技术领域technical field
本发明属于微流控技术与细胞生物学的交叉领域，具体涉及一种基于液滴微流控芯片的高通量自动捕获单细胞的方法。The invention belongs to the intersecting field of microfluidic technology and cell biology, and in particular relates to a method for automatically capturing single cells with high throughput based on a droplet microfluidic chip.
背景技术Background technique
微流控芯片实验室早期致力于芯片电泳的研究，并提出了微全分析系统(μ-TAS)的概念，研究工作主要集中在连续流微流控系统。近年来，微流控芯片领域出现了一个新的分支-非连续流微流控系统，即液滴微流控系统。液滴微流控系统使用不相溶的两相流体在微孔道界面处形成液滴，这类液滴的体积通常在纳升至皮升(10-9～10-12L)范围。相对于连续流系统，液滴具有体积小、低扩散、无交叉污染、快速的反应动力学等特点，并且具有高通量分析的潜力。经过几年的发展，液滴的制备技术已日趋成熟；同时，液滴的分裂、融合、混合、分选、存储和编码等丰富多样的操控技术也都有广泛的报道。The microfluidic chip laboratory devoted itself to the research of chip electrophoresis in the early stage, and proposed the concept of micro-total analysis system (μ-TAS), and the research work mainly focused on the continuous flow microfluidic system. In recent years, a new branch has emerged in the field of microfluidic chips - discontinuous flow microfluidic systems, namely droplet microfluidic systems. Droplet microfluidic systems use immiscible two-phase fluids to form droplets at the interface of micropores, and the volume of such droplets is usually in the range of nanoliters to picoliters (10-9 ~ 10-12 L). Compared with continuous flow systems, droplets have the characteristics of small size, low diffusion, no cross-contamination, fast reaction kinetics, and the potential for high-throughput analysis. After several years of development, the droplet preparation technology has become increasingly mature; at the same time, a variety of manipulation technologies such as droplet splitting, fusion, mixing, sorting, storage, and encoding have also been widely reported.
单细胞水平的生物物理特性表征，可有效阐明细胞的功能和状态，揭示细胞的单体差异性，对于细胞的分化和病理研究，以及疾病的早期临床诊断和治疗具有非常重要的意义，目前已成为研究的热点之一。由于细胞极小，直径一般为5～500μm，组分含量少，种类繁多，使得操纵和分析难度很大。早期的单细胞研究，主要采用膜片钳、细胞穿刺等技术，同时借助显微镜三维微操作器进行操作。毛细管电泳分离方法具有进样体积小、分离效率高、分离速度快等优点，已用于单细胞多组分的检测，取得了一些成果。但毛细管属一维结构，进样和溶膜操作复杂。传统的流式细胞仪虽已实现集成化和自动化，但同样具有仪器体积大、内部结构复杂、易损坏等缺点。The characterization of biophysical characteristics at the single-cell level can effectively clarify the function and state of cells, and reveal the differences of cell monomers. It is of great significance for cell differentiation and pathological research, as well as early clinical diagnosis and treatment of diseases. become one of the hotspots of research. Because the cells are extremely small, generally 5-500 μm in diameter, with few components and various types, it is very difficult to manipulate and analyze. Early single-cell research mainly used techniques such as patch clamping and cell puncture, and at the same time operated with the aid of a three-dimensional micromanipulator under a microscope. The capillary electrophoresis separation method has the advantages of small sample volume, high separation efficiency, and fast separation speed. It has been used in the detection of single-cell multi-components and achieved some results. However, the capillary has a one-dimensional structure, and the operation of sample injection and membrane dissolution is complicated. Although the traditional flow cytometer has been integrated and automated, it also has the disadvantages of large instrument volume, complex internal structure, and easy damage.
