WO2023168775A1 - 一种微粒子流动聚焦装置及方法 - Google Patents

一种微粒子流动聚焦装置及方法 Download PDF

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WO2023168775A1
WO2023168775A1 PCT/CN2022/085184 CN2022085184W WO2023168775A1 WO 2023168775 A1 WO2023168775 A1 WO 2023168775A1 CN 2022085184 W CN2022085184 W CN 2022085184W WO 2023168775 A1 WO2023168775 A1 WO 2023168775A1
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flow
microparticle
flow channel
sorting
focusing
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PCT/CN2022/085184
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English (en)
French (fr)
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陈华英
徐正
陈思远
陈震林
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珠海大略科技有限公司
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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
    • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules

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  • the invention relates to a microparticle flow focusing device and method, belonging to the field of biomedical analysis.
  • microspheres or cells in the sample are often studied, such as circulating tumor cells in blood samples or healthy cells in cell culture media. These particles often have special properties that can be used to detect and sort them out.
  • microfluidic flow cytometry is a technology for manipulating fluids in channels with dimensions in the range of tens of micrometers.
  • microfluidic flow cytometers are also composed of five major subsystems, of which sample pumping, focusing and sorting can mostly be completed on the microfluidic chip.
  • sample pumping, focusing and sorting can mostly be completed on the microfluidic chip.
  • the small size of microfluidic chips can greatly reduce the required sample and reagent volumes. And integrating it into the system can reduce the overall size and maintenance requirements of the equipment. Because of this, combining it with microfluidics is considered to be the further development direction of flow cytometers in the future.
  • the ability to detect single cells is one of the main features and capabilities of flow cytometry.
  • the sample When cells are injected into the flow cytometer, the sample needs to be focused into a narrow core flow so that it can pass through the detection area one by one at a certain rate. This is the pre-processing stage of the flow cytometer.
  • Focusing can be divided into active focusing and passive focusing. Passive focusing relies on the inherent characteristics of the fluid to achieve microparticle focusing, while active focusing often relies on external fields to generate driving force, including acoustoelectrophoresis (AP), dielectrophoresis (DEP), magnetic Electrophoresis (MP) and fluorescence activation separation methods.
  • AP acoustoelectrophoresis
  • DEP dielectrophoresis
  • MP magnetic Electrophoresis
  • Inertial microfluidics is a passive focusing method that uses inertial lift to drive particles to one or more equilibrium positions in a microchannel to achieve focusing function.
  • flow cytometry mostly uses sheath flow to focus microparticles, but its sheath flow increases the complexity of the microfluidic chip and requires a large amount of additional sheath fluid.
  • the high-speed flowing sheath fluid may also cause cell damage;
  • Active focusing equipment requires the introduction of external physical fields such as sound, light, electricity, magnetism, etc.
  • This focusing method greatly increases the complexity of focusing, and the intervention of external physical fields may also cause damage to the cells themselves, affecting their Active; although other passive focusing can do without introducing external physical fields, inertial microfluidics have low resolution and require high flow rates and long focusing lengths.
  • Other passive focusing methods such as DLD are difficult to combine with imaging, fluorescence and telecommunications to achieve deterministic sorting.
  • the present invention provides a microparticle flow focusing device and method, aiming to solve at least one of the technical problems existing in the prior art.
  • the technical solution of the present invention is a microparticle flow focusing device, which includes: a chip layer mechanism.
  • a microparticle focusing area is provided inside the chip layer mechanism.
  • the microparticle focusing area is provided with first flow channels on the upper and lower layers of the microparticle focusing area.
  • at least one row of micropore mechanisms corresponding to the bottom of the first flow channel, the micropore mechanism includes a plurality of micropores arranged in a row, and a particle inlet is provided at the beginning of the first flow channel.
  • microparticle sorting area the microparticle sorting area includes a plurality of sorting flow channels, and the ends of the sorting flow channels are connected with a sorting outlet; correspondingly arranged near each of the sorting flow channels.
  • Valve switching mechanism wherein the starting end of the sorting flow channel is connected with the end of the first flow channel.
  • micropore mechanism further includes a second flow channel, and the connected micropores are connected through the second flow channel.
  • microholes are mirror symmetrical to each other with respect to the central axis of the first flow channel.
  • a plurality of adjacent second flow channels and micropores are symmetrically arranged along the central axis of the flow direction, and the cross-sectional shapes of the micropores include circles, ellipses, triangles or rhombuses, etc. that can be converged.
  • One or more streamline shapes include circles, ellipses, triangles or rhombuses, etc.
  • the valve switching mechanism includes a pressure pump and a membrane layer.
  • a plurality of air passages are provided in the middle of the membrane layer. Both ends of the air passage are provided with air inlets and air outlets respectively.
  • the high-pressure outlet of the pressure pump and the low-pressure inlet are connected to the air inlet and the air outlet respectively, wherein the thin film layer is bonded and connected to the surface of the chip layer mechanism, and each of the air channels correspondingly spans above the beginning of the sorting flow channel, and the bottom surface of the air channel
  • the part that intersects with the separation flow channel is the switch layer.
