WO2022174471A1 - Puce microfluidique entièrement intégrée pour le test d'acide nucléique des agents pathogènes - Google Patents

Puce microfluidique entièrement intégrée pour le test d'acide nucléique des agents pathogènes Download PDF

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Publication number
WO2022174471A1
WO2022174471A1 PCT/CN2021/078003 CN2021078003W WO2022174471A1 WO 2022174471 A1 WO2022174471 A1 WO 2022174471A1 CN 2021078003 W CN2021078003 W CN 2021078003W WO 2022174471 A1 WO2022174471 A1 WO 2022174471A1
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layer
chamber
nucleic acid
reagent storage
liquid
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PCT/CN2021/078003
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English (en)
Chinese (zh)
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刘鹏
李保
李尚霖
林宝宝
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杭州梓晶生物有限公司
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Publication of WO2022174471A1 publication Critical patent/WO2022174471A1/fr

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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
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of biological analysis and detection, in particular to a fully integrated pathogen nucleic acid detection microfluidic chip.
  • nucleic acid detection methods such as Southern blot hybridization, agarose gel electrophoresis, etc.
  • Nucleic acid amplification methods such as polymerase chain reaction PCR, loop-mediated isothermal amplification of LAMP, recombinase polymerase amplification of RPA, etc.
  • Pathogen detection by nucleic acid amplification generally includes four steps: sample pretreatment, nucleic acid extraction, amplification, and detection.
  • Microfluidic chip technology integrates basic operation units such as sample preparation, reaction, separation, and detection in biological, chemical, and medical analysis processes into a micro-scale chip, and automatically completes the entire analysis process. Due to its great potential in biology, chemistry, medicine and other fields, it has developed into one of the important carrier tools for nucleic acid detection. Microfluidic chips have been cited many times in nucleic acid extraction and amplification detection, but most of them are still in the laboratory stage. The reason is that it requires various microvalves, micropumps and auxiliary equipment, which increases the complexity of chip applications, greatly increases the manufacturing difficulty and cost, limits its further clinical application, and is difficult to achieve fully integrated. Therefore, a fully integrated, high-sensitivity microfluidic chip is urgently needed to improve the nucleic acid detection process while reducing the detection time and the difficulty and cost of chip manufacturing.
  • a fully integrated pathogen nucleic acid detection microfluidic chip that can quickly and highly sensitively integrates nucleic acid capture and in-situ amplification is provided.
  • the present invention provides a fully integrated pathogen nucleic acid detection microfluidic chip, comprising a fluid pipeline layer, an elastic tape layer and a reagent storage layer.
  • a chamber, an in-situ extraction and amplification chamber with a capture filter paper is arranged in the fluid pipeline layer, the elastic tape layer is sandwiched between the fluid pipeline layer and the reagent storage layer, and the storage layer in the reagent storage layer is The collection solution, cleaning solution and reaction reagents flow through the elastic tape layer to the fluid conduit layer under the action of external force, and flow to the in-situ extraction and amplification chamber through the flow channel of the fluid conduit layer for reaction.
  • the elastic adhesive tape layer is a material with adhesive on both sides, which can seal and connect the reagent storage layers above the fluid pipeline layers below together.
  • the elastic adhesive tape layer may be a double-sided tape.
  • the fluid conduit layer corresponds to the chamber of the reagent storage layer, and the elastic tape layer between the upper and lower corresponding chambers is provided with a one-way valve communicating from top to bottom.
  • a collection liquid chamber, a cleaning liquid chamber, a mixing chamber and a waste liquid pool are sequentially arranged on the reagent storage layer, and a reaction reagent storage sleeve is detachably connected to the reagent storage layer.
  • the liquid, cleaning solution, and reaction reagent are sealed and stored in the collection solution chamber, the cleaning solution chamber, and the reaction reagent storage sleeve, respectively.
  • reaction reagent storage sleeve comprises a lower end spinner, a liquid storage chamber and an upper end spinner sequentially connected from bottom to top, the upper end spinner is connected with a push rod, and the rubber stopper ball is located in the liquid storage chamber. Between the bottom wall and the push rod, the reagent storage layer is provided with an interface matching the lower end of the liquid storage chamber.
  • an in-situ extraction and amplification chamber is provided with a capture filter paper fixing position on the fluid pipeline layer corresponding to the mixing chamber, and the capture filter paper used for capturing nucleic acid is connected to the capture filter paper fixing position;
  • the mixing chamber is provided with a capture filter paper fixing position;
  • the corresponding position of the uniform chamber is provided with an in-situ extraction and amplification chamber, and the elastic tape layer between the chambers is provided with a detackifying layer.
  • the detackifying layer with through holes is the detackifying layer of the elastic adhesive tape at the position between the corresponding chambers, and the detackifying elastic adhesive tape layer is provided with through holes.
  • the detackifying layer is connected with the in-situ extraction and amplification chamber; the fluid pipeline layer is provided with a first flow channel, one end of the first flow channel is respectively connected with the cleaning solution pool and the collection solution pool, and the other end can pass through the
