技术领域technical field
本发明涉及一种基于液滴寄存的肿瘤细胞筛查微流控装置及筛查方法，属于微流控技术领域。The invention relates to a microfluidic device and a screening method for tumor cell screening based on droplet storage, belonging to the field of microfluidic technology.
背景技术Background technique
微流控芯片以及广泛应用于疾病诊断、药物筛选、环境检测、食品安全等领域。微流控芯片的基本要点之一就是对微尺度下的流体进行操作和控制,而作为操作和控制的对象的流体量又极其微小，物质的很多表现形式和常量物质有所不同。肿瘤症是威胁人类生命健康最危险的疾病之一，其产生的根本原因是控制细胞生长增殖机制失常，即与细胞凋亡的异常有关，肿瘤细胞传统的医学检测方法对其检测效率较低所致。因此，如何快速、准确的对肿瘤细胞进行定量检测与分选是现代生物学研究的紧迫问题。Microfluidic chips are widely used in disease diagnosis, drug screening, environmental testing, food safety and other fields. One of the basic points of the microfluidic chip is to operate and control the fluid at the micro scale, and the amount of fluid as the object of operation and control is extremely small, and many manifestations of substances are different from constant substances. Tumor is one of the most dangerous diseases that threaten human life and health. The root cause of its occurrence is the abnormality of the mechanism of controlling cell growth and proliferation, which is related to the abnormality of cell apoptosis. The traditional medical detection methods for tumor cells have low detection efficiency. Sincerely. Therefore, how to quickly and accurately quantitatively detect and sort tumor cells is an urgent problem in modern biological research.
发明内容Contents of the invention
本发明要解决的技术问题是提供一种基于液滴寄存的肿瘤细胞筛查微流控装置及筛查方法，能够实现微量及恒量物质的输运以及高效检测。The technical problem to be solved by the present invention is to provide a microfluidic device and screening method for tumor cell screening based on droplet storage, which can realize the transportation and efficient detection of trace and constant substances.
本发明采用的技术方案：一种基于液滴寄存的肿瘤细胞筛查微流控装置，包括玻璃基片1和固定在其上的PDMS芯片2、控制器，PDMS芯片2上包含了连续相入口4、聚焦结构5、连续相出口7、分散相入口6、寄存器通道、出口3，聚焦结构5为十字形通道，聚焦结构5的两个入口端通过微通道分别与连续相入口4、分散相入口6连接，两个出口端通过微通道分别与连续相出口7及寄存器通道的入口连接，从分散相入口6进入的分散相与从连续相入口4进入的连续相经过共聚焦结构5生成包裹细胞液滴8，包裹细胞液滴8通过聚焦结构5与寄存器通道的入口之间的微通道进入寄存器通道，寄存器通道为向上连续折弯形且其内自前向后依次设有若干个结构相同、长度逐渐变短的寄存器，寄存器与控制器连接，相邻两个寄存器上下交错设在寄存器通道的折弯处，包裹细胞液滴8以固定的频率依次通过每个寄存器，每个寄存器可检测通过其的包裹细胞液滴8的个数，寄存器通道的出口通过微通道与出口3连接。The technical solution adopted in the present invention: a microfluidic device for tumor cell screening based on droplet storage, including a glass substrate 1, a PDMS chip 2 fixed on it, and a controller, and the PDMS chip 2 includes a continuous phase inlet 4. Focusing structure 5, continuous phase outlet 7, dispersed phase inlet 6, register channel, outlet 3, focusing structure 5 is a cross-shaped channel, and the two inlet ports of focusing structure 5 are respectively connected to continuous phase inlet 4 and dispersed phase through microchannels. The inlet 6 is connected, and the two outlets are respectively connected to the continuous phase outlet 7 and the inlet of the register channel through microchannels. The dispersed phase entering from the dispersed phase inlet 6 and the continuous phase entering from the continuous phase inlet 4 pass through the confocal structure 5 to generate parcels The cell droplet 8, the cell droplet 8 enters the register channel through the microchannel between the focusing structure 5 and the entrance of the register channel. The registers are gradually shortened in length, the registers are connected to the controller, and two adjacent registers are staggered up and down at the bend of the register channel, and the wrapped cell droplets 8 pass through each register in turn at a fixed frequency, and each register can be detected It is the number of wrapped cell droplets 8, and the outlet of the register channel is connected with the outlet 3 through the microchannel.
优选地，所述的出口3、连续相入口4、分散相入口6、连续相出口7整齐排列在PDMS芯片2的左侧，寄存器通道设置在PDMS芯片2的右侧。Preferably, the outlet 3 , the continuous phase inlet 4 , the dispersed phase inlet 6 , and the continuous phase outlet 7 are neatly arranged on the left side of the PDMS chip 2 , and the register channel is set on the right side of the PDMS chip 2 .
