WO2022174472A1 - Fully-integrated multi-index nucleic acid test microfluidic chip - Google Patents

Fully-integrated multi-index nucleic acid test microfluidic chip Download PDF

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Publication number
WO2022174472A1
WO2022174472A1 PCT/CN2021/078004 CN2021078004W WO2022174472A1 WO 2022174472 A1 WO2022174472 A1 WO 2022174472A1 CN 2021078004 W CN2021078004 W CN 2021078004W WO 2022174472 A1 WO2022174472 A1 WO 2022174472A1
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nucleic acid
pool
liquid
amplification
tube
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PCT/CN2021/078004
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French (fr)
Chinese (zh)
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刘鹏
林宝宝
耿智
李保
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杭州梓晶生物有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • B01L3/502738—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by integrated valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept

Definitions

  • the invention belongs to the field of life medical detection, and in particular relates to a fully integrated multi-index nucleic acid detection microfluidic chip.
  • nucleic acid Since the 1990s, as one of the most basic substances in organisms, nucleic acid has played an increasingly important role in modern biomedicine. It is characterized by strong detection specificity, very easy operation, and technological development. It is mature and has a very short detection time, so it is of great significance in the fields of infectious disease detection, food safety monitoring, and environmental protection. Therefore, rapid detection based on nucleic acid has been recognized as one of the important molecular detection methods.
  • nucleic acid-based rapid detection mainly includes the following two methods: polymerase chain reaction (PCR) and isothermal amplification technology.
  • PCR polymerase chain reaction
  • isothermal amplification technology As a mature nucleic acid amplification technology, PCR has become a technology that cannot be ignored in the field of life medical detection and disease diagnosis after decades of development.
  • the temperature cycler performs precise temperature control of each thermal cycling step.
  • Nucleic acid isothermal amplification technology only needs a single reaction temperature in the whole process, and has simpler reaction temperature conditions. It is simpler and more convenient than PCR technology in terms of actual operation and instrument requirements, and can get rid of the need for sophisticated equipment. It has shown its good application prospects in clinical and field rapid diagnosis.
  • the current nucleic acid detection is mainly based on traditional methods, that is, the first step is to manually extract the nucleic acid in the sample. This step is generally to purchase a commercial nucleic acid extraction kit, and then operate according to the product instructions. A series of complex biochemical tests are carried out in test tubes.
  • the following amplification process takes the commonly used polymerase chain reaction amplification as an example. The amplification needs to be manually added to the reaction mixture and then used for the final detection by a commercial instrument. The whole process is time-consuming and labor-intensive, and also requires professional laboratories and professional operators.
  • nucleic acid detection methods can no longer meet the needs of the growing business volume of nucleic acid detection.
  • some companies have launched commercial fully integrated nucleic acid analyzers, their detection throughput is low.
  • high-throughput detection is achieved by stacking multiple control and detection instruments, such as the FilmArray 2.0 system.
  • the fully integrated microfluidic system still needs to be improved in the adjustability of the flux.
  • the purpose of the present invention is to overcome the defects in the prior art, to solve the complex and precise fluid operations such as sample pretreatment, nucleic acid extraction, amplification and detection in nucleic acid detection based on microfluidic chips, and at the same time use an independent chamber as a reaction area Complete multi-index, high-throughput detection.
  • the present invention provides a multi-index nucleic acid detection microfluidic chip, comprising:
  • nucleic acid sample pretreatment assembly includes a base plate, an elastic mold and a guide plate, the base plate is provided with a plurality of liquid storage tubes for liquid storage, and the baffle plate is provided with a storage tube and a storage tube.
  • the liquid storage tank corresponding to the liquid pipe and the flow channel used to communicate with the liquid storage tank, an elastic mold is sandwiched between the bottom of the base plate and the top of the guide plate, and the elastic mold is deformed between the liquid storage pipe and the liquid storage tank.
  • the guide plate is provided with a sample pool for storing processed nucleic acid samples;
  • a multi-index nucleic acid amplification assembly includes a main channel and a plurality of amplification chambers, and each amplification chamber and the main channel are used to achieve isolation between different amplification chambers
  • the dispersing pipeline is communicated; the main channel is communicated with the sample pool.
  • a shunt column is arranged between the main channel and the amplification chamber, and the shunt column divides the space between the main channel and the amplification chamber into three dispersed flow channels, along the direction of the flow of the main channel liquid.
  • the dispersion flow channel between the shunt column and the outer wall of the amplification chamber is the first dispersion flow channel and the third dispersion flow channel
  • the second dispersion flow channel is between the shunt column and the main channel
  • the dispersion flow channel The relationship of the cross-sectional area is the first dispersion flow channel > the second dispersion flow channel > the third dispersion flow channel.
  • the cross-sectional area ratio of the first dispersion channel, the second dispersion channel, and the third dispersion channel is 7-12:3-5:1.
  • the cross-sectional area ratio of the first dispersion flow channel, the second dispersion flow channel, and the third dispersion flow channel is 10:4:1.
  • the shunt column 43 may be a column with a trapezoidal cross-section, the shorter base of the trapezoid faces the direction of liquid flow, and the distance between the side wall of the amplification chamber near the flow direction and the column is greater than The distance between the sidewall of the amplification chamber away from the flow direction and the column.
  • the liquid flow direction refers to the direction shown by the arrow in Fig. 2a.
  • the baffle plate is correspondingly provided with a lysis solution pool, a ventilation pool, a washing solution pool, an eluent pool, a first waste solution pool, a flow channel, a collection pool, an amplification reagent pool, and a sealing oil Pool and sample pool; wherein, the lysate tube and the lysate pool, the washing solution tube and the washing solution pool, the eluent tube and the eluent pool, the amplification reagent tube and the amplification reagent pool, the sealing oil tube and the sealing oil There is a liquid inlet one-way valve that flows from top to bottom between the pools; the first waste liquid pipe and the first waste liquid pool, the second waste liquid pipe and the sample pool are provided with a bottom
  • the liquid inlet check valve, the liquid outlet check valve and the liquid inlet and outlet check valve are realized by the deformation on the elastic film, and the function of the check valve is realized by applying pressure to control the deformation direction of the elastic film.
  • the liquid inlet check valve forms a downward one-way valve under the action of pressure;
  • the valve forms an upward one-way valve under the action of pressure; under the action of no external pressure, due to the elastic properties of the elastic membrane itself, it is in the state of a closed valve.
  • liquid inlet one-way valve and the liquid outlet one-way valve are both sheet-shaped elastic diaphragms including a unilateral fixed part and a first elastic movable part;
  • one end of the unilateral fixed part of the liquid inlet check valve is fixedly connected with the outer wall of the liquid storage pipe, and the other end of the unilateral fixed part is fixedly connected with the first elastic movable part.
  • the first elastic movable part moves downward and is separated from the liquid outlet of the liquid storage pipe, so that the liquid outlet and the flow channel on the guide plate are in a conducting state, that is, the The liquid inlet check valve is in the open state.
  • the first elastic movable part is attached to the liquid outlet, so that the liquid outlet is sealed and isolated. state, that is, the liquid inlet check valve is in a closed state;
  • One end of the unilateral fixed part of the liquid outlet check valve is fixedly connected to the outer wall of the liquid storage pipe, and the other end of the unilateral fixed part is fixedly connected to the first elastic movable part.
  • the first elastic movable part moves upward and is separated from the liquid inlet of the liquid storage tube, so that the liquid inlet and the flow channel on the baffle plate are in a conductive state , that is, the liquid outlet one-way valve is in an open state, and when the pressure in the liquid storage pipe is positive pressure, the first elastic movable part is attached to the liquid inlet, so that the liquid inlet is in a closed state. , the liquid outlet one-way valve is closed.
  • the liquid inlet and outlet check valve includes a liquid inlet check valve, a liquid outlet check valve and a fixing piece located between the liquid outlet and the liquid inlet, and the unilateral fixing part of the liquid inlet check valve is in phase with the fixing piece.
  • the unilateral fixed part of the liquid outlet check valve is fixedly connected to the side wall of the liquid storage pipe, and the liquid inlet check valve and the liquid outlet check valve are closed and opened in the same state as above.