近年来，随着液滴微流控技术的迅速发展，其在单细胞分析中的应用引起了越来越多的关注。液滴作为单细胞微反应器，能够有效控制扩散，提高检测灵敏度，已被成功应用于多种单细胞分析中。然而，在微流控系统内研究单细胞，首要解决的问题就是如何对单细胞进行捕获，以实现单细胞的移动、固定等，然后才能对其进行分析和检测。目前，已发展出多种单细胞捕获技术，其驱动力主要包括流体压力、电动力、磁力、光学陷阱以及机械力等。其中，由于外部电学原件、磁力控制部件、光学传感器和机械控制器结构复杂，成本偏高，不便广泛应用。利用物理屏障捕获单细胞简单且成本低廉，但容易造成细胞损伤且不便于加载动态生化信号刺激。因此迫切需要一种能够长期捕获悬浮培养单细胞、减少细胞损伤且便于加入药物刺激的单细胞捕获微流控芯片。In recent years, with the rapid development of droplet microfluidics, its application in single-cell analysis has attracted more and more attention. Droplets, as single-cell microreactors, can effectively control diffusion and improve detection sensitivity, and have been successfully applied in a variety of single-cell assays. However, to study single cells in a microfluidic system, the first problem to be solved is how to capture single cells to realize the movement and immobilization of single cells before they can be analyzed and detected. At present, a variety of single-cell capture technologies have been developed, and their driving forces mainly include fluid pressure, electrodynamic force, magnetic force, optical trap and mechanical force. Among them, due to the complex structure and high cost of external electrical components, magnetic control components, optical sensors and mechanical controllers, it is inconvenient to be widely used. The use of physical barriers to capture single cells is simple and low-cost, but it is easy to cause cell damage and it is not convenient to load dynamic biochemical signal stimulation. Therefore, there is an urgent need for a single-cell capture microfluidic chip that can capture single cells in suspension culture for a long time, reduce cell damage, and facilitate the addition of drug stimulation.
本发明利用液滴微流控技术和流体力学实现单细胞的高通量自动捕获，通过在芯片通道内部形成负压，将单细胞悬液自动吸入单细胞捕获流路通道，通过单细胞捕获阱后，单细胞截留于生成的液滴中，实现单细胞自动捕获；通过调节单细胞悬液流速和单细胞悬液密度控制单细胞捕获率。整个装置结构简单，无需任何复杂和昂贵的设备，可实现高通量，操作方便自动化。因此，本发明提供了一种灵活可控的高通量自动捕获单细胞的方法，并期望在细胞学、药物毒理学研究等领域得到应用。The present invention utilizes droplet microfluidic technology and fluid mechanics to realize high-throughput automatic capture of single cells. By forming a negative pressure inside the chip channel, the single-cell suspension is automatically sucked into the single-cell capture flow channel and passed through the single-cell capture trap. Finally, the single cells are trapped in the generated droplets to realize the automatic capture of single cells; the single cell capture rate is controlled by adjusting the flow rate and density of the single cell suspension. The whole device has a simple structure, does not need any complicated and expensive equipment, can realize high throughput, and is easy to operate automatically. Therefore, the present invention provides a flexible and controllable high-throughput method for automatically capturing single cells, and is expected to be applied in the fields of cytology, drug toxicology research and the like.
发明内容Contents of the invention
本发明的目的是提供一种基于液滴微流控芯片的高通量自动捕获单细胞的方法，本发明制备过程稳定，操作简单，可实现快速、高效、高通量、自动捕获单细胞。The purpose of the present invention is to provide a method for high-throughput automatic capture of single cells based on droplet microfluidic chips. The preparation process of the present invention is stable, easy to operate, and can achieve fast, efficient, high-throughput, and automatic capture of single cells.
一种液滴微流控芯片，所述芯片由两层组成，上层为流路出入口层；下层为流路控制层；A droplet microfluidic chip, the chip is composed of two layers, the upper layer is the flow path inlet and outlet layer; the lower layer is the flow path control layer;
所述流路出入口层有液体流路通道入口和液体流路通道出口；The flow channel entrance and exit layer has a liquid flow channel inlet and a liquid flow channel outlet;
所述流路控制层由单细胞捕获流路通道、气路通道、液滴生成单元(单细胞捕获阱)组成；The flow control layer is composed of a single cell capture flow channel, an air channel, and a droplet generation unit (single cell trap);
所述单细胞捕获流路通道是一条由多个交互连接的U型通道构成的长通道，长通道两端分别连接液体流路通道入口和液体流路通道出口，每个U型通道的直通道均有与之平行的气体流路通道，每个U型通道的直通道上分布着数个圆柱形凹陷小坑结构的液滴生成单元。The single-cell capture flow channel is a long channel composed of a plurality of U-shaped channels that are interconnected. There are gas flow channels parallel thereto, and several droplet generating units with a cylindrical concave pit structure are distributed on the straight channel of each U-shaped channel.