具体地，所述的寄存器通道内自前向后依次设有液滴寄存器Ⅰ9、液滴寄存器Ⅱ10、液滴寄存器Ⅲ11、液滴寄存器Ⅳ12、液滴寄存器Ⅴ13、液滴寄存器Ⅵ14，相邻液滴寄存器之间通过微通道连接，液滴寄存器Ⅴ13包括寄存腔、激光检测器13-5，激光检测器13-5与控制器连接，寄存腔包括寄存腔入口13-2、腔体、寄存腔出口13-4，寄存腔入口13-2与前端微通道连接，寄存腔出口13-4与折弯处的竖向微通道连接，激光检测器13-5安装在寄存腔正上方的微通道中，寄存腔的宽度大于前后连接的微通道的宽度，寄存腔的腔体内设有与腔体内壁间隔放置的长方形辅助结构Ⅰ13-1、长方形辅助结构Ⅱ13-3，长方形辅助结构Ⅱ13-3位于长方形辅助结构Ⅰ13-1的末端且其长度大于长方形辅助结构Ⅰ13-1的长度，长方形辅助结构Ⅰ13-1包括若干组上下对称且水平放置的长方形板，同一水平线上相邻两长方形辅助结构Ⅰ13-1等间隔设置，相对两长方形辅助结构Ⅰ13-1之间的宽度等于前端微通道的宽度，长方形辅助结构Ⅱ13-3包括相互垂直的一水平放置的长方形板和一垂直放置的长方形板，垂直放置的长方形辅助结构Ⅱ13-3位于水平放置的长方形辅助结构Ⅱ13-3的上方且垂直放置的长方形辅助结构Ⅱ13-3与上方的长方形辅助结构Ⅰ13-1的末端形成缺口，进入寄存腔内的包裹细胞液滴8从缺口挤出后从寄存腔出口13-4进入后端的微通道。Specifically, the register channel is provided with droplet register I9, droplet register II10, droplet register III11, droplet register IV12, droplet register V13, droplet register VI14, and adjacent droplet registers from front to back. The droplet register V13 includes a storage cavity, a laser detector 13-5, and the laser detector 13-5 is connected to the controller. The storage cavity includes a storage cavity entrance 13-2, a cavity body, and a storage cavity exit 13. -4, the storage cavity entrance 13-2 is connected to the front microchannel, the storage cavity exit 13-4 is connected to the vertical microchannel at the bend, and the laser detector 13-5 is installed in the microchannel directly above the storage cavity, and the storage cavity The width of the cavity is greater than the width of the microchannels connected before and after. The cavity of the storage cavity is provided with a rectangular auxiliary structure I13-1 and a rectangular auxiliary structure II13-3 placed at intervals from the inner wall of the cavity. The rectangular auxiliary structure II13-3 is located in the rectangular auxiliary structure The end of I13-1 and its length is greater than the length of the rectangular auxiliary structure I13-1. The rectangular auxiliary structure I13-1 includes several groups of vertically symmetrical and horizontally placed rectangular plates. Two adjacent rectangular auxiliary structures I13-1 are equally spaced on the same horizontal line It is set that the width between the two rectangular auxiliary structures I13-1 is equal to the width of the front-end microchannel, and the rectangular auxiliary structure II13-3 includes a horizontally placed rectangular plate and a vertically placed rectangular plate perpendicular to each other, and the vertically placed rectangular auxiliary Structure II13-3 is located above the horizontally placed rectangular auxiliary structure II13-3, and the vertically placed rectangular auxiliary structure II13-3 forms a gap with the end of the upper rectangular auxiliary structure I13-1, and enters the wrapped cell droplet in the storage cavity8 After extruding from the gap, enter the microchannel at the rear end from the storage cavity outlet 13-4.
具体地，所述PDMS芯片2长为120mm，宽为60mm，高为10mm；所有的微通道的宽为0.020mm，高为0.010mm。Specifically, the PDMS chip 2 has a length of 120 mm, a width of 60 mm, and a height of 10 mm; all microchannels have a width of 0.020 mm and a height of 0.010 mm.
具体地，所述液滴寄存器Ⅰ9、液滴寄存器Ⅱ10、液滴寄存器Ⅲ11、液滴寄存器Ⅳ12、液滴寄存器Ⅴ13、液滴寄存器Ⅵ14中寄存腔腔体在水平方向的长度分别为0.180mm、0.160mm、0.140mm、0.120mm、0.100mm、0.080mm；各寄存器寄存腔腔体的宽均为0.040mm，高均为0.010mm；长方形辅助结构Ⅰ13-1长为0.010mm，宽为0.003mm，数量为8个；长方形辅助结构Ⅱ13-3长为0.020mm，宽为0.003mm，同一水平线上相邻两长方形辅助结构Ⅰ13-1的间隔为0.005mm；长方形辅助结构Ⅰ13-1、长方形辅助结构Ⅱ13-3与对应寄存器腔体内壁之间的宽度均为0.005mm；垂直放置的长方形辅助结构Ⅱ13-3与上方的长方形辅助结构Ⅰ13-1的末端形成缺口的宽度为0.005mm。Specifically, the lengths of the storage cavities in the horizontal direction in the droplet register I9, droplet register II10, droplet register III11, droplet register IV12, droplet register V13, and droplet register VI14 are 0.180 mm and 0.160 mm respectively. mm, 0.140mm, 0.120mm, 0.100mm, 0.080mm; the width of each register storage cavity is 0.040mm, and the height is 0.010mm; the length of the rectangular auxiliary structure I13-1 is 0.010mm, and the width is 0.003mm. 8 pieces; the length of the rectangular auxiliary structure II13-3 is 0.020 mm, the width is 0.003 mm, and the distance between two adjacent rectangular auxiliary structures I13-1 on the same horizontal line is 0.005 mm; the rectangular auxiliary structure I13-1 and the rectangular auxiliary structure II13- The width between 3 and the inner wall of the corresponding register cavity is 0.005mm; the width of the gap between the vertically placed rectangular auxiliary structure II13-3 and the end of the upper rectangular auxiliary structure I13-1 is 0.005mm.