  • the switch layer When the high-pressure outlet of the pressure pump passes high-pressure gas into the air channel, the switch layer is extruded by the high-pressure gas and expands toward the bottom of the separation flow channel, causing the switch layer to block the separation flow channel.
  • the switch layer When the high-pressure gas flows out of the outlet of the pressure pump, the deformation recovery of the switch layer clears the sorting flow channel.
  • the particle focusing area further includes a transition flow channel, one end of the transition flow channel is connected from the starting end of the sorting flow channel to the first flow channel in the direction of the particle inlet, and the other end of the transition flow channel is connected with the first flow channel. End connections.
  • valve switching mechanism includes a pressure pump and a control fluid channel.
  • the control fluid channel is arranged inside the chip layer mechanism. One end of the control fluid channel is connected to the beginning of the sorting flow channel.
  • the pressure pump The pressure port is connected to the other end of the control fluid channel.
  • a microparticle flow focusing and active sorting method according to the working method of the above-mentioned device, wherein the device includes a detection mechanism provided at the end of the first flow channel, the method includes the following steps:
  • the detection mechanism controls the opening or closing of the valve switch mechanism to actively control the flow of microparticles with different properties into the corresponding sorting channels to complete the sorting of microparticles.
  • the above-mentioned micro-particle flow focusing device has good focusing effect and can achieve one-dimensional focusing of micro-particles.
  • the device of the present invention requires a shorter channel length, which can reduce the size of the device, and the device does not require a high flow rate to achieve focusing of particles, and can be used in many applications. In low flow rate scenarios, accurate detection and sorting of microparticles can be achieved.
  • microparticle sorting method can use different detection mechanisms for sorting based on the differences in size, fluorescence, and electrical signals of microparticles.
  • Figure 1 is a top view of a microparticle flow focusing device according to an embodiment of the present invention.
  • Figure 2 is a top view of a microparticle focusing area according to an embodiment of the present invention.
  • Figure 3 is a top view of the connection between the micropore and the second flow channel according to an embodiment of the present invention.
  • Figure 4 is a perspective view of the connection between the micropore and the second flow channel according to an embodiment of the present invention.
  • Figure 5 is a top view of the connection between microparticles of different shapes and the second flow channel according to an embodiment of the present invention.
  • Figure 6 is a cross-sectional view of the connection between the micropore and the first flow channel according to Embodiment 1 of the present invention.
  • Figure 7 is a cross-sectional view of the connection between the micropore and the first flow channel according to Embodiment 2 of the present invention.
  • Figure 8 is an overall schematic diagram of a microparticle flow sorting device according to an embodiment of the present invention.
  • Figure 9 is a schematic diagram of the connection between the microparticle focusing area and the microparticle sorting area according to an embodiment of the present invention.
  • Figure 10 is a schematic diagram of the external structure of a microparticle flow focusing and sorting connection valve according to an embodiment of the present invention.
  • Figure 11 is a schematic connection diagram of the valve switching mechanism and external structure according to the embodiment of the present invention.
  • Figure 12 is a cross-sectional view of the valve switching mechanism and external structure according to the embodiment of the present invention.
  • FIG. 13 is a partial enlarged view of part AA of FIG. 5 according to an embodiment of the present invention.
  • first, second, third, etc. may be used in this disclosure to describe various elements, these elements should not be limited to these terms. These terms are only used to distinguish elements of the same type from each other. For example, a first element could be termed a second element, and similarly, a second element could be termed a first element, without departing from the scope of the present disclosure.
  • the present invention discloses a microparticle flow sorting device, including: a chip layer mechanism 1000, and a microparticle focusing area 1100 is provided inside the chip layer mechanism 1000.
  • a section of the chip layer mechanism 1000 is a particle focusing area 1100 .
  • the upper and lower layers of the microparticle focusing area 1100 are respectively provided with a first flow channel 1110 and at least one row of microhole mechanisms 1120 corresponding to the bottom of the first flow channel 1110 .
  • the microparticle focusing area in Figure 4 it can be seen that the microparticles are gradually focused in the direction of the arrow, and the location of the three microparticles on the right is the first flow channel 1110.
  • the upper and lower layers of the microparticle focusing area 1100 are respectively provided with a first flow channel 1110 and a row of micropore mechanisms 1120 corresponding to the bottom of the first flow channel 1110 .
  • the micropore mechanism 1120 includes a plurality of micropores 1121 arranged in rows, and the connected micropores 1121 are connected through the second flow channel 1122 .
  • a particle inlet 1111 is provided at the beginning of the first flow channel 1110 .
  • microparticles to be focused are input with the liquid through the microparticle inlet 1111 at the beginning of the first flow channel 1110.