所述芯片抽真空后，气路通道处于负压状态，由于PDMS的透气性，气路带动周围的液路通道形成负压，可自动将单细胞悬液从液路通道入口吸入，用于单细胞进液和捕获。After the chip is evacuated, the air channel is in a negative pressure state. Due to the gas permeability of PDMS, the air channel drives the surrounding liquid channel to form a negative pressure, which can automatically suck the single-cell suspension from the inlet of the liquid channel. Cell influx and capture.
所述单细胞捕获流路通道宽100～300μm，U型通道的直通道长为40～50mm，直通道间距为0.5～1mm。The channel width of the single cell capture flow path is 100-300 μm, the straight channel length of the U-shaped channel is 40-50 mm, and the distance between the straight channels is 0.5-1 mm.
所述气路通道宽为200～500μm，长为40～50mm，间距为0.5～1mm。The air channel has a width of 200-500 μm, a length of 40-50 mm, and a distance of 0.5-1 mm.
所述单细胞捕获流路通道与气路通道间距为0.5～1.5mm。The distance between the single cell capture flow channel and the air channel is 0.5-1.5mm.
所述液滴生成单元为圆柱形凹陷小坑结构，共有6000～10000个，小坑直径为15～45μm，高80～200μm，小坑间距80～150μm。The droplet generating unit is a cylindrical concave pit structure, with a total of 6000-10000 pits with a diameter of 15-45 μm, a height of 80-200 μm, and a pitch of 80-150 μm.
一种液滴微流控芯片的制备方法，所述芯片制备步骤如下：A method for preparing a droplet microfluidic chip, the chip preparation steps are as follows:
(1)采用光刻和腐蚀方法制备出通道部分突起的SU-8模板：所述SU-8模板由双层结构组成，第一层由单细胞捕获流路通道和气路通道组成，第二层由液滴生成单元组成；SU-8模板的制备经过两次甩胶、前烘、曝光、后烘后，一次显影、坚膜得到；(1) The SU-8 template with channel part protrusions was prepared by photolithography and corrosion methods: the SU-8 template is composed of a double-layer structure, the first layer is composed of a single cell capture flow channel and an air channel, and the second layer It is composed of a droplet generating unit; the preparation of the SU-8 template is obtained through two times of glue rejection, pre-baking, exposure, and post-baking, and one development and film hardening;
(2)基于SU-8模板制得得的PDMS模块：将PDMS与引发剂以体积比10：1混合均匀，浇注于SU-8模板，75～85℃烘箱固化30～60min，将PDMS与SU-8模板剥离，得到带有结构的上层芯片；将上层芯片与带有流路出入口的下层芯片经过等离子体处理1～4min后封接。(2) PDMS module prepared based on SU-8 template: mix PDMS and initiator uniformly at a volume ratio of 10:1, cast on SU-8 template, cure in an oven at 75-85°C for 30-60min, and mix PDMS and SU -8 The template is peeled off to obtain an upper chip with a structure; the upper chip and the lower chip with a flow path inlet and outlet are treated with plasma for 1 to 4 minutes and then sealed.
一种基于液滴微流控芯片的高通量自动捕获单细胞的方法，采用上述液滴微流控芯片，按照以下步骤进行：A high-throughput method for automatically capturing single cells based on a droplet microfluidic chip, using the above-mentioned droplet microfluidic chip, according to the following steps:
将上述制备的微流控芯片经60～90％乙醇浸泡，紫外照射30～60min灭菌处理后，将液体流路通道出口堵住，置于真空培养箱抽真空5～10min，随后，快速将单细胞悬液以5×103cells/mL～5×106cells/mL的细胞密度加入芯片入口，控制单细胞悬液流速在0.01m/s～0.05m/s之间，待全部液滴生成单元都充满单细胞悬液时，打开液体流路通道出口，将液体流路通道中多余液体排除，待细胞粘附于芯片底面，补加新鲜的高糖DMEM培养基，置于37℃培养箱培养。The microfluidic chip prepared above was soaked in 60-90% ethanol, sterilized by ultraviolet irradiation for 30-60 minutes, the outlet of the liquid flow channel was blocked, and placed in a vacuum incubator to evacuate for 5-10 minutes. The single-cell suspension was added to the chip inlet at a cell density of 5×103 cells/mL to 5×106 cells/mL, and the flow rate of the single-cell suspension was controlled between 0.01m/s and 0.05m/s. When the generation units are all filled with single cell suspension, open the outlet of the liquid flow channel and drain the excess liquid in the liquid flow channel. After the cells adhere to the bottom of the chip, add fresh high-sugar DMEM medium and culture at 37°C. box culture.