一种所述的基于液滴寄存的肿瘤细胞筛查微流控装置的筛查方法，具体步骤如下：A screening method of a microfluidic device for screening tumor cells based on droplet registration, the specific steps are as follows:
Step1：向连续相入口4注入氟化油 HFE-7500，分散相入口6注入带有肿瘤细胞的血液，通过分别控制分散相入口6和连续相入口4的入口速度，当正常血红细胞进入十字型共聚焦结构5后，会稳定生成0.020mm直径的带有单个血红细胞的包裹细胞液滴8，当比血红细胞大或者小的肿瘤细胞进入十字型共聚焦结构5后，会生成比带有单个血红细胞的包裹细胞液滴8大或者小的包裹细胞液滴8，包裹细胞液滴8依次通过液滴寄存器Ⅰ9、液滴寄存器Ⅱ10、液滴寄存器Ⅲ11、液滴寄存器Ⅳ12、液滴寄存器Ⅴ13、液滴寄存器Ⅵ14，各个液滴寄存器能容纳包裹细胞液滴8的数量是固定的，每一个液滴寄存器中最后进入的超出容纳个数的包裹细胞液滴8会把最前面的一个液滴挤出该液滴寄存器，每个液滴寄存器中的激光检测器检测出经过各自液滴寄存器的包裹细胞液滴8的个数，并将检测结果传送给控制器，从最后一个液滴寄存器流出的包裹细胞液滴8从出口3流至外部；Step1: Inject fluorinated oil HFE-7500 into continuous phase inlet 4, and inject blood with tumor cells into dispersed phase inlet 6. By controlling the inlet speeds of dispersed phase inlet 6 and continuous phase inlet 4 respectively, when normal red blood cells enter the cross After the confocal structure 5, a wrapped cell droplet 8 with a diameter of 0.020mm with a single red blood cell will be stably generated. The wrapped cell droplet 8 of the red blood cell is a large or small wrapped cell droplet 8, and the wrapped cell droplet 8 sequentially passes through the droplet register Ⅰ9, the droplet register Ⅱ10, the droplet register Ⅲ11, the droplet register Ⅳ12, the droplet register Ⅴ13, Droplet register Ⅵ14, each droplet register can hold a fixed number of wrapped cell droplets 8, and the last droplet 8 that enters in each droplet register will squeeze the first droplet. Out of the droplet register, the laser detector in each droplet register detects the number of wrapped cell droplets 8 passing through the respective droplet register, and transmits the detection result to the controller, and the flow out of the last droplet register The wrapped cell droplet 8 flows from the outlet 3 to the outside;
Step2：由于血红细胞和肿瘤细胞的尺寸和柔软程度不同，每个液滴寄存器在挤压包裹不同细胞的包裹细胞液滴8时所需要的个数不同，控制器内存储有固定时间内全部为正常血红细胞通过时通过各液滴寄存器的包裹细胞液滴8的数量，控制器根据每个液滴寄存器中的激光检测器的实时检测结构判断出血液中是否含有肿瘤细胞，同一时间内，当通过各液滴寄存器的包裹细胞液滴8的数量与全部为正常血红细胞通过时通过各液滴寄存器的包裹细胞液滴8的数量不同，则认为存在肿瘤细胞。Step2: Due to the different sizes and softness of red blood cells and tumor cells, each droplet register requires different numbers of wrapped cell droplets 8 to squeeze and wrap different cells. The number of wrapped cell droplets 8 passing through each droplet register when normal red blood cells pass through, the controller judges whether there are tumor cells in the blood according to the real-time detection structure of the laser detector in each droplet register, and at the same time, when If the number of encapsulated cell droplets 8 passing through each droplet register is different from the number of encapsulated cell droplets 8 passing through each droplet register when all normal red blood cells pass through, it is considered that there are tumor cells.
液滴寄存器Ⅰ9、液滴寄存器Ⅱ10、液滴寄存器Ⅲ11、液滴寄存器Ⅳ12、液滴寄存器Ⅴ13、液滴寄存器Ⅵ14能容纳带有单个血红细胞的包裹细胞液滴8的数量分别为7个、6个、5个、4个、3个、2个。Droplet register Ⅰ9, droplet register Ⅱ10, droplet register Ⅲ11, droplet register Ⅳ12, droplet register Ⅴ13, and droplet register Ⅵ14 can hold 7 and 6 wrapped cell droplets 8 with a single red blood cell, respectively 1, 5, 4, 3, 2.
本发明的有益效果是：The beneficial effects of the present invention are:
1、本装置结构简单、操作方便、灵敏度高。1. The device has simple structure, convenient operation and high sensitivity.
