WO2022174472A1 - Puce microfluidique multi-index entièrement intégrée pour le test d'acide nucléique - Google Patents

Puce microfluidique multi-index entièrement intégrée pour le test d'acide nucléique 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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Prior art keywords
nucleic acid
pool
liquid
amplification
tube
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PCT/CN2021/078004
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English (en)
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

La présente invention concerne une puce microfluidique multi index entièrement intégrée pour le test d'acide nucléique, comprenant les éléments suivants : un ensemble de prétraitement d'échantillon d'acide nucléique, l'ensemble de prétraitement d'échantillon d'acide nucléique comprenant un substrat, un moule élastique et un plateau de guidage d'écoulement, une pluralité de tubes de stockage de liquide pour stocker du liquide étant disposés sur le substrat, des réservoirs de stockage de liquide correspondant aux tubes de stockage de liquide et des canaux d'écoulement pour communiquer avec les réservoirs de stockage de liquide sont disposés sur le plateau de guidage d'écoulement, le moule élastique est intercalé entre le fond du substrat et le haut du plateau de guidage d'écoulement, les tubes de stockage de liquide et les réservoirs de stockage de liquide sont en communication ou fermés par la déformation du moule élastique, et le plateau de guidage d'écoulement est muni d'un réservoir d'échantillon pour stocker un échantillon d'acide nucléique traité ; et un ensemble multi-index d'amplification d'acide nucléique, comprenant un canal d'écoulement principal et une pluralité de chambres d'amplification, chacune des chambres d'amplification étant en communication avec le canal d'écoulement principal par l'intermédiaire d'un conduit de dispersion pour assurer l'isolation entre différentes chambres d'amplification, et le canal d'écoulement principal étant en communication avec le réservoir d'échantillons.
PCT/CN2021/078004 2021-02-19 2021-02-26 Puce microfluidique multi-index entièrement intégrée pour le test d'acide nucléique WO2022174472A1 (fr)

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