一种基于液滴微流控芯片的高通量自动捕获单细胞的方法的应用，所捕获的单细胞可在芯片的液滴结构中长期培养，并可应用于细胞增殖、分化以及药物筛选等方面。Application of a high-throughput method for automatically capturing single cells based on a droplet microfluidic chip. The captured single cells can be cultured in the droplet structure of the chip for a long time, and can be applied to cell proliferation, differentiation, and drug screening, etc. aspect.
本发明利用液滴微流控技术和流体力学原理实现单细胞的高通量自动捕获，相比于传统的单细胞捕获方法，具有操作简便、灵活、高通量、无污染、适用范围广以及可扩展性强等优点。本发明期望提供一种灵活可控的单细胞捕获方法，并且在细胞学、药物毒理学研究等领域得到应用。The present invention utilizes droplet microfluidic technology and the principle of fluid mechanics to realize high-throughput automatic capture of single cells. Strong scalability and other advantages. The present invention expects to provide a flexible and controllable single cell capture method, which can be applied in the fields of cytology, drug toxicology research and the like.
附图说明：Description of drawings:
图1：芯片结构示意图；(a)整体结构图，(b)为局部放大图；Figure 1: Schematic diagram of chip structure; (a) overall structure diagram, (b) partial enlarged diagram;
图2：微流控芯片侧面局部放大图。Figure 2: Partial enlarged view of the side of the microfluidic chip.
其中：1为液体流路通道入口，2为液体流路通道出口，3为单细胞捕获流路通道，4为气体流路通道，5为液滴生成单元(单细胞捕获阱)，6为单细胞；Among them: 1 is the inlet of the liquid flow channel, 2 is the outlet of the liquid flow channel, 3 is the single cell capture flow channel, 4 is the gas flow channel, 5 is the droplet generation unit (single cell capture trap), and 6 is the single cell trap. cell;
图3：微流控芯片实物图。Figure 3: The physical picture of the microfluidic chip.
图4：单细胞悬液密度为5×104cells/mL时，芯片中的单细胞分布图。Figure 4: Distribution of single cells in the chip when the density of single cell suspension is 5×104 cells/mL.
图5：单细胞悬液密度为5×105cells/mL时，芯片中的单细胞分布图。Figure 5: Distribution of single cells in the chip when the density of single cell suspension is 5×105 cells/mL.
具体实施方式Detailed ways
一种高通量自动捕获单细胞的微流控芯片，如图1、图2、图3所示；所述芯片由两层组成，上层为流路出入口层；下层为流路控制层；A high-throughput microfluidic chip for automatically capturing single cells, as shown in Figure 1, Figure 2, and Figure 3; the chip is composed of two layers, the upper layer is the flow path inlet and outlet layer; the lower layer is the flow path control layer;
所述流路出入口层有液体流路通道入口1和液体流路通道出口2；The flow channel entrance and exit layer has a liquid flow channel inlet 1 and a liquid flow channel outlet 2;
所述流路控制层由单细胞捕获流路通道3、气路通道4、液滴生成单元(单细胞捕获阱)5组成；The flow control layer is composed of a single cell capture flow channel 3, an air channel 4, and a droplet generation unit (single cell trap) 5;
所述单细胞捕获流路通道3是一条由多个交互连接的U型通道构成的长通道，长通道两端分别连接液体流路通道入口1和液体流路通道出口2，每个U型通道的直通道均有与之平行的气体流路通道4，每个U型通道的直通道上分布着数个圆柱形凹陷小坑结构的液滴生成单元5。The single-cell capture channel 3 is a long channel composed of multiple interconnected U-shaped channels. The two ends of the long channel are respectively connected to the liquid flow channel inlet 1 and the liquid flow channel outlet 2. Each of the straight channels has a gas flow channel 4 parallel to it, and several droplet generating units 5 with a cylindrical concave pit structure are distributed on the straight channel of each U-shaped channel.