  • the on-off valve includes a fixed block connected with the flow channel and a second elastic movable part, the second elastic movable part is a part of the elastic mold, and the edge of the second elastic movable part is fixedly connected with the side outer wall of the on-off valve tube;
  • the switch valve tube When there is negative pressure in the switch valve tube, the upward protrusion of the second elastic movable part is separated from the fixed block, and the flow passages on both sides of the fixed block are connected; The runners on both sides of the fixed block are closed.
  • a guide groove is provided on the surface where the guide plate is attached to the elastic film, and the guide groove is used to communicate with each liquid storage tank on the guide plate.
  • the upper part of the liquid storage pipe is provided with a push rod; the bottom of the guide plate is also provided with a detection device.
  • each reaction chamber is provided with different primers in advance, so as to realize multi-index amplification.
  • the present invention also provides a multi-index nucleic acid amplification detection system, comprising the multi-index nucleic acid detection microfluidic chip.
  • the invention proposes a new rapid and automatic multi-index nucleic acid detection device based on a microfluidic chip, which is a rapid detection tool with simple operation and low cost, and is suitable for the detection of various types of detection samples in various detection occasions. Fast multi-index detection.
  • FIG. 1 is a schematic structural diagram of a multi-index nucleic acid detection microfluidic chip of the present invention, wherein Figure a is a schematic structural diagram, and Figure b is an exploded view;
  • Figure 2 is a schematic diagram of the principle structure of a multi-index amplification chip, wherein Figure a is a schematic diagram of a partial structure, and Figure b is a schematic diagram of the flow direction of the liquid over time;
  • Figure 3 is a schematic diagram of some valves, in which a is a liquid inlet check valve, b is a liquid outlet check valve, c is a liquid inlet and outlet check valve, and d is an on-off valve.
  • a microfluidic chip for multi-index nucleic acid detection based on a microfluidic chip proposed by the present invention includes:
  • the nucleic acid sample pretreatment assembly includes a substrate 1, an elastic mold 2 and a guide plate 3, the substrate 1 is provided with a plurality of liquid storage tubes for liquid storage, the guide plate 2 There is a liquid storage tank corresponding to the liquid storage pipe and a flow channel for connecting the liquid storage tank, an elastic mold 2 is sandwiched between the bottom of the base plate 1 and the top of the guide plate 3, and the liquid storage pipe is connected to the storage tank.
  • the liquid pools are connected or closed by the deformation of the elastic mold 2, and the guide plate 3 is provided with a sample pool for storing the processed nucleic acid samples.
  • the liquid storage pipes there are a plurality of the liquid storage pipes, as shown in FIG. 1 , along the direction from left to right are the lysing liquid pipe 11 , the vent pipe 12 , the washing liquid pipe 13 , the eluent pipe 14 , and the first waste liquid pipe. 15.
  • On-off valve pipe 16 collecting pipe 17, amplification reagent pipe 18, sealing oil pipe 19 and second waste liquid pipe 10; the baffle 2 is provided with a lysing solution pool 21, aeration pool 22, washing solution pool correspondingly 23.
  • a guide groove is provided on the surface where the guide plate is attached to the elastic film, the position of the guide groove corresponds to the through hole, and the guide groove is used to control the microfluid flowing out of the through hole.
  • Conduct drainage The upper part of the liquid storage pipe is provided with a push rod, which is used to adjust the pressure in the liquid storage tank and provide fluid forward power.
  • a detection device is also provided at the bottom of the deflector.
  • the liquid inlet check valve 5 and the liquid outlet check valve 6 are both sheet-shaped elastic diaphragms including a unilateral fixed portion and a first elastic movable portion.
  • One end of the unilateral fixed portion 51 of the liquid inlet check valve is fixedly connected to the outer wall of the liquid storage pipe, and the other end of the unilateral fixed portion is connected to the first elastic movable portion 52 of the liquid inlet check valve.
  • Fixed connection when there is a positive pressure in the liquid storage tube, the first elastic movable part moves downward and is separated from the liquid outlet of the liquid storage tube, so that the liquid outlet and the flow channel on the baffle plate are in a guiding position.
  • the liquid inlet one-way valve In the open state, that is, the liquid inlet one-way valve is in the open state, when the pressure in the liquid storage pipe is less than the flow channel pressure or negative pressure, the first elastic movable part is in contact with the liquid outlet, so that the The liquid outlet is in a closed and isolated state, that is, the liquid inlet check valve is in a closed state.
  • One end of the unilateral fixed part of the liquid outlet one-way valve 6 is fixedly connected to the outer wall of the liquid storage pipe, and the other end is fixedly connected to the first elastic movable part of the liquid outlet one-way valve.
  • the first elastic movable part moves upward and separates from the liquid inlet of the liquid storage pipe, so that the liquid inlet and the flow channel on the baffle plate are in a conducting state, that is, The liquid outlet one-way valve is in the open state
  • the first elastic movable part is attached to the liquid inlet, so that the liquid inlet is in a closed and isolated state, so The liquid one-way valve is closed.
  • the liquid inlet and outlet check valve 7 includes a liquid inlet check valve, a liquid outlet check valve and a fixing piece located between the liquid outlet and the liquid inlet.
  • the unilateral fixing part of the liquid inlet check valve is connected to the fixing piece.
  • the unilateral fixed part of the liquid outlet one-way valve is fixedly connected to the side wall of the liquid storage pipe, and the closing and opening states of the liquid inlet one-way valve and the liquid outlet one-way valve are the same as above.
  • the on-off valve 8 includes a fixed block 81 connected to the flow channel and a second elastic movable portion 82, the second elastic movable portion is a part of the elastic mold 2, and the edge of the second elastic movable portion is connected to the side of the on-off valve tube.
  • the outer wall is fixedly connected; when the inside of the switch valve tube is under negative pressure, the second elastic movable part protrudes upwards and separates from the fixed block, and the flow channels on both sides of the fixed block are connected; when the inside of the switch valve tube is under positive pressure, the second elastic movable part protrudes upwards It is in close contact with the fixed block, and the flow channels on both sides of the fixed block are closed.
  • the first waste liquid pool is provided with a silica gel membrane for collecting DNA; the silica gel membrane can realize the capture of nucleic acid in the sample, and after the capture, the elution of DNA can be realized by adding an eluent.
  • a multi-index nucleic acid amplification assembly 4 the multi-index nucleic acid amplification assembly 4 includes a main channel 41 and a plurality of amplification chambers 42, and each amplification chamber 42 and the main channel 41 pass through to achieve different amplifications
  • the isolated dispersion pipes between the chambers 42 communicate; the main channel 41 communicates with the sample cell.
  • a shunt column 43 is arranged between the main channel 41 and the amplification chamber 42 , and the shunt column divides the space between the main channel and the amplification chamber into three scattered channels, along the The flow direction of the main channel liquid, the dispersion channel between the distribution column 43 and the outer wall of the amplification chamber 42 is the first dispersion channel 44 and the third dispersion channel 46, between the distribution column 43 and the main channel 41 is the second dispersion flow channel 45, and the relationship of the cross-sectional area of the dispersion flow channel is the first dispersion flow channel>the second dispersion flow channel>the third dispersion flow channel.
  • cross-sectional area ratio of the first dispersion channel, the second dispersion channel, and the third dispersion channel may be 7-12:3-5:1, specifically 10:4:1.
  • the shunt column 43 can be a column with a trapezoidal cross-section, and the shorter side of the trapezoid faces the direction of liquid flow. As shown in FIG. 2a, the direction of the arrow indicates the flow direction of the liquid.
  • v1/v2/v3 represent the cross-sections of the first dispersion channel, the second dispersion channel, and the third dispersion channel, respectively.
  • the process of dispersing the sample liquid in the sample pool to each amplification chamber through the main channel is as follows, as shown in Figure 2b: S1.
  • S2. Since the resistance faced by the second dispersion channel > the resistance faced by the first dispersion channel, the sample liquid enters the amplification chamber along the first dispersion channel; S3.
