、原核细胞、单细胞生物、病毒颗粒、细胞器、生物大分子形成的颗粒、药物颗粒、药物载体颗粒、脂质体、多聚物粒子等。
本发明的第一方面，提供了一种微流控芯片，所述微流控芯片样品储存池，主微通道、以及副微通道，所述样品储存池为密闭中空立体结构，样品储存池通过副微通道与主微通道连通。
包括储油池、样品储存池，微通道、以及进样口，所述微通道两端分别与储油池及进样口连通，所述样品储存池为密闭中空立体结构，样品储存池的出口通过微通道支路与微通道连通。
所述储油池为中空立体结构。
所述储油池的表面为疏水亲油表面。
所述储油池的顶上为开口结构。
在一优选例中，所述储油池的直径为4～14mm,深度0.5～2mm；优选地，所述储油池的直径为6～10mm,深度0.5～1.5mm；更优选地，所述储油池的直径为6～10mm,深度1～1.5mm。
所述微通道为柱体结构。
在一优选例中，所述微通道为柱体结构。
在另一优选例中，所述微通道的宽度为10～100um，深度为10～100μm。
在另一优选例中，所述微通道的宽度为10～30μm，深度为10～30μm。
在另一优选例中，所述微通道的宽度为30～50μm，深度为30～50μm。
在另一优选例中，所述微通道的宽度为50～100μm，深度为50～100μm。
在另一优选例中，所述微通道上下通道壁为透明光学镜面。
在另一优选例中，所述微通道支路的深度与微通道的深度相同。
在一优选例中，所述样品储存池为密闭柱体空腔。
在另一优选例中，所述样品储存池的直径为30～1000μm；优选地，所述样品储存池的直径为30～100μm。
在另一优选中，所述样品储存池的深度为300～500μm。
所述微流控芯片的材质选自但不限于石英、PDMS(聚二甲基硅氧烷)、PMMA(聚甲基丙烯酸甲酯)、硼硅玻璃、氟化钙。
本发明的另一方面，提供了一种用于形成单个微粒包裹液滴的微流控芯片装置，该装置包括本发明第一方面提供的微流控芯片和液体进样装置，所述液体进样装置与所述进样口连通。
所述液体进样装置选自但不限于：重力驱动调节进样装置、注射器、蠕动泵、注射泵。
在一优选例中，所述重力驱动调节进样装置包含高度可调样品架、样品容器、导管，所述样品容器通过所述导管与所述进样口连通，所述样品容器可在所述高度可调样品架上上下移动。
在另一优选例中，所述样品容器上下移动的方式为手动调节。
在另一优选例中，所述样品容器上下移动的方式为电动调节。
在另一优选例中，所述高度可调样品架为滑轨设计，滑轨上具有可电动移动的滑块，用于固定样品容器；更佳的，所述高度可调样品架还包括高度调节控制器，所述高度调节控制器控制所述滑块在所述滑轨上进行上下移动。
本发明的另一方面，提供了一种用于形成单个微粒包裹液滴的微流控操作系统，包含本发明所提供的微流控芯片或微流控芯片装置，以及微粒捕获装置，所述微粒捕获装置选自光镊。
本发明采用的光镊装置，为本领域的现有技术。
本发明所述的捕获，是指利用包括光镊在内的微粒捕获装置固定住目标微粒，从而实现在移动本发明微流控芯片时、目标微粒不随芯片移动。
在另一优选例中，所述微流控操作系统进一步包括样品检测装置，所述样品检测装置包括但不限于拉曼检测装置、光学显微镜、荧光显微镜。
在另一优选例中，所述微流控操作系统进一步包括将单个微粒包裹液滴导出的装置，所述将单个微粒包裹液滴导出的装置选自毛细管、移液枪枪头。
本发明的另一方面，提供了一种单个微粒导出的方法，该方法利用本发明提供的微流控芯片或微流控芯片装置或微流控操作系统，并包括步骤：①将微粒相溶液连通至主微通道一端，将主通道另一端密封；②将所述微流控芯片及连通的样品溶液置于密封真空仓，抽真空(0.01至0.5个大气压)；③恢复真空仓至常压，微粒相溶液充满样品储存池；④将水相溶液通过微流控芯片的进样口注入到主微通道中冲刷主微通道，直至整个微通道部分没有残留微粒，所有芯片内的微粒都位于样品储存池内；⑤用微粒捕获装置捕获目标微粒经过副微通道拖拽到主微通道中，目标细胞随主通道水相流出收集，完成分选。