所述单细胞捕获流路通道宽100～300μm，U型通道的直通道长为40～50mm，直通道间距为0.5～1mm。The channel width of the single cell capture flow path is 100-300 μm, the straight channel length of the U-shaped channel is 40-50 mm, and the distance between the straight channels is 0.5-1 mm.
所述气路通道宽为200～500μm，长为40～50mm，间距为0.5～1mm。The air channel has a width of 200-500 μm, a length of 40-50 mm, and a distance of 0.5-1 mm.
所述单细胞捕获流路通道与气路通道间距为0.5～1.5mm。The distance between the single cell capture flow channel and the air channel is 0.5-1.5mm.
所述液滴生成单元为圆柱形凹陷小坑结构，共有6000～10000个，小坑直径为15～45μm，高80～200μm，小坑间距80～150μm。The droplet generating unit is a cylindrical concave pit structure, with a total of 6000-10000 pits with a diameter of 15-45 μm, a height of 80-200 μm, and a pitch of 80-150 μm.
当芯片抽真空后，气体流路通道4处于负压状态，由于PDMS的透气性，气路带动周围的液体流路通道3形成负压，MCF-7单细胞悬液6在负压作用下经通道入口1进入单细胞捕获流路通道3，通过液滴生成单元5后，单细胞6截留于生成的液滴中，剩余单细胞悬液经通道入口2流出，实现单细胞自动捕获。When the chip is evacuated, the gas channel 4 is in a negative pressure state. Due to the gas permeability of PDMS, the gas channel drives the surrounding liquid channel 3 to form a negative pressure. The channel inlet 1 enters the single cell capture flow channel 3, and after passing through the droplet generation unit 5, the single cell 6 is trapped in the generated droplet, and the remaining single cell suspension flows out through the channel inlet 2, realizing the automatic capture of single cells.
实施例1Example 1
一种高通量自动捕获单细胞的微流控芯片的制备方法如下：A method for preparing a high-throughput microfluidic chip that automatically captures single cells is as follows:
(1)高通量自动捕获单细胞的微流控芯片SU-8模板的制备(1) Preparation of SU-8 template for microfluidic chip with high-throughput automatic capture of single cells
微流控芯片采用光刻和腐蚀方法制备出通道部分突起的SU-8模板；首先，第一层甩SU-8胶厚度为100μm，95℃前烘20min，自然降温，将掩膜置于SU-8胶平板上面，紫外曝光30s，95℃后烘20min，自然降温；其次，基于第一层SU-8胶的基础，第二层甩SU-8胶厚度为150μm，95℃前烘30min，自然降温，将掩膜置于SU-8胶平板上面，紫外曝光40s，95℃后烘40min，自然降温；最后，采用乳酸乙酯将上述SU-8胶显影10min，180℃坚膜2h，自然降温备用。The microfluidic chip was prepared by photolithography and etching methods to prepare the SU-8 template with channel part protrusions; first, the thickness of the first layer of SU-8 glue was 100 μm, pre-baked at 95°C for 20 minutes, and the temperature was naturally lowered, and the mask was placed on the SU-8 template. -8 glue plate, UV exposure for 30s, 95°C post-baking for 20min, and natural cooling; secondly, based on the foundation of the first layer of SU-8 glue, the thickness of the second layer of SU-8 glue is 150μm, and 95°C for 30min before baking. Cool down naturally, put the mask on the SU-8 glue plate, expose to ultraviolet light for 40s, bake at 95°C for 40min, and cool down naturally; finally, use ethyl lactate to develop the above SU-8 glue for 10min, harden the film at 180°C for 2h, and naturally Cool down and set aside.