  • the microparticles gradually pass through at least one row of micropore mechanisms 1120 in the upper first flow channel 1110 in the lower layer. Since there are multiple micropores 1121 in the lower layer and pass through the second
  • the flow channels 1122 are connected, and the particles settle through the microholes 1121 under the action of gravity and streamlines, and then rise after passing through the second flow channel 1122. Each time the particles pass through a microhole 1121, they will gradually deflect toward the centerline until they are completely focused on the microhole 1121. the position of the central axis.
  • microparticles When the microparticles pass through the multi-row microparticle mechanism 1120, a large number of microparticles flowing from the upstream are focused into multiple rows of fine wire beams without changing the flow rate. The microparticles are then arranged through a row of downstream micropores 1121 and further converged into a beam. Finally, all the microparticles It can flow along a straight line to the end of the first flow channel 1110.
  • the microparticle sorting area 1200 includes a plurality of sorting flow channels 1220.
  • the end of the sorting flow channel 1220 is connected with a sorting outlet 1221, and both ends of the waste liquid channel 1210 All are connected to waste liquid outlet 1211.
  • valve switching mechanism 2000 controls whether the particles enter the specific sorting flow channel 1220.
  • the microparticles with different attributes are classified.
  • the microparticles with different properties enter the specific sorting flow channel 1220. This area completes the classification of the microparticles. .
  • the micropore mechanism 1120 also includes a second flow channel 1122.
  • the micropores 1121 are connected through the second flow channel 1122.
  • a plurality of adjacent second flow channels 1122 and the micropores are connected to each other through the second flow channel 1122.
  • the holes 1121 are symmetrically arranged along the central axis of the flow direction, which helps the microparticles to gradually focus during the flow of each microhole.
  • the cross-sectional shape of the micropore 1121 includes one or more of a circle, an ellipse, a triangle, or a rhombus.
  • the micropore 1121 can also be composed of other shapes of converging streamlines.
  • the entire device The shape, height and arrangement of the micropores can also be adjusted to adapt to specific application scenarios of microparticle focusing.
  • the valve switching mechanism 2000 includes a pressure pump 2100 and a film layer 2200 .
  • the film layer 2200 is a PDMS film with a thickness of 9 to 11 ⁇ m.
  • a plurality of air passages 2210 are provided in the middle of the film layer 2200, and air inlets 2211 and air outlets 2212 are provided at both ends of the air passages 2210.
  • a high pressure outlet 2110 and a low pressure inlet 2120 of the pressure pump 2100 are provided. Communicated with the air inlet 2211 and the air outlet 2212 respectively.
  • the thin film layer 2200 is bonded and connected to the surface of the chip layer mechanism 1000 , and each air channel 2210 corresponds to spanning above the beginning of the sorting flow channel 1220 .
  • the part where the bottom surface of the air channel 2210 intersects with the sorting flow channel 1220 is the switch layer 2213 .
  • the airway 2210 is connected to the pressure pump 2100, the syringe pump 4000 is connected to the particle inlet 1111 through a pipeline to input flowing liquid, and the end of the sorting channel 1220 is connected to a container to collect the liquid output by the syringe pump 4000.
  • the switch layer 2213 When the high-pressure outlet 2110 of the pressure pump 2100 passes high-pressure gas into the air channel 2210, the switch layer 2213 is extruded by the high-pressure gas and expands toward the bottom surface of the separation flow channel 1220, causing the switch layer 2213 to block the separation flow channel 1220.
  • the pressure pump When high-pressure gas flows out of the gas outlet 2212 of 2100, the deformation recovery of the switch layer 2213 clears the sorting flow channel 1220.
  • the microparticles enter the corresponding sorting flow channel as the switch layer 2213 opens and closes, and the sorting valve structure is simple to operate.
  • the microparticle focusing area 1100 also includes a transition flow channel 1130 .
  • the starting end of the sorting flow channel 1220 is connected to one end of the transition channel 1130 in the first flow channel 1110 in the direction of the particle inlet 1111, and the other end of the transition channel 1130 is connected to the end of the first flow channel 1110.
  • the particles flow at the end of the first flow channel 1110 by accommodating only one column of particles, ensuring that only a single particle enters the corresponding valve switching mechanism at the end of the transition channel 1130 and enters the specific sorting channel 1220.
  • the valve switching mechanism 2000 includes a pressure pump 2100 and a control fluid channel 2300.
  • the control fluid channel 2300 is disposed inside the chip layer mechanism 1000.
  • One end of the control fluid channel 2300 is connected to The starting end of the sorting flow channel 1220 is connected, and the pressure port of the pressure pump 2100 is connected with the other end of the control fluid channel 2300 .
  • An additional control fluid channel 2300 is provided at the beginning of the multiple sorted flow channels, and the flow of liquid in the changed channel is controlled by a pressure pump.
  • the pressure pump controls the fluid flow to push the microparticles into different sorting flow channels 1220.
  • the invention also discloses a particle flow sorting method, which includes: the above-mentioned microparticle flow sorting device.
  • the device also includes a detection mechanism 3000 set at the end of the first flow channel 1110.
  • the figure shows the structure of a microscope, which classifies microparticles based on their different visual attributes.