2、分散相采用氟化油HFE-7500 ，氟化油具有良好的生物兼容性，对液滴中包裹的细胞、病毒等生物颗粒没有毒性影响，保证液滴中生物反应能够正常进行。2. The dispersed phase uses fluorinated oil HFE-7500. The fluorinated oil has good biocompatibility and has no toxic effects on the cells, viruses and other biological particles wrapped in the droplets, ensuring that the biological reactions in the droplets can proceed normally.
3、本发明使用力学分析替代了传统医疗诊断技术所使用的血液生化检验分析，通过多个液滴寄存器的液滴通过的检测，可以实现极少样品量的高灵敏度实时检测。3. The present invention uses mechanical analysis to replace the blood biochemical analysis used in traditional medical diagnosis technology, and can realize high-sensitivity real-time detection with a very small amount of samples through the detection of droplet passing through multiple droplet registers.
4、本发明分析时间短、样品消耗量少、高灵敏度且成本较低，可快速的检测出是否有肿瘤细胞。4. The present invention has short analysis time, less sample consumption, high sensitivity and low cost, and can quickly detect whether there are tumor cells.
附图说明Description of drawings
图1为本发明的整体结构示意图；Fig. 1 is the overall structure schematic diagram of the present invention;
图2为图1中液滴寄存器Ⅴ的放大结构示意图；FIG. 2 is a schematic diagram of an enlarged structure of the droplet register V in FIG. 1;
图3为本发明实施例中各液滴寄存器存放A肿瘤细胞、正常血红细胞、B肿瘤细胞的数量分布图。FIG. 3 is a distribution diagram of the number of A tumor cells, normal red blood cells, and B tumor cells stored in each droplet register in the embodiment of the present invention.
图中各标号为：玻璃基片-1、PDMS芯片-2、出口-3、连续相入口-4、共聚焦结构-5、分散相入口-6、连续相出口-7、包裹细胞液滴-8、液滴寄存器Ⅰ-9、液滴寄存器Ⅱ-10、液滴寄存器Ⅲ-11、液滴寄存器Ⅳ-12、液滴寄存器Ⅴ-13、液滴寄存器Ⅵ-14、长方形辅助结构Ⅰ-13-1、寄存腔入口-13-2、长方形辅助结构Ⅱ-13-3、寄存腔出口-13-4，激光检测器-13-5。The labels in the figure are: glass substrate-1, PDMS chip-2, outlet-3, continuous phase inlet-4, confocal structure-5, dispersed phase inlet-6, continuous phase outlet-7, wrapped cell droplet- 8. Droplet Register Ⅰ-9, Droplet Register Ⅱ-10, Droplet Register Ⅲ-11, Droplet Register Ⅳ-12, Droplet Register Ⅴ-13, Droplet Register Ⅵ-14, Rectangular Auxiliary Structure Ⅰ-13 -1. Storage cavity entrance-13-2, rectangular auxiliary structure II-13-3, storage cavity exit-13-4, laser detector-13-5.
具体实施方式Detailed ways
下面结合附图和具体实施例，对本发明做进一步的说明。The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.
实施例1：一种基于液滴寄存的肿瘤细胞筛查微流控装置，包括玻璃基片1和固定在其上的PDMS芯片2、控制器，PDMS芯片2上包含了连续相入口4、聚焦结构5、连续相出口7、分散相入口6、寄存器通道、出口3，聚焦结构5为十字形通道，聚焦结构5的两个入口端通过微通道分别与连续相入口4、分散相入口6连接，两个出口端通过微通道分别与连续相出口7及寄存器通道的入口连接，从分散相入口6进入的分散相与从连续相入口4进入的连续相经过共聚焦结构5生成包裹细胞液滴8，包裹细胞液滴8通过聚焦结构5与寄存器通道的入口之间的微通道进入寄存器通道，寄存器通道为向上连续折弯形（如图1所示，为向上连续方波形折弯）且其内自前向后依次设有若干个结构相同、长度逐渐变短的寄存器，寄存器与控制器连接，相邻两个寄存器上下交错设在寄存器通道的折弯处，包裹细胞液滴8以固定的频率依次通过每个寄存器，每个寄存器可检测通过其的包裹细胞液滴8的个数，寄存器通道的出口通过微通道与出口3连接。Example 1: A microfluidic device for tumor cell screening based on droplet registration, including a glass substrate 1, a PDMS chip 2 fixed on it, and a controller. The PDMS chip 2 includes a continuous phase inlet 4, a focusing Structure 5, continuous phase outlet 7, dispersed phase inlet 6, register channel, outlet 3, focusing structure 5 is a cross-shaped channel, and the two inlet ports of focusing structure 5 are respectively connected to continuous phase inlet 4 and dispersed phase inlet 6 through microchannels , the two outlets are respectively connected to the continuous phase outlet 7 and the inlet of the register channel through microchannels, the dispersed phase entering from the dispersed phase inlet 6 and the continuous phase entering from the continuous phase inlet 4 pass through the confocal structure 5 to generate encapsulating cell droplets 8. The encapsulated cell droplet 8 enters the register channel through the microchannel between the focusing structure 5 and the entrance of the register channel. The register channel is in an upward continuous bending shape (as shown in Figure 1, it is an upward continuous square waveform bending) and its There are a number of registers with the same structure and gradually shortening length in sequence from front to back. The registers are connected to the controller, and two adjacent registers are staggered up and down at the bend of the register channel to wrap the cell droplet 8 at a fixed frequency. Through each register in turn, each register can detect the number of wrapped cell droplets 8 passing through it, and the outlet of the register channel is connected with the outlet 3 through the microchannel.