所述调节微通道内液体流动的方式包括但不限于重力驱动调节法、注射泵驱动调节法、蠕动泵驱动调节法。
在一优选例中，所述微粒捕获装置从所述样品储存池中捕获目标微粒。
在另一优选例中，所述油相选自矿物油、硅油、氟碳油、植物油、石油醚的一种或多种。
在另一优选例中，所述微粒相含有微粒和与储油池中的油相液体互不相溶的液体；较佳的，所述微粒相为非有机相；更佳的，所述微粒相为水相或非有机缓冲液。
在另一优选例中，所述形成单个微粒包裹液滴并导出的方法还包括样品检测步骤，所述样品检测步骤位于步骤⑤之前，所述样品检测步骤采用的方法选自但不限于拉曼光谱分析、荧光检测、光学显微镜检测、电导检测；
在另一优选例中，所述将包裹有单个目标微粒的液滴导出的方法包括毛细管导出法、移液枪导出法；
在另一优选例中，所述形成单个微粒包裹液滴并导出的方法还包括对导出的目标微粒进行进一步操作，所述操作包含单细胞测序、单细胞形态分析、单细胞培养。
在另一优选例中，所述在样品通道内采集单个微粒的特征信号的方法选自拉曼信号采集、荧光检测、光学显微镜检测；更佳的，采集单个微粒的特征信号的位置为检测池。
在另一优选例中，所述光镊的激光波长为1064nm。
本发明的另一方面，提供了微流控芯片、微流控芯片装置或微流控操控系统的应用，包括单个微粒筛选、单个微粒包裹液滴的形成或单个微粒包裹液滴的导出。
在一优选例中，所述微粒为细胞，包括但不限于细菌、真菌、哺乳动物细胞。
本发明具有以下技术优势：
1.适用于各尺寸微粒，如数十微米的酵母细胞和1微米左右细菌细胞的富集检测。
2.芯片中的样品储存池可在分选过程中保持为静态流场，保证了分选的稳定性和准确性，并且静态流场中成像无曝光时间限制，提高了成像质量。
3.实现了单个细胞的挑选并分离导出，该过程对细胞活性影响低，能成功与下游单细胞测序对接。
4.单个微粒筛选移取速度快，单个微粒从芯片内到试管中移取操作时间为15秒左右。
5.操作简便。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对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微流控芯片设计示意图；
图2微流控芯片分选单细胞示意图。
A、待分选细胞悬液全部位于样品储存池(2)中。
B、目标细胞被光镊移动到微通道(3)中。
C、目标细胞随水相进入储油池(1)中，形成单细胞液滴。
主要附图标记：储油池(1)，样品储存池(2)，微通道(3)，进样口(4)，空腔(5)，微通道支路(6)。
具体实施方式
为了使本领域技术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。
实施例1 微流控芯片的制备：
微流控芯片设计如附图1所示，其通道设计主要包括储油池(1)、样品储存池(2)、微通道(3)、进样口(4)，空腔(5)，微通道支路(6)。其中储油池(1)上层无盖片，为半开放式结构。微通道(3)、样品储存池(2)为封闭结构。样品储存池(2)周围有数个密闭空腔(5)，空腔(5)各个独立，与样品储存池不连通。整个芯片结构通过传统微流控领域光刻法制备，由一层带有通道结构的PDMS与一片双层镜面玻璃键合而成。进样装置为上下高度可调的样品支架，通过液面差将纯水向通入芯片微通道。光镊装置采用1064nm激光。
储油池(1)直径6～10mm,深度1mm左右；右边有微通道连通，微通道(3)的尺寸为10到100μm宽左右，深度为10到100μm(通道尺寸依据所分选细胞决定，细菌这种小尺寸细胞使用10～30μm通道，酵母这种中等尺寸细胞使用30～50μm通道，哺乳动物细胞一般使用50～100μm通道)；微通道支路(6)上连通是样品储存池(2)，样品储存池的体积依据需要分选的数量而定，一般设计为100～1000μm，如果将样品储存池作为单细胞培养用，则为30～100μm直径左右，样品储存池(2)周围分布有密封的空腔(5)；微通道(3)的另一端连通进样口(4)。