  • the microparticle sorting system is based on the Python development language and mainly consists of image acquisition and image recognition. , data processing, hardware control and visual GUI interactive page and other parts. Rapid image collection is achieved by taking quick screenshots of the detection area (35ms/frame).
  • OpenCV is used to convert the collected images from RGB space to HSV space and perform grayscale operations.
  • Gaussian blur is used to denoise, expand, corrode, and contour detection. and contour drawing to detect color and size, and determine whether the particles in adjacent frames are the same particles by setting a time threshold.
  • the detection results will be displayed in real time on the GUI interactive interface developed using PyQt5, and corresponding instructions will be transmitted to control the pressure pump based on the detection results.
  • the valve switching structure is controlled to realize automated sorting of microparticles based on color or size.
  • the time from collecting to sending instructions for each frame of image is controlled at about 40ms. No additional hardware support is needed to collect images, which is efficient and convenient.
  • the entire process from particle focusing to detection and sorting is fully automated.
  • Other detection equipment can also be set up here, and the detection equipment will sort according to the difference standards of particle size, fluorescence, and electrical signals.
  • S200 Focus the microparticles along a straight line from the first flow channel 1110 through the plurality of microholes 1121 arranged in at least one row at the end of the microparticle focusing area 1100.
  • the detection mechanism 3000 controls the opening or closing of the valve switch mechanism 2000 to actively control the flow of microparticles with different properties into the corresponding sorting flow channel 1220 to complete the sorting of the microparticles.
  • the above method focuses on sorting microparticles including cells.
  • microparticle flow sorting device and method a series of microparticles are passively focused, avoiding the use of external physical fields or sheath flow, effectively simplifying the complexity of chip production and specific operations, and will not damage the microparticles.
  • micro-particle flow focusing device has a good focusing effect and can achieve one-dimensional focusing of micro-particles.