(2)高通量自动捕获单细胞的PDMS芯片的制备(2) Preparation of high-throughput PDMS chip for automatic capture of single cells
将PDMS与引发剂以体积比10：1混合均匀，浇注于前期制备的SU-8模板，80℃烘箱固化30min，将PDMS与SU-8模板剥离，得到带有结构的微流控芯片；将PDMS与引发剂以体积比10：1混合均匀，浇注于空白玻璃板，80℃烘箱固化30min，将PDMS与SU-8模板剥离，得到空白PDMS芯片(2)；用打孔器在芯片(2)上打两个孔，分别对应流路出入口，将芯片1与芯片2经过等离子体处理120s后封接备用。Mix PDMS and initiator uniformly at a volume ratio of 10:1, cast on the previously prepared SU-8 template, cure in an oven at 80°C for 30 minutes, and peel PDMS and SU-8 template to obtain a microfluidic chip with structure; PDMS and initiator were mixed uniformly at a volume ratio of 10:1, poured on a blank glass plate, cured in an oven at 80°C for 30 minutes, and the PDMS and SU-8 template were peeled off to obtain a blank PDMS chip (2); ) to punch two holes corresponding to the inlets and outlets of the flow path respectively, and chip 1 and chip 2 are sealed after plasma treatment for 120s for later use.
实施例2：Example 2:
单细胞悬液密度为5×104cells/mL时，芯片自动捕获单细胞实验When the single cell suspension density is 5×104 cells/mL, the chip automatically captures single cell experiments
上述制备的微流控芯片经75％乙醇浸泡，紫外照射1h灭菌处理后，将液体流路通道出口堵住，置于真空培养箱抽真空10min，随后，快速将经细胞膜红色荧光探针标记的乳腺癌细胞(MCF-7)单细胞悬液以5×104cells/mL的细胞密度加入芯片入口，此时单细胞悬液的流速为0.027m/s，待全部液滴生成单元都充满单细胞悬液时，打开液体流路通道出口，将液体流路通道中多余液体排除，待细胞粘附于芯片底面，补加新鲜的高糖DMEM培养基，置于37℃培养箱培养。芯片中的单细胞分布图如图4所示。细胞密度为5×104cells/mL、单细胞悬液流速为0.027m/s时，芯片捕获单细胞的明场和荧光图片；根据荧光图片统计数据可知，芯片的单细胞捕获效率为42％。The microfluidic chip prepared above was soaked in 75% ethanol, sterilized by ultraviolet irradiation for 1 h, the outlet of the liquid flow channel was blocked, and placed in a vacuum incubator to evacuate for 10 min. The breast cancer cell (MCF-7) single cell suspension was added to the chip inlet at a cell density of 5×104 cells/mL. At this time, the flow rate of the single cell suspension was 0.027m/s. For single-cell suspension, open the outlet of the liquid flow channel, and drain the excess liquid in the liquid flow channel. After the cells adhere to the bottom of the chip, add fresh high-glucose DMEM medium and culture in a 37°C incubator. The distribution of single cells in the chip is shown in Figure 4. When the cell density is 5×104 cells/mL and the flow rate of single cell suspension is 0.027m/s, the chip captures the bright field and fluorescence pictures of single cells; according to the statistical data of fluorescence pictures, the single cell capture efficiency of the chip is 42% .
实施例3：Example 3:
单细胞悬液密度为5×105cells/mL时，芯片自动捕获单细胞实验When the single cell suspension density is 5×105 cells/mL, the chip automatically captures single cell experiments
上述制备的微流控芯片经75％乙醇浸泡，紫外照射1h灭菌处理后，将液体流路通道出口堵住，置于真空培养箱抽真空10min，随后，快速将经细胞膜红色荧光探针标记的乳腺癌细胞(MCF-7)单细胞悬液以5×105cells/mL的细胞密度加入芯片入口，此时单细胞悬液的流速为0.027m/s，待全部液滴生成单元都充满单细胞悬液时，打开液体流路通道出口，将液体流路通道中多余液体排除，待细胞粘附于芯片底面，补加新鲜的高糖DMEM培养基，置于37℃培养箱培养。芯片中的单细胞分布图如图5所示。细胞密度为5×105cells/mL、单细胞悬液流速为0.027m/s时，芯片捕获单细胞的明场和荧光图片；根据荧光图片统计数据可知，芯片的单细胞捕获效率为66％。The microfluidic chip prepared above was soaked in 75% ethanol, sterilized by ultraviolet irradiation for 1 h, the outlet of the liquid flow channel was blocked, and placed in a vacuum incubator to evacuate for 10 min. The breast cancer cell (MCF-7) single cell suspension was added to the chip inlet at a cell density of 5×105 cells/mL. At this time, the flow rate of the single cell suspension was 0.027m/s. For single-cell suspension, open the outlet of the liquid flow channel, and drain the excess liquid in the liquid flow channel. After the cells adhere to the bottom of the chip, add fresh high-glucose DMEM medium and culture in a 37°C incubator. The distribution of single cells in the chip is shown in Figure 5. When the cell density is 5×105 cells/mL and the flow rate of single cell suspension is 0.027m/s, the chip captures the bright field and fluorescence pictures of single cells; according to the statistical data of fluorescence pictures, the single cell capture efficiency of the chip is 66% .