进一步地，所述的出口3、连续相入口4、分散相入口6、连续相出口7整齐排列在PDMS芯片2的左侧，寄存器通道设置在PDMS芯片2的右侧。出口3、连续相入口4、分散相入口6、连续相出口7整齐排列在芯片的左侧是为了方便插入毛细管通入液体。由于各寄存器被挤出去的包裹细胞液滴8出口向上，所以将各个寄存器交替向上串联排列在PDMS芯片2的右侧。Further, the outlet 3, the continuous phase inlet 4, the dispersed phase inlet 6, and the continuous phase outlet 7 are neatly arranged on the left side of the PDMS chip 2, and the register channel is set on the right side of the PDMS chip 2. The outlet 3, the continuous phase inlet 4, the dispersed phase inlet 6, and the continuous phase outlet 7 are neatly arranged on the left side of the chip for the convenience of inserting the capillary to feed the liquid. Since the outlet of the encapsulated cell droplet 8 extruded by each register is upward, the registers are alternately arranged upward in series on the right side of the PDMS chip 2 .
进一步地，所述的寄存器通道内自前向后依次设有液滴寄存器Ⅰ9、液滴寄存器Ⅱ10、液滴寄存器Ⅲ11、液滴寄存器Ⅳ12、液滴寄存器Ⅴ13、液滴寄存器Ⅵ14，相邻液滴寄存器之间通过微通道连接，如图2所示，由于各液滴寄存器结构相同，仅以液滴寄存器Ⅴ13为例，对个液滴寄存器的结构进行详细说明：液滴寄存器Ⅴ13包括寄存腔、激光检测器13-5，激光检测器13-5与控制器连接，寄存腔包括寄存腔入口13-2、腔体、寄存腔出口13-4，寄存腔入口13-2与前端微通道连接，寄存腔出口13-4与折弯处的竖向微通道连接，激光检测器13-5安装在寄存腔正上方的微通道中，寄存腔的宽度大于前后连接的微通道的宽度，寄存腔的腔体内设有与腔体内壁间隔放置的长方形辅助结构Ⅰ13-1、长方形辅助结构Ⅱ13-3，长方形辅助结构Ⅱ13-3位于长方形辅助结构Ⅰ13-1的末端且其长度大于长方形辅助结构Ⅰ13-1的长度，长方形辅助结构Ⅰ13-1包括若干组上下对称且水平放置的长方形板，同一水平线上相邻两长方形辅助结构Ⅰ13-1等间隔设置（是因为因为从寄存腔通过的除了包裹细胞液滴8还有连续相液体，有间隔方便连续相液体通过），相对两长方形辅助结构Ⅰ13-1之间的宽度等于前端微通道的宽度，长方形辅助结构Ⅱ13-3包括相互垂直的一水平放置的长方形板和一垂直放置的长方形板，垂直放置的长方形辅助结构Ⅱ13-3位于水平放置的长方形辅助结构Ⅱ13-3的上方且垂直放置的长方形辅助结构Ⅱ13-3与上方的长方形辅助结构Ⅰ13-1的末端形成缺口，进入寄存腔内的包裹细胞液滴8从缺口挤出后从寄存腔出口13-4进入后端的微通道，从缺口挤出的包裹细胞液滴8如图2所示椭圆形液滴。Further, the register channel is sequentially provided with droplet register I9, droplet register II10, droplet register III11, droplet register IV12, droplet register V13, droplet register VI14, and adjacent droplet registers from front to back. They are connected through micro-channels, as shown in Figure 2. Since the structure of each droplet register is the same, only the droplet register V13 is taken as an example to describe the structure of each droplet register in detail: the droplet register V13 includes a register cavity, a laser The detector 13-5, the laser detector 13-5 is connected with the controller, the storage cavity includes the storage cavity entrance 13-2, the cavity body, and the storage cavity exit 13-4, the storage cavity entrance 13-2 is connected with the front-end microchannel, and the storage cavity The cavity outlet 13-4 is connected with the vertical microchannel at the bend, and the laser detector 13-5 is installed in the microchannel directly above the storage cavity. The width of the storage cavity is greater than the width of the microchannels connected before and after. There are rectangular auxiliary structures I13-1 and rectangular auxiliary structures II13-3 spaced from the inner wall of the cavity in the body. The rectangular auxiliary structures II13-3 are located at the end of the rectangular auxiliary structures I13-1 and their length is longer than that of the rectangular auxiliary structures I13-1. Length, the rectangular auxiliary structure Ⅰ13-1 includes several groups of vertically symmetrical and horizontally placed rectangular plates, and two adjacent rectangular auxiliary structures Ⅰ13-1 are arranged at equal intervals on the same horizontal line (because the cells passing through the storage cavity except for the wrapped cell droplet 8 There is also a continuous phase liquid, there is an interval to facilitate the passage of the continuous phase liquid), the width between the two rectangular auxiliary structures I13-1 is equal to the width of the front microchannel, and the rectangular auxiliary structure II13-3 includes a horizontally placed rectangular plate perpendicular to each other and a vertically placed rectangular plate, the vertically placed rectangular auxiliary structure II13-3 is located above the horizontally placed rectangular auxiliary structure II13-3 and the vertically placed rectangular auxiliary structure II13-3 is connected to the end of the upper rectangular auxiliary structure I13-1 A gap is formed, and the encapsulated cell droplet 8 entering the storage cavity is extruded from the gap and enters the microchannel at the rear end from the storage cavity outlet 13-4, and the encapsulated cell droplet 8 extruded from the gap is an elliptical droplet as shown in Figure 2 .