  • S3. When After the amplification chamber is full, the sample liquid encounters the third dispersion flow channel. At this time, since the resistance faced by the third dispersion flow channel > the resistance faced by the second dispersion flow channel, the sample liquid will pass through the second dispersion flow channel along the way.
  • the channel flows forward; S4.
  • a section of air column is left at the third dispersion flow channel of the amplification chamber, and the existence of the air column makes the samples in different amplification chambers stably separated; S5.
  • Pressure is applied to the sealed oil pool 29 for The oil separating the samples flows into the main channel. Due to the good wettability between the oil and the chip, the oil can drive out the air column stored in the third dispersion flow and realize the isolation between different amplification chambers.
  • step (3) Apply positive pressure to the vent pipe while keeping the on-off valve v6 closed, and introduce air into the pipe for 40s to completely remove the residual liquid on the silica gel membrane, and the residual liquid flows to the first waste liquid pipe along the same path in step (2);
  • Multi-index amplification can be achieved by pre-spotting different primers in each amplification chamber.
  • different primers can be pre-spotted in each amplification chamber. Since each amplification chamber does not interfere with each other, after nucleic acid amplification, it is possible to accurately determine which bacteria caused pneumonia; the judgment is marked as if it is pre-pointed with the amplification chamber. If the primers can be amplified, it means that it contains the pathogenic bacteria corresponding to the primers. If the primers on the amplification chamber cannot be amplified, it means that it does not contain the pathogenic bacteria corresponding to the primers. bacteria.
  • the invention proposes a new rapid and automatic multi-index nucleic acid detection device based on a microfluidic chip, which is a rapid detection tool with simple operation and low cost, and is suitable for the detection of various types of detection samples in various detection occasions.
  • Fast multi-index detection Multi-target amplification can be achieved by pre-spotting different primers in each amplification chamber.

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Abstract

Disclosed is a fully-integrated multi-index nucleic acid test microfluidic chip, comprising a nucleic acid sample pretreatment assembly, wherein the nucleic acid sample pretreatment assembly comprises a substrate, an elastic mold and a flow guide plate, a plurality of liquid storage tubes for storing liquid are arranged on the substrate, liquid storage tanks corresponding to the liquid storage tubes and flow channels for communicating with the liquid storage tanks are arranged on the flow guide plate, the elastic mold is sandwiched between the bottom of the substrate and the top of the flow guide plate, the liquid storage tubes and the liquid storage tanks are in communication or closed by the deformation of the elastic mold, and the flow guide plate is provided with a sample tank for storing a treated nucleic acid sample; and a multi-index nucleic acid amplification assembly, comprising a main flow channel and a plurality of amplification chambers, wherein each of the amplification chambers is in communication with the main flow channel through a dispersion pipeline for realizing isolation between different amplification chambers, and the main flow channel is in communication with the sample tank.

Description

一种全集成多指标核酸检测微流控芯片A fully integrated multi-index nucleic acid detection microfluidic chip 技术领域technical field
本发明属于生命医学检测领域，具体而言，涉及一种全集成多指标核酸检测微流控芯片。The invention belongs to the field of life medical detection, and in particular relates to a fully integrated multi-index nucleic acid detection microfluidic chip.
背景技术Background technique
自上个世纪九十年代来，作为生物体中最基本的物质之一，核酸在现代生物医学中扮演着越来越重要的角色，其特点在于：检测特异性强，非常易于操作，技术发展成熟，检测时间极短，因而其在传染病检测、食品安全监测、环境保护等领域都具有重大意义。因此，基于核酸进行快速检测己被公认为重要的分子检测手段之一。Since the 1990s, as one of the most basic substances in organisms, nucleic acid has played an increasingly important role in modern biomedicine. It is characterized by strong detection specificity, very easy operation, and technological development. It is mature and has a very short detection time, so it is of great significance in the fields of infectious disease detection, food safety monitoring, and environmental protection. Therefore, rapid detection based on nucleic acid has been recognized as one of the important molecular detection methods.
其中，基于核酸进行快速检测主要含有以下两种方式：聚合酶链式反应技术(PCR)和等温扩增技术。PCR作为一种发展成熟的核酸扩增技术经过近几十年的发展，已经成为生命医学检测和疾病诊断领域中的一种不可忽视的技术，其所涉及到的多个不同的反应温度，可用温度循环仪去进行精确的控制每个热循环步骤温度。核酸等温扩增技术在整个过程中则只需单一的反应温度，拥有更为简单的反应温度条件，无论是在实际操作还是仪器要求方面，都比PCR技术更为简单方便，可摆脱对精良设备的依赖，在临床和现场快速诊断中显示了其良好的应用前景。Among them, nucleic acid-based rapid detection mainly includes the following two methods: polymerase chain reaction (PCR) and isothermal amplification technology. As a mature nucleic acid amplification technology, PCR has become a technology that cannot be ignored in the field of life medical detection and disease diagnosis after decades of development. The temperature cycler performs precise temperature control of each thermal cycling step. Nucleic acid isothermal amplification technology only needs a single reaction temperature in the whole process, and has simpler reaction temperature conditions. It is simpler and more convenient than PCR technology in terms of actual operation and instrument requirements, and can get rid of the need for sophisticated equipment. It has shown its good application prospects in clinical and field rapid diagnosis.
但是，无论是PCR还是等温扩增技术，都需要完成样本前处理、核酸提取、扩增及检测等步骤。目前的核酸检测主要还是基于传统的方法，即首先是手工提取样本中的核酸，这一步一般是购买商业化的核酸提取试剂盒，再按照产品说明书进行操作，在试管内进行一系列复杂的生化反应来完成的；接下来的扩增流程以常用的聚合酶链式反应扩增为例，扩增需要手工加入反应混合液之后再通过商用的仪器来进行最后的检测。整个过程耗时耗力，而且还需要专业的实验室与专业的操作人员。可见，传统的核酸检测手段已不能满足日益增长的基核酸检测业务量需要。虽然目前已有公司推出商业化的全集成核酸分析仪，但其检测通量低，其一般是是通过堆叠多个控制与检测仪器来实现高通量的检测，如FilmArray 2.0系统。为了进一步提高检测效率并降低成本，全集成微流控系统在通量的可调节性上仍需改进。However, whether it is PCR or isothermal amplification technology, steps such as sample pretreatment, nucleic acid extraction, amplification and detection need to be completed. The current nucleic acid detection is mainly based on traditional methods, that is, the first step is to manually extract the nucleic acid in the sample. This step is generally to purchase a commercial nucleic acid extraction kit, and then operate according to the product instructions. A series of complex biochemical tests are carried out in test tubes. The following amplification process takes the commonly used polymerase chain reaction amplification as an example. The amplification needs to be manually added to the reaction mixture and then used for the final detection by a commercial instrument. The whole process is time-consuming and labor-intensive, and also requires professional laboratories and professional operators. It can be seen that traditional nucleic acid detection methods can no longer meet the needs of the growing business volume of nucleic acid detection. Although some companies have launched commercial fully integrated nucleic acid analyzers, their detection throughput is low. Generally, high-throughput detection is achieved by stacking multiple control and detection instruments, such as the FilmArray 2.0 system. In order to further improve the detection efficiency and reduce the cost, the fully integrated microfluidic system still needs to be improved in the adjustability of the flux.
发明公开Invention Disclosure
本发明的目的在于克服现有技术中的缺陷，解决基于微流控芯片进行核酸检测时的样本前处理、核酸提取、扩增及检测等复杂精密的流体操作，同时利用独立腔室作为反应区域完成对多指标、高通量的检测。The purpose of the present invention is to overcome the defects in the prior art, to solve the complex and precise fluid operations such as sample pretreatment, nucleic acid extraction, amplification and detection in nucleic acid detection based on microfluidic chips, and at the same time use an independent chamber as a reaction area Complete multi-index, high-throughput detection.