由此可知，当单细胞悬液流速不变，细胞密度在一定范围内时，细胞密度越大，捕获单细胞的效率越高。此单细胞捕获芯片可实现高效率单细胞捕获，同时，可实现单细胞在芯片内原位长期培养以及后期的分析评价。It can be seen that when the flow rate of the single cell suspension is constant and the cell density is within a certain range, the greater the cell density, the higher the efficiency of capturing single cells. This single cell capture chip can realize high-efficiency single cell capture, and at the same time, it can realize long-term in situ culture of single cells in the chip and later analysis and evaluation.
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。The above-mentioned embodiments are only to illustrate the technical concept and characteristics of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and not to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention shall fall within the protection scope of the present invention.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611061683.7ACN108117968A (en) | 2016-11-28 | 2016-11-28 | A kind of single celled method of high-throughput automatic capture based on drop micro-fluidic chip |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611061683.7ACN108117968A (en) | 2016-11-28 | 2016-11-28 | A kind of single celled method of high-throughput automatic capture based on drop micro-fluidic chip |
| Publication Number | Publication Date |
|---|---|
| CN108117968Atrue CN108117968A (en) | 2018-06-05 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611061683.7APendingCN108117968A (en) | 2016-11-28 | 2016-11-28 | A kind of single celled method of high-throughput automatic capture based on drop micro-fluidic chip |
| Country | Link |
|---|---|
| CN (1) | CN108117968A (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110878250A (en)* | 2018-09-05 | 2020-03-13 | 上海新微技术研发中心有限公司 | dPCR in-situ chip based on through hole structure and microfluidics and manufacturing method |
| CN113101989A (en)* | 2021-03-30 | 2021-07-13 | 苏州大学 | Cell capturing and stretching integrated arrayed microfluidic chip |
| CN113462543A (en)* | 2021-06-22 | 2021-10-01 | 东南大学 | Micro-fluidic chip for quantitatively detecting cancer cells in blood |
| CN113755332A (en)* | 2021-10-08 | 2021-12-07 | 临沂大学 | Single droplet replacement capture microchip system with high temporal resolution and its applications |
| CN114177955A (en)* | 2020-09-14 | 2022-03-15 | 中国科学院大连化学物理研究所 | A kind of microfluidic chip, template and preparation method |
| CN114247485A (en)* | 2020-09-25 | 2022-03-29 | 中国科学院青岛生物能源与过程研究所 | Micro-fluidic chip for particle screening and separation |
| CN114426922A (en)* | 2022-01-24 | 2022-05-03 | 国科温州研究院(温州生物材料与工程研究所) | High-throughput microfluidic chip for measuring cell mechanical parameters with wide pressure range and its application |
| CN115612613A (en)* | 2021-07-12 | 2023-01-17 | 上海新微技术研发中心有限公司 | Integrated micro-fluidic chip and single cell culture, screening and export method thereof |
| CN116218654A (en)* | 2023-03-21 | 2023-06-06 | 中国科学院深圳先进技术研究院 | A microfluidic chip, preparation method and detection method of bacterial drug resistance |
| CN118268057A (en)* | 2024-05-09 | 2024-07-02 | 东北大学 | Microflow control chip, system and method for single extracellular vesicle separation analysis |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103611584A (en)* | 2013-10-29 | 2014-03-05 | 武汉斯坦姆赛尔生物技术有限公司 | Micro-fluidic chip and micro-fluidic chip based cell counting method |
| CN103894248A (en)* | 2014-04-09 | 2014-07-02 | 国家纳米科学中心 | Micro-fluidic chip and micro-fluidic chip system for single cell analysis and single cell analyzing method |