进一步地，所述PDMS芯片2长为120mm，宽为60mm，高为10mm；所有的微通道的宽为0.020mm，高为0.010mm。Further, the PDMS chip 2 has a length of 120mm, a width of 60mm, and a height of 10mm; all microchannels have a width of 0.020mm and a height of 0.010mm.
进一步地，所述液滴寄存器Ⅰ9、液滴寄存器Ⅱ10、液滴寄存器Ⅲ11、液滴寄存器Ⅳ12、液滴寄存器Ⅴ13、液滴寄存器Ⅵ14中寄存腔腔体在水平方向的长度分别为0.180mm、0.160mm、0.140mm、0.120mm、0.100mm、0.080mm；各寄存器寄存腔腔体的宽均为0.040mm，高均为0.010mm；长方形辅助结构Ⅰ13-1长为0.010mm，宽为0.003mm，数量为8个；长方形辅助结构Ⅱ13-3长为0.020mm，宽为0.003mm，同一水平线上相邻两长方形辅助结构Ⅰ13-1的间隔为0.005mm；长方形辅助结构Ⅰ13-1、长方形辅助结构Ⅱ13-3与对应寄存器腔体内壁之间的宽度均为0.005mm；垂直放置的长方形辅助结构Ⅱ13-3与上方的长方形辅助结构Ⅰ13-1的末端形成缺口的宽度为0.005mm。Further, the lengths of the storage cavities in the horizontal direction in the droplet register I9, droplet register II10, droplet register III11, droplet register IV12, droplet register V13, and droplet register VI14 are 0.180mm and 0.160mm respectively. mm, 0.140mm, 0.120mm, 0.100mm, 0.080mm; the width of each register storage cavity is 0.040mm, and the height is 0.010mm; the length of the rectangular auxiliary structure I13-1 is 0.010mm, and the width is 0.003mm. 8 pieces; the length of the rectangular auxiliary structure II13-3 is 0.020 mm, the width is 0.003 mm, and the distance between two adjacent rectangular auxiliary structures I13-1 on the same horizontal line is 0.005 mm; the rectangular auxiliary structure I13-1 and the rectangular auxiliary structure II13- The width between 3 and the inner wall of the corresponding register cavity is 0.005mm; the width of the gap between the vertically placed rectangular auxiliary structure II13-3 and the end of the upper rectangular auxiliary structure I13-1 is 0.005mm.
一种所述的基于液滴寄存的肿瘤细胞筛查微流控装置的筛查方法，具体步骤如下：A screening method of a microfluidic device for screening tumor cells based on droplet registration, the specific steps are as follows:
Step1：向连续相入口4注入氟化油 HFE-7500，分散相入口6注入带有肿瘤细胞的血液，通过分别控制分散相入口6和连续相入口4的入口速度，当正常血红细胞进入十字型共聚焦结构5后，会稳定生成0.020mm直径的带有单个血红细胞的包裹细胞液滴8，当比血红细胞大或者小的肿瘤细胞进入十字型共聚焦结构5后，会生成比带有单个血红细胞的包裹细胞液滴8大或者小的包裹细胞液滴8，包裹细胞液滴8依次通过液滴寄存器Ⅰ9、液滴寄存器Ⅱ10、液滴寄存器Ⅲ11、液滴寄存器Ⅳ12、液滴寄存器Ⅴ13、液滴寄存器Ⅵ14，各个液滴寄存器能容纳包裹细胞液滴8的数量是固定的，每一个液滴寄存器中最后进入的超出容纳个数的包裹细胞液滴8会把最前面的一个液滴挤出该液滴寄存器，每个液滴寄存器中的激光检测器检测出经过各自液滴寄存器的包裹细胞液滴8的个数，并将检测结果传送给控制器，从最后一个液滴寄存器流出的包裹细胞液滴8从出口3流至外部；Step1: Inject fluorinated oil HFE-7500 into continuous phase inlet 4, and inject blood with tumor cells into dispersed phase inlet 6. By controlling the inlet speeds of dispersed phase inlet 6 and continuous phase inlet 4 respectively, when normal red blood cells enter the cross After the confocal structure 5, a wrapped cell droplet 8 with a diameter of 0.020mm with a single red blood cell will be stably generated. The wrapped cell droplet 8 of the red blood cell is a large or small wrapped cell droplet 8, and the wrapped cell droplet 8 sequentially passes through the droplet register Ⅰ9, the droplet register Ⅱ10, the droplet register Ⅲ11, the droplet register Ⅳ12, the droplet register Ⅴ13, Droplet register Ⅵ14, each droplet register can hold a fixed number of wrapped cell droplets 8, and the last droplet 8 that enters in each droplet register will squeeze the first droplet. Out of the droplet register, the laser detector in each droplet register detects the number of wrapped cell droplets 8 passing through the respective droplet register, and transmits the detection result to the controller, and the flow out of the last droplet register The wrapped cell droplet 8 flows from the outlet 3 to the outside;