本发明提供一种多指标核酸检测微流控芯片，包括：The present invention provides a multi-index nucleic acid detection microfluidic chip, comprising:
核酸样品前处理组件，所述核酸样品前处理组件包括基板、弹性模和导流板，所述基板上设有多个用于储液的储液管，所述导流板上设有与储液管对应 的储液池和用于连通储液池的流道，所述基板的底部与导流板的顶部之间夹设有弹性模，储液管与储液池之间通过弹性模的形变连通或封闭，所述导流板上设有用于存储处理好的核酸样品的样品池；Nucleic acid sample pretreatment assembly, the nucleic acid sample pretreatment assembly includes a base plate, an elastic mold and a guide plate, the base plate is provided with a plurality of liquid storage tubes for liquid storage, and the baffle plate is provided with a storage tube and a storage tube. The liquid storage tank corresponding to the liquid pipe and the flow channel used to communicate with the liquid storage tank, an elastic mold is sandwiched between the bottom of the base plate and the top of the guide plate, and the elastic mold is deformed between the liquid storage pipe and the liquid storage tank. Connected or closed, the guide plate is provided with a sample pool for storing processed nucleic acid samples;
多指标核酸扩增组件，所述多指标核酸扩增组件包括主流道和多个扩增腔室，每个扩增腔室与主流道之间通过用于实现不同扩增腔室之间的隔离的分散管道相连通；所述主流道和样品池相连通。A multi-index nucleic acid amplification assembly, the multi-index nucleic acid amplification assembly includes a main channel and a plurality of amplification chambers, and each amplification chamber and the main channel are used to achieve isolation between different amplification chambers The dispersing pipeline is communicated; the main channel is communicated with the sample pool.
进一步，所述主流道与扩增腔室之间设有分流柱，所述分流柱将主流道与扩增腔室之间的空间分成三个分散流道，沿着主流道液体的流动的方向，分流柱与扩增腔室外壁之间的分散流道为第一分散流道和第三分散流道，所述分流柱与主流道之间的为第二分散流道，所述分散流道的的横截面积的关系为第一分散流道>第二分散流道>第三分散流道。Further, a shunt column is arranged between the main channel and the amplification chamber, and the shunt column divides the space between the main channel and the amplification chamber into three dispersed flow channels, along the direction of the flow of the main channel liquid. , the dispersion flow channel between the shunt column and the outer wall of the amplification chamber is the first dispersion flow channel and the third dispersion flow channel, and the second dispersion flow channel is between the shunt column and the main channel, and the dispersion flow channel The relationship of the cross-sectional area is the first dispersion flow channel > the second dispersion flow channel > the third dispersion flow channel.
进一步，所述第一分散流道、第二分散流道、第三分散流道的横截面积比为7～12：3～5：1。Further, the cross-sectional area ratio of the first dispersion channel, the second dispersion channel, and the third dispersion channel is 7-12:3-5:1.
进一步，所述第一分散流道、第二分散流道、第三分散流道的横截面积比为10：4：1。Further, the cross-sectional area ratio of the first dispersion flow channel, the second dispersion flow channel, and the third dispersion flow channel is 10:4:1.
进一步，所述分流柱43可以为横截面为梯形的柱体，所述梯形的较短底边朝向液体流动的方向，所述扩增腔室靠近流动方向侧壁与柱体之间的距离大于扩增腔室远离流动方向侧壁与柱体之间的距离。所述液体流动方向是指如图2a中箭头所示的方向，主流道中的液体流动时先与流体柱较短底边对应的侧面相接触。Further, the shunt column 43 may be a column with a trapezoidal cross-section, the shorter base of the trapezoid faces the direction of liquid flow, and the distance between the side wall of the amplification chamber near the flow direction and the column is greater than The distance between the sidewall of the amplification chamber away from the flow direction and the column. The liquid flow direction refers to the direction shown by the arrow in Fig. 2a. When the liquid in the main channel flows, it first contacts the side surface corresponding to the shorter bottom side of the fluid column.
进一步，所述储液管为多个，依次为裂解液管、通气管、洗涤液管、洗脱液管、第一废液管、开关阀管、收集管、扩增试剂管、密封油管和第二废液管；所述导流板上对应设置有裂解液池、通气池、洗涤液池、洗脱液池、第一废液池、流道、收集池、扩增试剂池、密封油池和样品池；其中，所述裂解液管与裂解液池、洗涤液管与洗涤液池、洗脱液管与洗脱液池、扩增试剂管与扩增试剂池、密封油管与密封油池之间设有由上向下流通的进液单向阀；所述第一废液管与第一废液池、第二废液管与样品池之间设有由下向上流通的出液单向阀；所述收集管与收集池之间设有可以控制进出方向的进液出液单向阀，所述开关阀管与流道之间设有开关阀。Further, there are a plurality of said liquid storage tubes, which are lysate tube, vent tube, washing liquid tube, eluent tube, first waste liquid tube, switch valve tube, collection tube, amplification reagent tube, sealed oil tube and The second waste liquid pipe; the baffle plate is correspondingly provided with a lysis solution pool, a ventilation pool, a washing solution pool, an eluent pool, a first waste solution pool, a flow channel, a collection pool, an amplification reagent pool, and a sealing oil Pool and sample pool; wherein, the lysate tube and the lysate pool, the washing solution tube and the washing solution pool, the eluent tube and the eluent pool, the amplification reagent tube and the amplification reagent pool, the sealing oil tube and the sealing oil There is a liquid inlet one-way valve that flows from top to bottom between the pools; the first waste liquid pipe and the first waste liquid pool, the second waste liquid pipe and the sample pool are provided with a bottom-to-up flow one-way liquid outlet. valve; between the collection pipe and the collection tank, a liquid inlet and outlet check valve that can control the direction of inflow and outflow is arranged, and an on-off valve is arranged between the on-off valve pipe and the flow channel.
进一步，所述进液单向阀、出液单向阀和进液出液单向阀通过弹性膜上的形变实现，通过施压控制弹性膜的形变方向实现单向阀的功能，当向储液管实施正压时，进液单向阀在压力的作用下形成向下的单向阀；当向储液管实施负压、或者是向导流板的流道施压时，出液单向阀在压力的作用下形成向上的单向阀；而在没有外界压力的作用下，由于弹性膜自身的弹性性质，处于关闭的阀门的状态。Further, the liquid inlet check valve, the liquid outlet check valve and the liquid inlet and outlet check valve are realized by the deformation on the elastic film, and the function of the check valve is realized by applying pressure to control the deformation direction of the elastic film. When positive pressure is applied to the liquid pipe, the liquid inlet check valve forms a downward one-way valve under the action of pressure; The valve forms an upward one-way valve under the action of pressure; under the action of no external pressure, due to the elastic properties of the elastic membrane itself, it is in the state of a closed valve.
进一步，所述进液单向阀和出液单向阀均为片状弹性膜片包括单边固定部和第一弹性活动部；Further, the liquid inlet one-way valve and the liquid outlet one-way valve are both sheet-shaped elastic diaphragms including a unilateral fixed part and a first elastic movable part;
其中，所述进液单向阀的单边固定部的一端与所述储液管的外壁固定连接，所述单边固定部的另一端与所述第一弹性活动部固定连接，当储液管内为正压时，所述第一弹性活动部向下移动与储液管的出液口分离，使得所述出液口与所述导流板上的流道处于导通状态，即所述进液单向阀处于开启状态，当储液管内的压力小于流道压力或者为负压时，所述第一弹性活动部与所述出液口贴合，使得所述出液口处于密闭隔绝状态，即所述进液单向阀处于闭合状态；Wherein, one end of the unilateral fixed part of the liquid inlet check valve is fixedly connected with the outer wall of the liquid storage pipe, and the other end of the unilateral fixed part is fixedly connected with the first elastic movable part. When the inside of the pipe is under positive pressure, the first elastic movable part moves downward and is separated from the liquid outlet of the liquid storage pipe, so that the liquid outlet and the flow channel on the guide plate are in a conducting state, that is, the The liquid inlet check valve is in the open state. When the pressure in the liquid storage pipe is lower than the flow channel pressure or negative pressure, the first elastic movable part is attached to the liquid outlet, so that the liquid outlet is sealed and isolated. state, that is, the liquid inlet check valve is in a closed state;
所述出液单向阀的单边固定部的一端与所述储液管的外壁固定连接，所述单边固定部的另一端与所述第一弹性活动部固定连接，当储液管内的压力小于流道压力或者为负压时，所述第一弹性活动部向上移动与储液管的进液口分离，使得所述进液口与所述导流板上的流道处于导通状态，即所述出液单向阀处于开启状态，当储液管内的压力为正压时，所述第一弹性活动部与所述进液口贴合，使得所述进液口处于密闭隔绝状态，所述出液单向阀处于关闭状态。One end of the unilateral fixed part of the liquid outlet check valve is fixedly connected to the outer wall of the liquid storage pipe, and the other end of the unilateral fixed part is fixedly connected to the first elastic movable part. When the pressure is lower than the flow channel pressure or negative pressure, the first elastic movable part moves upward and is separated from the liquid inlet of the liquid storage tube, so that the liquid inlet and the flow channel on the baffle plate are in a conductive state , that is, the liquid outlet one-way valve is in an open state, and when the pressure in the liquid storage pipe is positive pressure, the first elastic movable part is attached to the liquid inlet, so that the liquid inlet is in a closed state. , the liquid outlet one-way valve is closed.