芯片制备方法参考传统PDMS微流控芯片制备方法：用CAD软件做出芯片结构图，打印出掩膜。按照软光刻的方法制备出SU-8芯片模具。将PDMS倒入模具，80摄氏度一小时，PDMS固化。将固化后的PDMS从模具上取下，这时PDMS上会留下设计好的通道结构。之后将PDMS层与玻璃通过氧等离子体轰击键合，完成芯片制备。
实施例2 荧光大肠杆菌单细胞分选
1.分选开始前，需将荧光大肠杆菌悬液导入芯片的样品储存池(2)中。
利用了PDMS的透气性，首先将芯片置入真空腔同真空泵连接的干燥器内中抽真空，一定时间后取出芯片，将细胞悬液用移液枪点在储油池(1)中的微通道出口处，依靠PDMS残留的负压，尤其是空腔(5)提供的负压，细胞悬液会慢慢充满样品储存池(2)，如图2A所示。之后将纯水接入芯片进样口(4)，连续冲刷微通道(3)。最终，整个微通道(3)部分将没有残留细胞，所有芯片内的细胞都位于样品储存池(2)内。
2.将储油池(1)内的水相取走，替换成油相。此时通过调节液体进样装置高度，使微通道(3)内的水相与储油池(1)的油相达到平衡，界面停留在微通道(3)靠近储油池(1)的位置。
3.开始分选。
荧光大肠杆菌为绿色荧光蛋白，打开荧光光源，切换shutter至蓝光激发。可在视野中观察到发射绿色荧光的大肠杆菌大细胞。利用光镊装置将样品储存池(2)中的目标荧光大肠杆菌单细胞，捕获拖拽到微通道(3)中，如图2B所示。升高进样装置高度，光镊释放，细胞随纯水相流入储油池(1)，被产生的油包水液滴包裹，如图2C所示。用毛细管将所有产生的液滴取出，完成单细胞分离。
整个微流控芯片通过流体力学计算，微通道内纯水相的流动不会干扰到与之相连通的样品储存池，因此除光镊捕获拖拽的目标细胞外，无其他细胞被引入充满纯水相的微通道，保证里单细胞分离的成功率。

Claims (7)

  1. 一种微流控芯片，其特征在于，所述微流控芯片包括样品储存池，主微通道、以及副微通道，所述样品储存池为密闭中空立体结构，样品储存池通过副微通道与主微通道连通。
  2. 如权利要求1所述的微流控芯片，其特征在于，所述样品储存池，主微通道、以及副微通道的上下通道壁中靠近显微镜头一面为透明光学镜面。
  3. 一种微流控芯片装置，其特征在于，所述微流控芯片装置包括如权利要求1所述的微流控芯片和液体进样装置，所述液体进样装置与所述进样口连通。
  4. 一种微流控操作系统，其特征在于，所述微流控操作系统包括如权利要求1所述的微流控芯片或权利要求3所述的微流控芯片装置，以及微粒捕获装置。
  5. 如权利要求4所述的微流控操作系统，其特征在于，所述微粒捕获装置为光镊。
  6. 如权利要求3所述的微流控操作系统，其特征在于，所述微流控操作系统进一步包括样品检测装置。
  7. 一种形成单个微粒包裹液滴并导出的方法，其特征在于，所述方法利用如权利要求1所述的微流控芯片或权利要求3所述的微流控芯片装置或权利要求4所述的微流控操作系统，包括步骤：①将微粒相溶液连通至主微通道一端，将主通道另一端密封；②将所述微流控芯片及连通的样品溶液置于密封真空仓，抽真空(0.01至0.5个大气压)；③恢复真空仓至常压，微粒相溶液充满样品储存池；④将水相溶液通过微流控芯片的进样口注入到主微通道中冲刷主微通道，直至整个微通道部分没有残留微粒，所有芯片内的微粒都位于样品储存池内；⑤用微粒捕获装置捕获目标微粒经过副微通道拖拽到主微通道中，目标细胞随主通道水相流出收集，完成分选。
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