  • the device of the present invention requires a shorter channel length and can reduce the size of the device.
  • the device can achieve focusing of particles without requiring a high flow rate, and can be used in many low flow rate scenarios to achieve accurate detection and sorting of single particles.
  • microparticle sorting method can use different detection mechanisms for sorting based on differences in size, fluorescence, and electrical signals of microparticles.

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Abstract

一种微粒子流动聚焦装置及方法，微粒子流动聚焦装置包括：芯片层机构（1000），芯片层机构（1000）的内部设置微粒子聚焦区域（1100），微粒子聚焦区域（1100）的上层及下层分别设置第一流道（1110）及与第一流道（1110）下方对应的至少一行微孔机构（1120），微孔机构（1120）包括成行排列的多个微孔（1121），第一流道（1110）的始端设置有微粒子入口（1111）。微粒子流动分选装置及方法不需要鞘流就可以实现对微粒子的一维聚焦，而且在低流速时聚焦效果更好，因此对哺乳动物细胞的活性无损伤。微粒子流动聚焦装置及方法还可用于微量样品的聚焦，比传统的基于鞘流的聚焦具有明显优势。

Description

一种微粒子流动聚焦装置及方法 技术领域
本发明涉及一种微粒子流动聚焦装置及方法，属于生物医学分析领域。
背景技术
在生物医学分析过程中，往往只对样本中特定的微粒(微球或者细胞)进行研究，比如血液样本中的循环肿瘤细胞，或者细胞培养基中的健康细胞。这些微粒通常具有特殊的性质，因此可以利用这些性质将其进行检测并分选出来。
在过去的20年里，流式细胞术由于其检测和分选细胞的能力，已经被广泛用作许多疾病的强大分析工具。其利用激光或者基于阻抗的技术来进行细胞计数或分类，通过将细胞悬浮在流体中通过光学或电器件测试设备，并在下游进行下一步的分选工作，是生物分析的重要工具。传统的流式细胞仪体积庞大，价格昂贵且操作复杂，为了克服这些缺点，研究人员将微流体的概念引入到流式细胞术当中，称之为微流控流式细胞术。微流控是一种在尺寸几十微米范围内的通道中操纵流体的技术。与传统的流式细胞仪类似，微流控流式细胞仪也由五大子系统组成，其中样品泵送、聚焦和分选大多都可以在微流控芯片上完成。微流控芯片的尺寸很小，可以大大地减少所需样品和试剂的体积。并且将其集成在系统中，可以减少设备的总尺寸和维护要求，也正因为如此，和微流控相结合被认为是流式细胞仪未来进一步的发展方向。
单个细胞的检测能力是流式细胞仪的主要特征和能力之一。当细胞被注入流式细胞仪时，需要将样品聚焦到一个狭窄的核心流当中，使其能够以一定速率逐个通过检测区域，这是流式细胞仪的前处理阶段。聚焦可以分为主动聚焦和被动聚焦，被动聚焦依赖于流体的固有特性来实现微粒子聚焦，而主动聚焦常常依赖于外部场来产生驱动力，包括声电泳(AP)、介电泳(DEP)、磁电泳(MP)和荧光活化分离法。主动聚焦常常需要引入外部设备，这就增加了设备的复杂度，且可能会对细胞造成损伤。比如传统的流体细胞仪通过鞘流来实现样品流的聚焦，在微流控芯片出现以后，通过巧妙的结构设计来引入鞘层流动。鞘流从四面八方将样品流压缩到中间，其虽可以实现对样品流的精确控制，但是需要大量的鞘流试剂，并且高速流动的鞘流也有可能损伤细胞。惯性微流控是一种利用惯性升力将微粒驱动到微通道的一个或多个平衡位置实现聚焦功能的被动聚焦方法。其中，有研究人员利用二次流和惯性升力设计了一种弧形槽阵列微流控装置，但这种装 置需要很长的长度，且需要很高的速度才可以做到聚焦，并且其聚焦的准确度比较差，没办法和检测系统结合实现单细胞的高精度分选。又有研究人员设计了一种螺旋微流控装置，这种装置可以根据大小直接将微粒聚焦在不同的位置，然后直接进行分选。为了避免较大的螺旋布局，再有研究人员利用惯性微流控设计了一种包含收缩扩张阵列的直通道微流控芯片。但还是依然无法避免惯性微流控带来的需要高流速，且聚焦分辨率低等一系列问题。其他被动式聚焦，比如确定性横向位移侧向位移(DLD)，和惯性微流控一样都很难在后续通过检测微粒来做到确定性分选。
综上所述，流式细胞术大多采用鞘流聚焦微粒子，但是其鞘流增加了微流控芯片的复杂度，且需要大量额外的鞘液，高速流动的鞘液也可能会造成细胞损伤；主动式聚焦设备需要引入外部物理场比如声、光、电、磁等等，这种聚焦方式极大地增加了聚焦的复杂性，且外部物理场的干预也有可能会对细胞本身造成损伤，影响其活性；其他被动式聚焦虽然可以不引入外部物理场，但惯性微流控分辨率低，且需要很高的流速和很长的聚焦长度。其他的被动式聚焦比如DLD则很难和图像、荧光以及电信结合起来做到确定性分选。
发明内容
本发明提供一种微粒子流动聚焦装置及方法，旨在至少解决现有技术中存在的技术问题之一。