进液出液单向阀包括进液单向阀、出液单向阀和位于出液口和进液口之间的固定件，所述进液单向阀的单边固定部与固定件相连接，所述出液单向阀的单边固定部与储液管的侧壁固定连接，进液单向阀和出液单向阀关闭开合状态与上相同。The liquid inlet and outlet check valve includes a liquid inlet check valve, a liquid outlet check valve and a fixing piece located between the liquid outlet and the liquid inlet, and the unilateral fixing part of the liquid inlet check valve is in phase with the fixing piece. The unilateral fixed part of the liquid outlet check valve is fixedly connected to the side wall of the liquid storage pipe, and the liquid inlet check valve and the liquid outlet check valve are closed and opened in the same state as above.
所述开关阀包括与流道相连接的固定块和第二弹性活动部，所述第二弹性活动部为弹性模的一部分，第二弹性活动部的边缘与开关阀管的侧外壁固定连接；当开关阀管内为负压时，第二弹性活动部向上突起与固定块分离，固定块两侧的流道连通；当开关阀管内为正压时，第二弹性活动部向上突起与固定块紧贴，固定块两侧的流道关闭。The on-off valve includes a fixed block connected with the flow channel and a second elastic movable part, the second elastic movable part is a part of the elastic mold, and the edge of the second elastic movable part is fixedly connected with the side outer wall of the on-off valve tube; When there is negative pressure in the switch valve tube, the upward protrusion of the second elastic movable part is separated from the fixed block, and the flow passages on both sides of the fixed block are connected; The runners on both sides of the fixed block are closed.
进一步，所述导流板与所述弹性膜贴合的面上设有导流槽，所述导流槽用于连通导流板上的各储液池。Further, a guide groove is provided on the surface where the guide plate is attached to the elastic film, and the guide groove is used to communicate with each liquid storage tank on the guide plate.
进一步，所述储液管上部设有推杆；所述导流板底部还设有检测装置。Further, the upper part of the liquid storage pipe is provided with a push rod; the bottom of the guide plate is also provided with a detection device.
进一步，所述每一个反应腔室内设有预先点入不同的引物，用以实现多指标的扩增。Further, each reaction chamber is provided with different primers in advance, so as to realize multi-index amplification.
本发明还提供一种多指标核酸扩增检测系统，包括所述的多指标核酸检测微流控芯片。The present invention also provides a multi-index nucleic acid amplification detection system, comprising the multi-index nucleic acid detection microfluidic chip.
本发明提出了一种新的基于微流控芯片进行快速自动的多指标核酸检测装置，是一种操作简单，成本低的快速检测工具，适用于多种检测场合下对多种类型检测样品的快速多指标检测。The invention proposes a new rapid and automatic multi-index nucleic acid detection device based on a microfluidic chip, which is a rapid detection tool with simple operation and low cost, and is suitable for the detection of various types of detection samples in various detection occasions. Fast multi-index detection.
附图说明Description of drawings
图1为本发明的多指标核酸检测微流控芯片的结构示意图，其中图a为结构示意图，图b为爆炸图；1 is a schematic structural diagram of a multi-index nucleic acid detection microfluidic chip of the present invention, wherein Figure a is a schematic structural diagram, and Figure b is an exploded view;
图2为多指标扩增芯片原理结构示意图，其中，图a为局部结构示意图，图b为随时间液体的流动方向示意图；Figure 2 is a schematic diagram of the principle structure of a multi-index amplification chip, wherein Figure a is a schematic diagram of a partial structure, and Figure b is a schematic diagram of the flow direction of the liquid over time;
图3为部分阀门的原理图，其中a为进液单向阀，b为出液单向阀，c为进液出液单向阀，d为开关阀。Figure 3 is a schematic diagram of some valves, in which a is a liquid inlet check valve, b is a liquid outlet check valve, c is a liquid inlet and outlet check valve, and d is an on-off valve.
实施发明的最佳方式Best way to implement your invention
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.
本发明提出的一种基于微流控芯片进行多指标核酸检测微流控芯片，包括：A microfluidic chip for multi-index nucleic acid detection based on a microfluidic chip proposed by the present invention includes:
核酸样品前处理组件，所述核酸样品前处理组件包括基板1、弹性模2和导流板3，所述基板1上设有多个用于储液的储液管，所述导流板2上设有与储液管对应的储液池和用于连通储液池的流道，所述基板1的底部与导流板3的顶部之间夹设有弹性模2，储液管与储液池之间通过弹性模2的形变连通或封闭，所述导流板3上设有用于存储处理好的核酸样品的样品池。Nucleic acid sample pretreatment assembly, the nucleic acid sample pretreatment assembly includes a substrate 1, an elastic mold 2 and a guide plate 3, the substrate 1 is provided with a plurality of liquid storage tubes for liquid storage, the guide plate 2 There is a liquid storage tank corresponding to the liquid storage pipe and a flow channel for connecting the liquid storage tank, an elastic mold 2 is sandwiched between the bottom of the base plate 1 and the top of the guide plate 3, and the liquid storage pipe is connected to the storage tank. The liquid pools are connected or closed by the deformation of the elastic mold 2, and the guide plate 3 is provided with a sample pool for storing the processed nucleic acid samples.
所述储液管为多个，如图1所示，沿着从左至右的方向依次为裂解液管11、通气管12、洗涤液管13、洗脱液管14、第一废液管15、开关阀管16、收集管17、扩增试剂管18、密封油管19和第二废液管10；所述导流板2上对应设置有裂解液池21、通气池22、洗涤液池23、洗脱液池24、第一废液池25、流道26、收集池27、扩增试剂池28、密封油池29和样品池20；其中，所述裂解液管与裂解液池、洗涤液管与洗涤液池、洗脱液管与洗脱液池、扩增试剂管与扩增试剂池、密封油管与密封油池之间设有由上向下流通的进液单向阀5；所述第一废液池与第二废液池之间设有由下向上流通的出液单向阀6；所述收集管与收集池之间设有可以控制进出方向的进液出液单向阀7，所述开关阀管16与流道26之间设有开关阀8。There are a plurality of the liquid storage pipes, as shown in FIG. 1 , along the direction from left to right are the lysing liquid pipe 11 , the vent pipe 12 , the washing liquid pipe 13 , the eluent pipe 14 , and the first waste liquid pipe. 15. On-off valve pipe 16, collecting pipe 17, amplification reagent pipe 18, sealing oil pipe 19 and second waste liquid pipe 10; the baffle 2 is provided with a lysing solution pool 21, aeration pool 22, washing solution pool correspondingly 23. The eluent pool 24, the first waste liquid pool 25, the flow channel 26, the collection pool 27, the amplification reagent pool 28, the sealed oil pool 29 and the sample pool 20; wherein, the lysate tube and the lysate pool, Between the washing liquid pipe and the washing liquid pool, the eluent pipe and the eluent pool, the amplification reagent tube and the amplification reagent pool, the sealing oil pipe and the sealing oil pool, a liquid inlet check valve 5 which flows from top to bottom is arranged; Between the first waste liquid pool and the second waste liquid pool, there is an outlet check valve 6 that flows from bottom to top; between the collection pipe and the collection pool, there is a liquid inlet and outlet check valve that can control the direction of inflow and outflow. 7. An on-off valve 8 is provided between the on-off valve pipe 16 and the flow channel 26 .