Step2：由于血红细胞和肿瘤细胞的尺寸和柔软程度不同，每个液滴寄存器在挤压包裹不同细胞的包裹细胞液滴8时所需要的个数不同，控制器内存储有固定时间内全部为正常血红细胞通过时通过各液滴寄存器的包裹细胞液滴8的数量，控制器根据每个液滴寄存器中的激光检测器的实时检测结构判断出血液中是否含有肿瘤细胞，同一时间内，当通过各液滴寄存器的包裹细胞液滴8的数量与全部为正常血红细胞通过时通过各液滴寄存器的包裹细胞液滴8的数量不同，则认为存在肿瘤细胞。Step2: Due to the different sizes and softness of red blood cells and tumor cells, each droplet register requires different numbers of wrapped cell droplets 8 to squeeze and wrap different cells. The number of wrapped cell droplets 8 passing through each droplet register when normal red blood cells pass through, the controller judges whether there are tumor cells in the blood according to the real-time detection structure of the laser detector in each droplet register, and at the same time, when If the number of encapsulated cell droplets 8 passing through each droplet register is different from the number of encapsulated cell droplets 8 passing through each droplet register when all normal red blood cells pass through, it is considered that there are tumor cells.
液滴寄存器Ⅰ9、液滴寄存器Ⅱ10、液滴寄存器Ⅲ11、液滴寄存器Ⅳ12、液滴寄存器Ⅴ13、液滴寄存器Ⅵ14能容纳带有单个血红细胞的包裹细胞液滴8的数量分别为7个、6个、5个、4个、3个、2个。Droplet register Ⅰ9, droplet register Ⅱ10, droplet register Ⅲ11, droplet register Ⅳ12, droplet register Ⅴ13, and droplet register Ⅵ14 can hold 7 and 6 wrapped cell droplets 8 with a single red blood cell, respectively 1, 5, 4, 3, 2.
本发明的工作原理是：由于肿瘤细胞和血细胞的尺寸和柔软度差异，通过液滴寄存器实现对包裹肿肿瘤细胞液滴通过的频率进行控制，利用激光检测液滴通过的频率，从而检测出是否有肿瘤细胞。The working principle of the present invention is: due to the difference in size and softness between tumor cells and blood cells, the droplet register is used to control the passing frequency of droplets wrapped in tumor cells, and the laser is used to detect the passing frequency of droplets, thereby detecting whether There are tumor cells.
本发明中的液滴寄存器Ⅰ9、液滴寄存器Ⅱ10、液滴寄存器Ⅲ11、液滴寄存器Ⅳ12、液滴寄存器Ⅴ13、液滴寄存器Ⅵ14分别能够同时容纳7个到2个直径为0.020mm的包裹细胞液滴8，如液滴寄存器Ⅰ9所示，当第八个包裹细胞液滴8进入后，会把最前面的一个包裹细胞液滴8挤出液滴寄存器Ⅰ9，如图2中的椭圆形液滴就是即将被挤出去的包裹细胞液滴8，由于血红细胞和肿瘤细胞的尺寸和柔软程度不同，每个寄存器在挤压包裹不同细胞的液滴时所需要的个数不同，从而判断出血液中是否含有肿瘤细胞。包裹细胞液滴8从寄存器出口13-4出来后经过激光检测13-5，从而得到包裹细胞液滴8通过的频率，出来的包裹细胞液滴8依次经过液滴寄存器Ⅱ10、液滴寄存器Ⅲ11、液滴寄存器Ⅳ12、液滴寄存器Ⅴ13、液滴寄存器Ⅵ14，原理与液滴寄存器Ⅰ9一样，不过寄存器容纳的液滴数量根据包裹细胞尺寸不同所容纳的个数不同。Droplet register I9, droplet register II10, droplet register III11, droplet register IV12, droplet register V13, and droplet register VI14 in the present invention can respectively hold 7 to 2 wrapped cell fluids with a diameter of 0.020mm at the same time Droplet 8, as shown in the droplet register Ⅰ9, when the eighth cell droplet 8 enters, the first cell droplet 8 will be squeezed out of the droplet register Ⅰ9, as shown in the elliptical droplet in Figure 2 It is the encapsulating cell droplet 8 that is about to be extruded. Due to the different sizes and softness of red blood cells and tumor cells, each register needs a different number when extruding the encapsulating droplets of different cells, thus judging the blood contain tumor cells. After the wrapped cell droplet 8 comes out from the register outlet 13-4, it passes through the laser detection 13-5, so as to obtain the passing frequency of the wrapped cell droplet 8, and the wrapped cell droplet 8 passes through the droplet register II10, droplet register III11, Droplet register IV12, droplet register V13, and droplet register VI14 have the same principle as droplet register I9, but the number of droplets contained in the registers varies according to the size of the wrapped cells.