本发明的技术方案为一种微粒子流动聚焦装置，其包括：芯片层机构，所述芯片层机构的内部设置微粒子聚焦区域，微粒子聚焦区域，所述微粒子聚焦区域的上层及下层分别设置第一流道及与所述第一流道下方对应的至少一行微孔机构，所述微孔机构包括成行排列的多个微孔，所述第一流道的始端设置有微粒子入口。
进一步，还包括：微粒子分选区域，所述微粒子分选区域包括多条分选流道，所述分选流道的末端连通有分选出口；对应设置在各个所述分选流道附近的阀门开关机构，其中，所述分选流道的始端与所述第一流道的末端连通。
进一步，所述微孔机构还包括第二流道，所述相连所述微孔之间通过第二流道连通。
进一步，所述微孔分别与所述第一流道的中轴线互为镜像对称。
进一步，多个相邻的所述第二流道及所述微孔沿流动的方向的中轴线对称设置，所述微孔的横截面的形状包括圆形、椭圆形、三角形或菱形等能够汇聚流线形状的一种或多种。
进一步，所述阀门开关机构包括压力泵及薄膜层，所述薄膜层的中部设有多条气道，所述气道的两端分别设有入气口及出气口，所述压力泵的高压出口及低压入口分别与入气口及出气口连通，其中，所述薄膜层键合连接在芯片层机构的表面，各个所述气道对应横跨分选流道始端的上方，所述气道的底面与分选流道相交的部分为开关层，当压力泵的高压出口通入高压气体到气道时，所述开关层受到高压气体挤压向分选流道的底面膨胀，使开关层堵塞分选流道，当压力泵的出气口流出高压气体时，所述开关层的形变复原使分选流道疏通。
进一步，所述微粒子聚焦区域还包括过渡流道，所述分选流道的始端向微粒子入口方向的第一流道内连通有过渡流道的一端，所述过渡流道的另一端与第一流道的末端连接。
进一步，所述阀门开关机构包括压力泵及控制流体通道，所述控制流体通道设置在所述芯片层机构的内部，所述控制流体通道的一端与分选流道的始端连通，所述压力泵的压力口与控制流体通道的另一端连通。
一种微粒子流动聚焦和主动分选方法，根据上述的装置的工作方法，其中，所述装置包括设置在所述第一流道末端的检测机构，所述方法包括以下步骤：
S100、通过微粒子聚焦区域的始端通入液体及微粒子，微粒子从微粒子聚焦区域的始端到末端流经第一流道；
S200、使微粒子从所述第一流道依次经过至少一行排列的多个微孔沿直线聚焦在微粒子聚焦区域的末端；
S300、检测机构通过识别微粒子的属性后，控制开启或关闭阀门开关机构以主动控制流入不同属性的微粒子到对应的分选流道，完成微粒子的分选。
本发明的有益效果为：
1、上述的微粒子流动聚焦装置，聚焦效果好，可以实现对微粒子的一维聚焦。
2、上述的装置和惯性微流控相比，本发明的装置所需的通道长度短，可以减少装置的尺寸，并且该装置不需要很高的流速即可以实现微粒的聚焦，可应用在很多低流速的场景，可实现微粒子的准确性检测和分选。
3、上述的微粒子分选方法可基于微粒子的大小、荧光、电信号的差异选用不同的检测机构进行分选。
附图说明
图1是根据本发明实施例的微粒子流动聚焦装置的俯视图。
图2是根据本发明实施例的微粒子聚焦区域的俯视图。
图3是根据本发明实施例的微孔及第二流道连接的俯视图。
图4是根据本发明实施例的微孔及第二流道连接的立体图。
图5是根据本发明实施例的不同形状微粒子及第二流道连接的俯视图。
图6是根据本发明实施例一的微孔及第一流道连接的剖视图。
图7是根据本发明实施例二的微孔及第一流道连接的剖视图。
图8是根据本发明实施例的微粒子流动分选装置的总体示意图。
图9是根据本发明实施例的微粒子聚焦区域及微粒子分选区域连接的示意图。
图10是根据本发明实施例的微粒子流动聚焦和分选连接阀门外部结构的示意图。
图11是根据本发明实施例的阀门开关机构及外部结构的连接示意图。
图12是根据本发明实施例的阀门开关机构及外部结构的剖视图。
图13是根据本发明实施例的图5的AA部局部放大图。
具体实施方式
以下将结合实施例和附图对本发明的构思、具体结构及产生的技术效果进行清楚、完整的描述，以充分地理解本发明的目的、方案和效果。需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。
需要说明的是，如无特殊说明，当某一特征被称为“固定”、“连接”在另一个特征，它可以直接固定、连接在另一个特征上，也可以间接地固定、连接在另一个特征上。此外，本发明中所使用的上、下、左、右、顶、底等描述仅仅是相对于附图中本发明各组成部分的相互位置关系来说的。
此外，除非另有定义，本文所使用的所有的技术和科学术语与本技术领域的技术人员通常理解的含义相同。本文说明书中所使用的术语只是为了描述具体的实施例，而不是为了限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的组合。
应当理解，尽管在本公开可能采用术语第一、第二、第三等来描述各种元件，但这些元件不应限于这些术语。这些术语仅用来将同一类型的元件彼此区分开。例如，在不脱离本公开范围的情况下，第一元件也可以被称为第二元件，类似地，第二元件也可以被称为第一元件。
参照图1至13，在一些实施例中，本发明公开了一种微粒子流动分选装置，包括：芯片层机构1000，所述芯片层机构1000的内部设置微粒子聚焦区域1100。参照图1所示，芯片层机构1000中一段为微粒子聚焦区域1100。
参照图1至图4所示的微粒子聚焦区域1100，微粒子聚焦区域1100的上层及下层分别设置第一流道1110及与第一流道1110下方对应的至少一行的微孔机构1120。图4中的微粒子聚焦区域中看出微粒子沿箭头方向逐渐聚焦，最右边三个微粒子所在位置为第一流道1110。参照图5所示，微粒子聚焦区域1100的上层及下层分别设置第一流道1110及与第一流道1110下方对应的一行的微孔机构1120。