所述导流板与所述弹性膜贴合的面上设有导流槽，所述导流槽的位置与所述通孔对应，所述导流槽用于对从通孔流出的微流体进行引流。所述储液管上部设有推杆，用于调整所述储液池中压力并提供流体前进动力。所述导流板底部还设有检测装置。A guide groove is provided on the surface where the guide plate is attached to the elastic film, the position of the guide groove corresponds to the through hole, and the guide groove is used to control the microfluid flowing out of the through hole. Conduct drainage. The upper part of the liquid storage pipe is provided with a push rod, which is used to adjust the pressure in the liquid storage tank and provide fluid forward power. A detection device is also provided at the bottom of the deflector.
如图3所示，所述进液单向阀5和出液单向阀6均为片状弹性膜片包括单边固定部和第一弹性活动部。As shown in FIG. 3 , the liquid inlet check valve 5 and the liquid outlet check valve 6 are both sheet-shaped elastic diaphragms including a unilateral fixed portion and a first elastic movable portion.
其中，所述进液单向阀的单边固定部51的一端与所述储液管的外壁固定连接，所述单边固定部的另一端与进液单向阀的第一弹性活动部52固定连接，当储液管内为正压时，所述第一弹性活动部向下移动与储液管的出液口分离，使得所述出液口与所述导流板上的流道处于导通状态，即所述进液单向阀处于开启状态，当储液管内的压力小于流道压力或者为负压时，所述第一弹性活动部与所述出液口贴合，使得所述出液口处于密闭隔绝状态，即所述进液单向阀处 于闭合状态。One end of the unilateral fixed portion 51 of the liquid inlet check valve is fixedly connected to the outer wall of the liquid storage pipe, and the other end of the unilateral fixed portion is connected to the first elastic movable portion 52 of the liquid inlet check valve. Fixed connection, when there is a positive pressure in the liquid storage tube, the first elastic movable part moves downward and is separated from the liquid outlet of the liquid storage tube, so that the liquid outlet and the flow channel on the baffle plate are in a guiding position. In the open state, that is, the liquid inlet one-way valve is in the open state, when the pressure in the liquid storage pipe is less than the flow channel pressure or negative pressure, the first elastic movable part is in contact with the liquid outlet, so that the The liquid outlet is in a closed and isolated state, that is, the liquid inlet check valve is in a closed state.
所述出液单向阀6的单边固定部的一端与所述储液管的外壁固定连接，另一端与出液单向阀的第一弹性活动部固定连接，当储液管内的压力小于流道压力或者为负压时，所述第一弹性活动部向上移动与储液管的进液口分离，使得所述进液口与所述导流板上的流道处于导通状态，即所述出液单向阀处于开启状态，当储液管内的压力为正压时，所述第一弹性活动部与所述进液口贴合，使得所述进液口处于密闭隔绝状态，所述出液单向阀处于关闭状态。One end of the unilateral fixed part of the liquid outlet one-way valve 6 is fixedly connected to the outer wall of the liquid storage pipe, and the other end is fixedly connected to the first elastic movable part of the liquid outlet one-way valve. When the pressure in the liquid storage pipe is less than When the flow channel pressure or negative pressure, the first elastic movable part moves upward and separates from the liquid inlet of the liquid storage pipe, so that the liquid inlet and the flow channel on the baffle plate are in a conducting state, that is, The liquid outlet one-way valve is in the open state, when the pressure in the liquid storage pipe is positive pressure, the first elastic movable part is attached to the liquid inlet, so that the liquid inlet is in a closed and isolated state, so The liquid one-way valve is closed.
进液出液单向阀7包括进液单向阀、出液单向阀和位于出液口和进液口之间的固定件，所述进液单向阀的单边固定部与固定件相连接，所述出液单向阀的单边固定部与储液管的侧壁固定连接，进液单向阀和出液单向阀关闭开合状态与上相同。The liquid inlet and outlet check valve 7 includes a liquid inlet check valve, a liquid outlet check valve and a fixing piece located between the liquid outlet and the liquid inlet. The unilateral fixing part of the liquid inlet check valve is connected to the fixing piece. The unilateral fixed part of the liquid outlet one-way valve is fixedly connected to the side wall of the liquid storage pipe, and the closing and opening states of the liquid inlet one-way valve and the liquid outlet one-way valve are the same as above.
所述开关阀8包括与流道相连接的固定块81和第二弹性活动部82，所述第二弹性活动部为弹性模2的一部分，第二弹性活动部的边缘与开关阀管的侧外壁固定连接；当开关阀管内为负压时，第二弹性活动部向上突起与固定块分离，固定块两侧的流道连通；当开关阀管内为正压时，第二弹性活动部向上突起与固定块紧贴，固定块两侧的流道关闭。The on-off valve 8 includes a fixed block 81 connected to the flow channel and a second elastic movable portion 82, the second elastic movable portion is a part of the elastic mold 2, and the edge of the second elastic movable portion is connected to the side of the on-off valve tube. The outer wall is fixedly connected; when the inside of the switch valve tube is under negative pressure, the second elastic movable part protrudes upwards and separates from the fixed block, and the flow channels on both sides of the fixed block are connected; when the inside of the switch valve tube is under positive pressure, the second elastic movable part protrudes upwards It is in close contact with the fixed block, and the flow channels on both sides of the fixed block are closed.
所述第一废液池内设有用于收集DNA的硅胶膜；所述硅胶膜可以实现对样本中的核酸的捕获，在捕获后，通过加入洗脱液实现对DNA的洗脱。The first waste liquid pool is provided with a silica gel membrane for collecting DNA; the silica gel membrane can realize the capture of nucleic acid in the sample, and after the capture, the elution of DNA can be realized by adding an eluent.
多指标核酸扩增组件4，所述多指标核酸扩增组件4包括主流道41和多个扩增腔室42，每个扩增腔室42与主流道41之间通过用于实现不同扩增腔室42之间的隔离的分散管道相连通；所述主流道41和样品池相连通。A multi-index nucleic acid amplification assembly 4, the multi-index nucleic acid amplification assembly 4 includes a main channel 41 and a plurality of amplification chambers 42, and each amplification chamber 42 and the main channel 41 pass through to achieve different amplifications The isolated dispersion pipes between the chambers 42 communicate; the main channel 41 communicates with the sample cell.
如图2所示，所述主流道41与扩增腔室42之间设有分流柱43，所述分流柱将主流道与扩增腔室之间的空间分成三个分散流道，沿着主流道液体的流动的方向，分流柱43与扩增腔室42外壁之间的分散流道为第一分散流道44和第三分散46流道，所述分流柱43与主流道41之间的为第二分散流道45，所述分散流道的的横截面积的关系为第一分散流道>第二分散流道>第三分散流道。As shown in FIG. 2 , a shunt column 43 is arranged between the main channel 41 and the amplification chamber 42 , and the shunt column divides the space between the main channel and the amplification chamber into three scattered channels, along the The flow direction of the main channel liquid, the dispersion channel between the distribution column 43 and the outer wall of the amplification chamber 42 is the first dispersion channel 44 and the third dispersion channel 46, between the distribution column 43 and the main channel 41 is the second dispersion flow channel 45, and the relationship of the cross-sectional area of the dispersion flow channel is the first dispersion flow channel>the second dispersion flow channel>the third dispersion flow channel.
进一步，所述第一分散流道、第二分散流道、第三分散流道的横截面积比可为7～12：3～5：1具体可为10：4：1。Further, the cross-sectional area ratio of the first dispersion channel, the second dispersion channel, and the third dispersion channel may be 7-12:3-5:1, specifically 10:4:1.
具体而言，为了实现上述方案，所述分流柱43可以为横截面为梯形的柱体，所述梯形的较短一边朝向液体流动的方向，如图2a所示，箭头方向表示液体的流动方向，v1/v2/v3分别表示第一分散流道、第二分散流道、第三分散流道的截面。Specifically, in order to realize the above solution, the shunt column 43 can be a column with a trapezoidal cross-section, and the shorter side of the trapezoid faces the direction of liquid flow. As shown in FIG. 2a, the direction of the arrow indicates the flow direction of the liquid. , v1/v2/v3 represent the cross-sections of the first dispersion channel, the second dispersion channel, and the third dispersion channel, respectively.