| US20160144362A1 (en)* | 2013-05-23 | 2016-05-26 | The Regents Of The University Of California | Proximal degas driven microfluidic actuation |
| CN206512222U (en)* | 2016-11-28 | 2017-09-22 | 中国科学院大连化学物理研究所 | A kind of single celled micro-fluidic chip of high flux automatic capture |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160144362A1 (en)* | 2013-05-23 | 2016-05-26 | The Regents Of The University Of California | Proximal degas driven microfluidic actuation |
| CN103611584A (en)* | 2013-10-29 | 2014-03-05 | 武汉斯坦姆赛尔生物技术有限公司 | Micro-fluidic chip and micro-fluidic chip based cell counting method |
| CN103894248A (en)* | 2014-04-09 | 2014-07-02 | 国家纳米科学中心 | Micro-fluidic chip and micro-fluidic chip system for single cell analysis and single cell analyzing method |
| CN206512222U (en)* | 2016-11-28 | 2017-09-22 | 中国科学院大连化学物理研究所 | A kind of single celled micro-fluidic chip of high flux automatic capture |
| Title |
|---|
| JIANBO SHAO等: "A microfluidic chip for permeability assays", 《BIOMED MICRODEVICES》* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110878250A (en)* | 2018-09-05 | 2020-03-13 | 上海新微技术研发中心有限公司 | dPCR in-situ chip based on through hole structure and microfluidics and manufacturing method |
| CN114177955A (en)* | 2020-09-14 | 2022-03-15 | 中国科学院大连化学物理研究所 | A kind of microfluidic chip, template and preparation method |
| CN114247485A (en)* | 2020-09-25 | 2022-03-29 | 中国科学院青岛生物能源与过程研究所 | Micro-fluidic chip for particle screening and separation |
| CN114247485B (en)* | 2020-09-25 | 2022-12-30 | 中国科学院青岛生物能源与过程研究所 | Micro-fluidic chip for particle screening and separation |
| CN113101989A (en)* | 2021-03-30 | 2021-07-13 | 苏州大学 | Cell capturing and stretching integrated arrayed microfluidic chip |
| CN113462543A (en)* | 2021-06-22 | 2021-10-01 | 东南大学 | Micro-fluidic chip for quantitatively detecting cancer cells in blood |
| CN113462543B (en)* | 2021-06-22 | 2024-02-02 | 东南大学 | Microfluidic chip for quantitatively detecting cancer cells in blood |
| CN115612613A (en)* | 2021-07-12 | 2023-01-17 | 上海新微技术研发中心有限公司 | Integrated micro-fluidic chip and single cell culture, screening and export method thereof |
| CN113755332B (en)* | 2021-10-08 | 2023-10-31 | 临沂大学 | Single drop replacement capture microchip system with high time resolution and application thereof |
| CN113755332A (en)* | 2021-10-08 | 2021-12-07 | 临沂大学 | Single droplet replacement capture microchip system with high temporal resolution and its applications |
| CN114426922A (en)* | 2022-01-24 | 2022-05-03 | 国科温州研究院(温州生物材料与工程研究所) | High-throughput microfluidic chip for measuring cell mechanical parameters with wide pressure range and its application |
| CN114426922B (en)* | 2022-01-24 | 2023-12-22 | 国科温州研究院(温州生物材料与工程研究所) | High-flux micro-fluidic chip for measuring cell mechanical parameters in wide pressure range and application |
| CN116218654A (en)* | 2023-03-21 | 2023-06-06 | 中国科学院深圳先进技术研究院 | A microfluidic chip, preparation method and detection method of bacterial drug resistance |
| CN118268057A (en)* | 2024-05-09 | 2024-07-02 | 东北大学 | Microflow control chip, system and method for single extracellular vesicle separation analysis |
| Publication | Publication Date | Title |
|---|---|---|
| CN108117968A (en) | A kind of single celled method of high-throughput automatic capture based on drop micro-fluidic chip | |
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