参照图2结合4所示，微孔机构1120包括成行排列的多个微孔1121，相连微孔1121之间通过第二流道1122连通。参照图1，第一流道1110的始端设置有微粒子入口1111。
待聚焦的众多微粒子通过第一流道1110始端的微粒子入口1111随液体输入，微粒子在上层的第一流道1110逐渐下层经过至少一行的微孔机构1120，由于下层有多个微孔1121及通过第二流道1122连通，微粒在重力及流线的作用下经过微孔1121中发生沉降，经过第二流道1122再上升，微粒子每经过一个微孔1121都会逐渐向中线偏转直到完全聚焦到微孔1121的中轴线位置。微粒子还经过多行微粒机构1120时，上游流过来的大量微粒子在不改变流速的情况下聚焦成多行细小的线束，微粒子再通过下游的一行微孔1121排列再进一步汇聚成一束，最后所有微粒子能沿一条直线流向第一流道1110末端。
参照图1结合图8所示的微粒子分选区域1200，微粒子分选区域1200包括多条分选流道1220，分选流道1220的末端连通有分选出口1221，废液通道1210的两端均连通有废液出口1211。
参照图9所示，对应设置在各个分选流道1220附近的阀门开关机构2000，图中从第一流道1110中引出两条分选流道1220，然后两个分选流道1220上设置各自的阀门开关机构2000，控制微粒子是否进入特定的分选流道1220中。
对于微粒子聚焦区域末端的直线留微粒子，对不同属性的微粒子进行分类，通过控制阀门开关机构2000的开启及关闭使不同属性的微粒子进入特定的分选流道1220中，该区域完成对微粒子的分类。
参照图1至5、结合图13，微孔机构1120还包括第二流道1122，相连所述微孔1121之间通过第二流道1122连通，多个相邻的第二流道1122及微孔1121沿流动的方向的中轴线对称设置，有助于微粒子在每个微孔流动的过程中逐步实 现聚焦。
参照图5至图7所示，微孔1121的横截面的形状包括圆形、椭圆形、三角形或菱形的一种或多种，微孔1121的也可以其他汇聚流线的形状组成，整个装置还可以通过调整微孔的形状、高度及排列方式来适应微粒子聚焦的具体的应用场景。
参照图9至图12所示，阀门开关机构2000包括压力泵2100及薄膜层2200，薄膜层2200的厚度为9至11μm的PDMS薄膜。
参照图9至图12所示，薄膜层2200的中部设有多条气道2210，气道2210的两端分别设有入气口2211及出气口2212，压力泵2100的高压出口2110及低压入口2120分别与入气口2211及出气口2212连通。
参照图9，薄膜层2200键合连接在芯片层机构1000的表面，各个气道2210对应横跨分选流道1220始端的上方。参照图13，气道2210的底面与分选流道1220相交的部分为开关层2213。
参照图10所示的实施例，气道2210连通压力泵2100，注射泵4000通过管道连通微粒子入口1111，输入流动液体，分选流道1220末端连通容器收集注射泵4000输出的液体。
当压力泵2100的高压出口2110通入高压气体到气道2210时，开关层2213受到高压气体挤压向分选流道1220的底面膨胀，使开关层2213堵塞分选流道1220，当压力泵2100的出气口2212流出高压气体时，开关层2213的形变复原使分选流道1220疏通。微粒子随开关层2213的开启及关闭进入到对应的分选流道中，分选的阀门结构操作简单。
参照图1至2，微粒子聚焦区域1100还包括过渡流道1130。分选流道1220的始端向微粒子入口1111方向的第一流道1110内连通有过渡流道1130的一端，过渡流道1130的另一端与第一流道1110的末端连接。微粒子在第一流道1110的末端通过只容纳一列微粒子进行流动，保证在过渡流道1130的末端只有单个微粒子进入对应的阀门开关机构进入特定的分选流道1220。
参照图1至2的第二中阀门开关机构的实施例，阀门开关机构2000包括压力泵2100及控制流体通道2300，控制流体通道2300设置在芯片层机构1000的内部，控制流体通道2300的一端与分选流道1220的始端连通，压力泵2100的压力口与控制流体通道2300的另一端连通。在多个分选的流道始端设置一个额外的控制流体通道2300，，通过压力泵控制改通道液体的流动。当特定的微粒子通过微粒子聚焦区域时，压力泵控制流体流量从而将微粒子推入到不同的分选流 道1220中。
本发明还公开了一种粒子流动分选方法，包括：根据上述的微粒子流动分选装置。
参照图8，该装置还包括设置在第一流道1110末端的检测机构3000，图中为显微镜结构，通过微粒子不同的视觉属性进行分类，微粒子分选系统基于Python开发语言主要由图像采集、图像识别、数据处理、硬件控制以及可视化GUI交互页面等部分组成。通过对检测区域快速截屏(35ms/帧)实现了图像的快速采集，利用opencv将采集的图像从RGB空间转化为hsv空间并进行灰度化操作，通过高斯模糊去噪、膨胀、腐蚀、轮廓检测及轮廓绘制进行颜色和大小检测，并通过设定时间阈值判断相邻帧的微粒子是否为同一微粒子，检测结果会实时显示在利用PyQt5开发的GUI交互界面上，根据检测结果传输相应指令控制压力泵控制阀门开关结构从而实现了基于颜色或者大小的微粒子自动化分选。每一帧图像从采集到发送指令时间控制在40ms左右，采集图像无需额外硬件支持，高效便捷，整个过程从微粒子聚焦到检测分选完全自动化。该处还可以设置其他的检测设备，检测设备根据微粒子大小、荧光、电信号的差异标准进行过分选。
S100、通过微粒子聚焦区域1100的始端通入液体及微粒子，微粒子从微粒子聚焦区域1100的始端到末端流经第一流道1110。
S200、使微粒子从所述第一流道1110依次经过至少一行排列的多个微孔1121沿直线聚焦在微粒子聚焦区域1100的末端。