所述样品池中的样品液体通过主流道分散到各个扩增腔室过程如下，如图2b所示，：S1.样品液体流经扩增腔室的入口时，遇到第一分散流道和第二分散流道的分流；S2.由于通过第二分散流道面临的阻力>通过第一分散流道面临的阻力，样品液体沿着第一分散流道，进入扩增腔室；S3.当扩增腔室充满后，样品液体遇到第三分散流道，此时，由于通过第三分散流道面临的阻力>通过第二 分散流道面临的阻力，样品液体沿着通过第二分散流道向前流动；S4.在扩增腔室的第三分散流道处存留一段空气柱，空气柱的存在使得不同扩增腔室内的样本稳定分隔；S5.对密封油池29施压使用于分隔样品的油通入主流道，由于油与芯片的浸润性好，油可将之前第三分散流处保存的空气柱赶出，并实现不同扩增腔室之间的隔离。The process of dispersing the sample liquid in the sample pool to each amplification chamber through the main channel is as follows, as shown in Figure 2b: S1. When the sample liquid flows through the inlet of the amplification chamber, it encounters the first dispersion channel and Diversion of the second dispersion channel; S2. Since the resistance faced by the second dispersion channel > the resistance faced by the first dispersion channel, the sample liquid enters the amplification chamber along the first dispersion channel; S3. When After the amplification chamber is full, the sample liquid encounters the third dispersion flow channel. At this time, since the resistance faced by the third dispersion flow channel > the resistance faced by the second dispersion flow channel, the sample liquid will pass through the second dispersion flow channel along the way. The channel flows forward; S4. A section of air column is left at the third dispersion flow channel of the amplification chamber, and the existence of the air column makes the samples in different amplification chambers stably separated; S5. Pressure is applied to the sealed oil pool 29 for The oil separating the samples flows into the main channel. Due to the good wettability between the oil and the chip, the oil can drive out the air column stored in the third dispersion flow and realize the isolation between different amplification chambers.
使用时：when using it:
(1)将待测样本加入裂解液管，密封后，对裂解液管处加热至60℃，在裂解液管中完成裂解；随后对裂解液管施加正压，同时对开关阀施加正压关闭开关阀v6，使裂解后的混合液通过进液单向阀流向裂解液池并通过流道流至洗涤液池流过硅胶膜；(1) Add the sample to be tested into the lysate tube, after sealing, heat the lysate tube to 60°C, and complete the lysis in the lysate tube; then apply positive pressure to the lysate tube, and at the same time apply positive pressure to the switch valve to close Switch valve v6, so that the mixed solution after lysis flows to the lysis solution pool through the inlet check valve and flows through the flow channel to the washing solution pool and flows through the silica gel membrane;
(2)对洗涤液管施加正压，关闭阀门v6，使洗涤液管中的800μL洗涤液流过硅胶膜(c1)，除去硅胶膜上的蛋白等其他杂质，洗涤液通过通过流道流至废液池进一步通过出液单向阀流向第一废液管；(2) Apply positive pressure to the washing solution tube, close the valve v6, and make 800 μL washing solution in the washing solution tube flow through the silica gel membrane (c1) to remove other impurities such as proteins on the silica gel membrane, and the washing solution flows through the flow channel to The waste liquid pool further flows to the first waste liquid pipe through the liquid outlet check valve;
(3)对通气管施加正压，同时保持开关阀v6关闭，向管道内通入空气40s，彻底去除硅胶膜上残留的液体，残留液体沿步骤(2)相同路径流向第一废液管；(3) Apply positive pressure to the vent pipe while keeping the on-off valve v6 closed, and introduce air into the pipe for 40s to completely remove the residual liquid on the silica gel membrane, and the residual liquid flows to the first waste liquid pipe along the same path in step (2);
(4)对洗脱液管施加正压，同时打开开关阀v6，用正压封闭第一废液管，使200μL洗脱液流过滤膜，洗脱的DNA溶液进入收集管；(4) Apply positive pressure to the eluent tube, open the switch valve v6 at the same time, close the first waste liquid tube with positive pressure, make 200 μL of the eluent flow through the filter membrane, and the eluted DNA solution enters the collection tube;
(5)对收集管施加正压，使DNA溶液进入扩增试剂管，溶解扩增试剂管内的LAMP反应试剂；可以分多次将DNA溶液打入，保证干粉试剂充分溶解；(5) Apply positive pressure to the collection tube, so that the DNA solution enters the amplification reagent tube, and dissolves the LAMP reaction reagent in the amplification reagent tube; the DNA solution can be injected in multiple times to ensure that the dry powder reagent is fully dissolved;
(6-7)对收集管和扩增试剂管同时施加正压，反应试剂进入扩增芯片，自动填充进每个扩增反应池；(6-7) Apply positive pressure to the collection tube and the amplification reagent tube at the same time, and the reaction reagent enters the amplification chip and automatically fills each amplification reaction pool;
(8)为防止污染，对密封油管施加正压，密封油进入扩增芯片，排出多余的气体，防止扩增过程中产生气溶胶污染。(8) In order to prevent pollution, positive pressure is applied to the sealed tubing, the sealing oil enters the amplification chip, and excess gas is discharged to prevent aerosol pollution during the amplification process.
(9)将芯片放在相匹配的的扩增仪和检测平台上上进行核酸的扩增与检测。(9) Put the chip on the matched amplification instrument and detection platform to perform nucleic acid amplification and detection.
通过在每一个扩增腔室内预先点入不同的引物可以实现多指标的扩增，例如：对于肺炎，其可能会由多种病菌所致，可以在每一个扩增腔室内预先点上对应不同的肺炎致病菌的引物，由于每一个扩增腔室之间互不干扰，经过核酸扩增，可以准确判断出是哪种病菌所导致肺炎；判断标注为如果与扩增腔室内预先点上的引物能够发生扩增反应，即表示含有此种引物对应的致病菌，若，不能与扩增腔室内预先点上的引物能够发生扩增反应，即表示不含有此种引物对应的致病菌。Multi-index amplification can be achieved by pre-spotting different primers in each amplification chamber. For example, for pneumonia, which may be caused by a variety of bacteria, different primers can be pre-spotted in each amplification chamber. Since each amplification chamber does not interfere with each other, after nucleic acid amplification, it is possible to accurately determine which bacteria caused pneumonia; the judgment is marked as if it is pre-pointed with the amplification chamber. If the primers can be amplified, it means that it contains the pathogenic bacteria corresponding to the primers. If the primers on the amplification chamber cannot be amplified, it means that it does not contain the pathogenic bacteria corresponding to the primers. bacteria.
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已 知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the present invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.
工业应用Industrial application
本发明提出了一种新的基于微流控芯片进行快速自动的多指标核酸检测装置，是一种操作简单，成本低的快速检测工具，适用于多种检测场合下对多种类型检测样品的快速多指标检测。通过在每一个扩增腔室内预先点入不同的引物可以实现多指标的扩增。The invention proposes a new rapid and automatic multi-index nucleic acid detection device based on a microfluidic chip, which is a rapid detection tool with simple operation and low cost, and is suitable for the detection of various types of detection samples in various detection occasions. Fast multi-index detection. Multi-target amplification can be achieved by pre-spotting different primers in each amplification chamber.