正常血红细胞的长大概在0.008mm左右，而肿瘤细胞大小不一，有比血红细胞大的，也有比血红细胞小的，假设正常包裹血红细胞的液滴，通过液滴寄存器Ⅰ9、液滴寄存器Ⅱ10、液滴寄存器Ⅲ11、液滴寄存器Ⅳ12、液滴寄存器Ⅴ13、液滴寄存器Ⅵ14挤出去需要的数量分别为8、7、6、5、4、3，挤出一个液滴寄存器Ⅰ9、液滴寄存器Ⅱ10、液滴寄存器Ⅲ11、液滴寄存器Ⅳ12、液滴寄存器Ⅴ13、液滴寄存器Ⅵ14需要的时间为1s，一个包裹细胞液滴8从进入到出去的时间大概需要33s；设比血红细胞小一倍左右肿瘤细胞的包裹细胞液滴8为A，比血红细胞大一倍左右肿瘤细胞的包裹细胞液滴8为B，当生成的包裹细胞液滴8中包含有一个细胞A或者B，可以得到如下表格的每个寄出器通过的个数：The length of normal red blood cells is about 0.008mm, while tumor cells vary in size, some are larger than red blood cells, and some are smaller than red blood cells. Ⅱ10, droplet register Ⅲ11, droplet register Ⅳ12, droplet register Ⅴ13, droplet register Ⅵ14. Droplet register Ⅱ10, droplet register Ⅲ11, droplet register Ⅳ12, droplet register Ⅴ13, droplet register Ⅵ14 takes 1s, and the time for a wrapped cell droplet 8 from entering to exiting takes about 33s; suppose it is smaller than red blood cells The encapsulating cell droplet 8 of about twice the size of the tumor cell is A, and the encapsulating cell droplet 8 of the tumor cell is about twice as large as the red blood cell. When the generated encapsulating cell droplet 8 contains a cell A or B, it can be Get the number of each sender passed in the following table:
检测之后，将每个寄存器通过包裹细胞液滴8的个数组合在一起形成这个细胞的固有的特征组合，可以得出图3的内容，从而可以判断出是否有A或者B肿瘤细胞，其他大小的肿瘤细胞检测原理一样，这样可以缩短检测时间，实现精确的检测。After detection, each register is combined by the number of wrapped cell droplets 8 to form the inherent characteristic combination of this cell, and the content in Figure 3 can be obtained, so that it can be judged whether there are A or B tumor cells, and other sizes The principle of tumor cell detection is the same, which can shorten the detection time and achieve accurate detection.
液滴微流控技术以其高通量、速度快的特征已成为近年来微流控技术的主流科研热点，它在生物颗粒的精确定量与快速检测方面独具优势。本发明以液滴微流控技术为出发点，面向生物颗粒的高通量、快速检测等关键科学技术问题开展研究，以期实现对肿瘤细胞等生物样品的精准检测，为疾病的早期诊断、快速治疗等提供理论依据与技术支撑。这种新方法将帮助医生更好地进行诊断和治疗。Droplet microfluidic technology has become the mainstream research focus of microfluidic technology in recent years due to its high throughput and fast speed. It has unique advantages in the precise quantification and rapid detection of biological particles. The present invention starts from droplet microfluidic technology, and conducts research on key scientific and technical issues such as high-throughput and rapid detection of biological particles, in order to realize accurate detection of biological samples such as tumor cells, and provide early diagnosis and rapid treatment of diseases. Provide theoretical basis and technical support. This new method will help doctors to better diagnose and treat.
本发明实现了将流体采样、驱动、操控、运输及检测分析集成于微流控芯片上，实现液体流动可控、微量及痕量物质的输运以及高效检测，制造成本低、便于携带、消耗试剂极少、分析速度快、检测效率高。The invention realizes the integration of fluid sampling, driving, manipulation, transportation and detection and analysis on the microfluidic chip, realizes controllable liquid flow, transportation of micro and trace substances and efficient detection, low manufacturing cost, easy to carry and consumes The reagents are few, the analysis speed is fast, and the detection efficiency is high.
以上结合附图对本发明的具体实施方式作了详细说明，但是本发明并不限于上述实施方式，在本领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。The specific embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Variations.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN110967513A (en)* | 2019-12-18 | 2020-04-07 | 京东方科技集团股份有限公司 | Sample prescreening chip, sample detection method and screening chip device |
| CN110967513B (en)* | 2019-12-18 | 2024-04-05 | 京东方科技集团股份有限公司 | Sample screening chip, sample detection method and screening chip device |
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| CN111019805A (en)* | 2019-12-20 | 2020-04-17 | 南通大学 | Microfluidic chip device for fixing single cell and performing medical analysis in situ and application thereof |
| CN111019805B (en)* | 2019-12-20 | 2023-01-24 | 南通大学 | Microfluidic chip device for fixing single cell and performing medical analysis in situ and application thereof |
| CN110982882B (en)* | 2019-12-20 | 2023-06-20 | 南通大学 | Microfluidic chip for single cell immobilization-isolation and in-situ nucleic acid amplification and application thereof |
| CN112557261A (en)* | 2020-12-07 | 2021-03-26 | 昆明理工大学 | Erythrocyte separation detection device and separation detection method based on C-shaped microcolumn |
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