S300、检测机构3000通过识别微粒子的属性后，控制开启或关闭阀门开关机构2000以主动控制流入不同属性的微粒子到对应的分选流道1220，完成微粒子的分选。上述方法聚焦分选的微粒子包括细胞。
上述的微粒子流动分选装置及方法，一系列的微粒子通过被动聚焦的方式，避免外加物理场或者鞘流的使用，有效地简化了芯片制作和具体操作的复杂度，并且不会损伤微粒子。
上述的微粒子流动聚焦装置，聚焦效果好，可以实现对微粒子的一维聚焦。
上述的装置和惯性微流控相比，本发明的装置所需的通道长度短，可以减少装置的尺寸。并且该装置不需要很高的流速即可以实现微粒的聚焦，可应用在很多低流速的场景，可实现单个微粒子的准确性检测和分选。
上述的微粒子分选方法可基于微粒子的大小、荧光、电信号的差异选用不同的检测机构进行分选。
以上所述，只是本发明的较佳实施例而已，本发明并不局限于上述实施方式，只要其以相同的手段达到本发明的技术效果，凡在本公开的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本公开保护的范围之内。都应属于本发明的保护范围。在本发明的保护范围内其技术方案和/或实施方式可以有各种不同的修改和变化。

Claims (9)

  1. 一种微粒子流动聚焦装置，其特征在于，包括：
    芯片层机构(1000)，所述芯片层机构(1000)的内部设置微粒子聚焦区域(1100)，
    微粒子聚焦区域(1100)，所述微粒子聚焦区域(1100)的上层及下层分别设置第一流道(1110)及与所述第一流道(1110)下方对应的至少一行微孔机构(1120)，所述微孔机构(1120)包括成行排列的多个微孔(1121)，所述第一流道(1110)的始端设置有微粒子入口(1111)。
  2. 根据权利要求1所述的微粒子流动聚焦装置，其特征在于，还包括：
    微粒子分选区域(1200)，所述微粒子分选区域(1200)包括多条分选流道(1220)，所述分选流道的末端连通有分选出口(1221)；
    对应设置在各个所述分选流道(1220)附近的阀门开关机构(2000)，
    其中，所述分选流道(1220)的始端与所述第一流道(1110)的末端连通。
  3. 根据权利要求2所述的微粒子流动聚焦装置，其特征在于，
    所述微孔机构(1120)还包括第二流道(1122)，所述相连所述微孔(1121)之间通过第二流道(1122)连通。
  4. 根据权利要求3所述的微粒子流动聚焦装置，其特征在于，
    所述微孔(1121)分别与所述第一流道(1110)的中轴线互为镜像对称。
  5. 根据权利要求2所述的微粒子流动分选装置，其特征在于，
    多个相邻的所述第二流道(1122)及所述微孔(1121)沿流动的方向的中轴线对称设置，所述微孔(1121)的横截面的形状包括圆形、椭圆形、三角形、菱形或汇聚流线形状中的一种或多种。
  6. 根据权利要求1所述的微粒子流动聚焦装置，其特征在于，
    所述阀门开关机构(2000)包括压力泵(2100)及薄膜层(2200)，所述薄膜层(2200)的中部设有多条气道(2210)，所述气道(2210)的两端分别设有入气口(2211)及出气口(2212)，所述压力泵(2100)的高压出口(2110)及低压入口(2120)分别与入气口(2211)及出气口(2212)连通，
    其中，所述薄膜层(2200)键合连接在芯片层机构(1000)的表面，各个所述气道(2210)对应横跨分选流道(1220)始端的上方，所述气道(2210)的底面与分选流道(1220)相交的部分为开关层(2213)，当压力泵(2100)的高压 出口(2110)通入高压气体到气道(2210)时，所述开关层(2213)受到高压气体挤压向分选流道(1220)的底面膨胀，使开关层(2213)堵塞分选流道(1220)，当压力泵(2100)的出气口(2212)流出高压气体时，所述开关层(2213)的形变复原使分选流道(1220)疏通。
  7. 根据权利要求1所述的微粒子流动聚焦装置，其特征在于，
    所述微粒子聚焦区域(1100)还包括过渡流道(1130)，
    所述分选流道(1220)的始端向微粒子入口(1111)方向的第一流道(1110)内连通有过渡流道(1130)的一端，所述过渡流道(1130)的另一端与第一流道(1110)的末端连接。
  8. 根据权利要求1所述的微粒子流动聚焦装置，其特征在于，
    所述阀门开关机构(2000)包括压力泵(2100)及控制流体通道(2300)，所述控制流体通道(2300)设置在所述芯片层机构(1000)的内部，所述控制流体通道(2300)的一端与分选流道(1220)的始端连通，所述压力泵(2100)的压力口与控制流体通道(2300)的另一端连通。
  9. 一种微粒子流动聚焦和主动分选方法，根据权利要求1至8中的任一权利要求所述的装置的工作方法，其中，所述装置包括设置在所述第一流道(1110)末端的检测机构(3000)，其特征在于，所述方法包括以下步骤：
    S100、通过微粒子聚焦区域(1100)的始端通入液体及微粒子，微粒子从微粒子聚焦区域(1100)的始端到末端流经第一流道(1110)；
    S200、使微粒子从所述第一流道(1110)依次经过至少一行排列的多个微孔(1121)沿直线聚焦在微粒子聚焦区域(1100)的末端；
    S300、检测机构(3000)通过识别微粒子的属性后，控制开启或关闭阀门开关机构(2000)以主动控制流入不同属性的微粒子到对应的分选流道(1220)，完成微粒子的分选。
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