Claims (10)

  1. 一种全集成多指标核酸检测微流控芯片，其特征在于，包括：A fully integrated multi-index nucleic acid detection microfluidic chip, characterized by comprising:
    核酸样品前处理组件，所述核酸样品前处理组件包括基板、弹性模和导流板，所述基板上设有多个用于储液的储液管，所述导流板上设有与储液管对应的储液池和用于连通储液池的流道，所述基板的底部与导流板的顶部之间夹设有弹性模，储液管与储液池之间通过弹性模的形变连通或封闭，所述导流板上设有用于存储处理好的核酸样品的样品池；Nucleic acid sample pretreatment assembly, the nucleic acid sample pretreatment assembly includes a base plate, an elastic mold and a guide plate, the base plate is provided with a plurality of liquid storage tubes for liquid storage, and the baffle plate is provided with a storage tube and a storage tube. The liquid storage tank corresponding to the liquid pipe and the flow channel used to communicate with the liquid storage tank, an elastic mold is sandwiched between the bottom of the base plate and the top of the guide plate, and the elastic mold is deformed between the liquid storage pipe and the liquid storage tank. Connected or closed, the guide plate is provided with a sample pool for storing processed nucleic acid samples;
    多指标核酸扩增组件，所述多指标核酸扩增组件包括主流道和多个扩增腔室，每个扩增腔室分别与主流道连通，不通过的扩增腔室之间相互隔离；所述主流道和样品池相连通。A multi-index nucleic acid amplification component, the multi-index nucleic acid amplification component includes a main channel and a plurality of amplification chambers, each amplification chamber is respectively connected with the main channel, and the amplification chambers that do not pass are isolated from each other; The main flow channel is communicated with the sample cell.
  2. 根据权利要求1所述的全集成多指标核酸检测微流控芯片，其特征在于：所述主流道与每个扩增腔室之间均设有分流柱，所述分流柱将主流道与扩增腔室之间的空间分成三个分散流道，沿着主流道液体的流动的方向，分流柱与扩增腔室外壁之间的分散流道为第一分散流道和第三分散流道，所述分流柱与主流道之间的分散流道为第二分散流道，所述分散流道的的横截面积的关系为第一分散流道>第二分散流道>第三分散流道。The fully integrated multi-index nucleic acid detection microfluidic chip according to claim 1, wherein a split column is provided between the main channel and each amplification chamber, and the split column separates the main channel and the amplification chamber. The space between the amplification chambers is divided into three dispersion flow channels. Along the flow direction of the main channel liquid, the dispersion flow channels between the distribution column and the outer wall of the amplification chamber are the first dispersion flow channel and the third dispersion flow channel. , the dispersion flow channel between the distribution column and the main channel is the second dispersion flow channel, and the relationship of the cross-sectional area of the dispersion flow channel is the first dispersion flow channel > the second dispersion flow channel > the third dispersion flow channel road.
  3. 根据权利要求2所述的全集成多指标核酸检测微流控芯片，其特征在于：所述第一分散流道、第二分散流道、第三分散流道的横截面积比为7～12：3～5：1。The fully integrated multi-index nucleic acid detection microfluidic chip according to claim 2, wherein the cross-sectional area ratio of the first dispersion channel, the second dispersion channel, and the third dispersion channel is 7-12 : 3 to 5:1.
  4. 根据权利要求3所述的全集成多指标核酸检测微流控芯片，其特征在于：所述第一分散流道、第二分散流道、第三分散流道的横截面积比为10：4：1。The fully integrated multi-index nucleic acid detection microfluidic chip according to claim 3, wherein the cross-sectional area ratio of the first dispersion channel, the second dispersion channel, and the third dispersion channel is 10:4 :1.
  5. 根据权利要求4所述的全集成多指标核酸检测微流控芯片，其特征在于：所述分流柱可以为横截面为梯形的柱体，所述梯形的较短一底边朝向液体流动的方向，所述扩增腔室靠近流动方向侧壁与柱体之间的距离大于扩增腔室远离流动方向侧壁与柱体之间的距离。The fully integrated multi-index nucleic acid detection microfluidic chip according to claim 4, wherein the shunt column can be a column with a trapezoidal cross-section, and the shorter bottom side of the trapezoid faces the direction of liquid flow , the distance between the side wall of the amplification chamber close to the flow direction and the cylinder is greater than the distance between the side wall and the cylinder away from the flow direction of the amplification chamber.
  6. 根据权利要求4所述的全集成多指标核酸检测微流控芯片，其特征在于，每一个所述扩增腔室内设有不同的引物，用以实现多指标的扩增。The fully integrated multi-index nucleic acid detection microfluidic chip according to claim 4, wherein each of the amplification chambers is provided with different primers for realizing multi-index amplification.
  7. 根据权利要求6所述的全集成多指标核酸检测微流控芯片，其特 征在于：所述储液管为多个，依次为裂解液管、通气管、洗涤液管、洗脱液管、第一废液管、开关阀管、收集管、扩增试剂管、密封油管和样品池；所述导流板上对应设置有裂解液池、通气池、洗涤液池、洗脱液池、第一废液池、收集池、扩增试剂池、密封油池和样品池；其中，所述裂解液管与裂解液池、洗涤液管与洗涤液池、洗脱液管与洗脱液池、扩增试剂管与扩增试剂池、密封油管与密封油池之间设有由上向下流通的进液单向阀；所述第一废液管与第一废液池、第二废液管与样品池之间设有由下向上流通的出液单向阀；所述收集管与收集池直接设有可以控制进出方向的进液出液单向阀，所述开关阀管与流道之间设有开关阀。The fully integrated multi-index nucleic acid detection microfluidic chip according to claim 6, characterized in that: there are a plurality of said liquid storage tubes, which are in sequence lysate tube, vent tube, washing solution tube, eluent tube, first tube a waste liquid pipe, an on-off valve pipe, a collection pipe, an amplification reagent pipe, a sealed oil pipe and a sample pool; the baffle plate is correspondingly provided with a lysate pool, a ventilation pool, a washing solution pool, an eluent pool, a first Waste liquid pool, collection pool, amplification reagent pool, sealed oil pool and sample pool; wherein, the lysate tube and the lysate pool, the washing solution tube and the washing solution pool, the eluent tube and the eluent pool, the A liquid inlet one-way valve that flows from top to bottom is arranged between the increasing reagent tube and the amplification reagent pool, the sealing oil tube and the sealing oil pool; the first waste liquid tube and the first waste liquid pool and the second waste liquid tube are connected to There is an outlet check valve that flows from bottom to top between the sample pools; the collection pipe and the collection pool are directly provided with a liquid inlet and outlet check valve that can control the direction of in and out, and the on-off valve pipe and the flow channel are provided with a one-way valve. There is an on-off valve.
  8. 根据权利要求7所述的全集成多指标核酸检测微流控芯片，其特征在于：所述进液单向阀、出液单向阀和进液出液单向阀通过弹性膜上的形变实现，通过施压控制弹性膜的形变方向实现单向阀的功能，当向储液管实施正压时，进液单向阀在压力的作用下形成向下的单向阀；当向储液管实施负压、或者是向导流板的流道施压时，出液单向阀在压力的作用下形成向上的单向阀；而在没有外界压力的作用下，由于弹性膜自身的弹性性质，处于关闭的阀门的状态。The fully integrated multi-index nucleic acid detection microfluidic chip according to claim 7, wherein the liquid inlet check valve, the liquid outlet check valve and the liquid inlet and outlet check valve are realized by deformation on the elastic membrane , the function of the one-way valve is realized by applying pressure to control the deformation direction of the elastic membrane. When positive pressure is applied to the liquid storage pipe, the liquid inlet one-way valve forms a downward one-way valve under the action of pressure; When negative pressure is applied or pressure is applied to the flow channel of the deflector, the liquid outlet one-way valve forms an upward one-way valve under the action of pressure; and under the action of no external pressure, due to the elastic properties of the elastic membrane itself, in the state of a closed valve.
  9. 根据权利要求8所述的全集成多指标核酸检测微流控芯片，其特征在于：所述储液管上部设有推杆；所述导流板底部还设有检测装置。The fully integrated multi-index nucleic acid detection microfluidic chip according to claim 8, wherein a push rod is arranged on the upper part of the liquid storage tube; and a detection device is also arranged at the bottom of the guide plate.
  10. 一种多指标核酸扩增检测系统，其特征在于，包括权利要求1-9任一所述的全集成多指标核酸检测微流控芯片。A multi-index nucleic acid amplification detection system, characterized in that it comprises the fully integrated multi-index nucleic acid detection microfluidic chip according to any one of claims 1-9.
PCT/CN2021/078004 2021-02-19 2021-02-26 Fully-integrated multi-index nucleic acid test microfluidic chip WO2022174472A1 (en)

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