  • the detackifying layer corresponding to the extrusion is communicated with the in-situ extraction and amplification chamber; the side of the in-situ extraction and amplification chamber close to the waste liquid pool is provided with a second flow channel communicated with the waste liquid pool, the A one-way valve communicating from bottom to top is arranged between the second flow channel and the waste liquid pool.
  • the one-way valve communicated from bottom to top is a detackifying layer with a through hole, the through hole is communicated with the waste liquid pool, and the liquid outlet of the second flow channel and the through hole are relative to the detackifying layer.
  • the layers are staggered.
  • the de-viscosity layer deforms upwards so that the second flow channel is connected with the through hole, and the liquid flows to the waste liquid pool; and when the liquid flows from top to bottom , because the through hole and the liquid outlet of the second flow channel are arranged in a staggered manner, the de-viscosity layer is pressed downward to close the through hole, and the one-way valve is closed at this time.
  • the waste liquid pool is provided with a waste liquid pool exhaust hole
  • the outer wall of the reagent storage layer is connected with a gas film
  • the gas mold and the outer wall of the reagent storage layer form a closed accommodating space, and the accommodating space is connected to the outer wall of the reagent storage layer.
  • the exhaust hole of the waste liquid pool is connected.
  • the reagent storage layer is provided with a handle.
  • the fully integrated pathogen nucleic acid detection microfluidic chip of the present invention can integrate pathogen sample processing, nucleic acid extraction, and in-situ amplification into the same microfluidic chip device, thereby getting rid of the tedious operations and professional requirements when actually performing pathogen nucleic acid detection. laboratory dependence.
  • Using the fully integrated pathogen nucleic acid detection microfluidic chip provided by the present invention can truly realize fully integrated, rapid and highly sensitive pathogen nucleic acid detection.
  • FIG. 2 is a front view of a fully integrated pathogen nucleic acid detection microfluidic chip of the present invention
  • FIG. 4 is a schematic structural diagram of the reagent storage sleeve of the fully integrated pathogen nucleic acid detection microfluidic chip of the present invention
  • the present invention provides a fully integrated pathogen nucleic acid detection microfluidic chip, which includes a fluid pipeline layer 1, an elastic tape layer 2 and a reagent storage layer 3 as shown in FIG. Operation; reconstitution chamber, which can be used for reconstituting lyophilized reagents; nucleic acid capture filter paper fixed position, which can fix the filter paper for nucleic acid capture; reaction chamber, which can be used as a nucleic acid amplification chamber for the original nucleic acid captured by the filter paper. bit amplification.
  • a double-sided adhesive base material is used to bond the fluid pipeline layer and the reagent storage layer to encapsulate the chip.
  • One side is provided with a magnesium ion redissolving chamber 15, one side of the magnesium ion redissolving chamber 15 is communicated with the reaction pool 14, and the other end can be connected to the in-situ extraction and amplification chamber by extruding the correspondingly arranged detackifying layer. 17 is connected.
  • the first flow channel 11, one end of the first flow channel 11 is respectively connected to the cleaning solution pool and the collection solution pool, and the other end can be communicated with the in-situ extraction and amplification chamber 17 by squeezing the correspondingly provided detackifying layer.
  • the side of the in-situ extraction and amplification chamber 17 close to the waste liquid pool is provided with a second flow channel 16 communicating with the waste liquid pool, and a de-viscosity layer is provided between the second flow channel 16 and the waste liquid pool 36 , the de-sticking layer is provided with a one-way valve which communicates from bottom to top.
  • the one-way valve that communicates from top to bottom is a detackifying layer with through holes, the flow channels on the through-hole fluid pipeline layer or each reaction cell are connected, and the through holes are connected with the reagent storage layer chamber.
  • the liquid outlet of the chamber is staggered. When the liquid flows to the de-viscosity layer, the de-viscosity layer is deformed downward, so that it can form a passage through the through holes; The ports are staggered, and the de-adhesive layer is squeezed upward to close the through holes, and the one-way valve is closed at this time.
  • the one-way valve communicated from bottom to top is a detackifying layer with a through hole, the through hole is communicated with the waste liquid pool, and the liquid outlet and the through hole of the second flow channel are staggered relative to the detackifying layer.
  • the reagent storage layer also includes a sealing valve chamber 35.
  • a sealing valve chamber 35 When the reaction reagent is injected into the in-situ extraction and amplification chamber to start the reaction, the lowest end of the inner chamber of the sealing valve chamber 35 will be plugged with a circular hole with a diameter slightly larger than the inner diameter of the sealing valve. The diameter of the rubber stopper, the rubber stopper squeezes the double-sided tape at both ends of the in-situ extraction and amplification chamber by extrusion, so as to seal the two ends of the reaction chamber for sealing.
  • the reaction reagent storage sleeve 34 includes a lower end spinner 341, a liquid storage chamber 342, and an upper end spinner 343 sequentially connected from bottom to top.
  • the upper end spinner 343 is connected with a push rod 344, and the liquid storage chamber
  • a rubber stopper ball 345 is arranged in the chamber 342.
  • the rubber stopper ball is located between the push rod 344 and the bottom wall of the liquid storage chamber 342.
  • the reagent storage layer 3 is provided with an interface matching the lower end of the liquid storage chamber 342.
  • the lower end of the liquid storage chamber 342 can be directly connected to the interface after removing the lower end cap 341 .
  • the lower end of the liquid storage chamber 342 is directly connected with the interface in a sealed manner.
  • the reagent storage layer is provided with a handle 31 .
  • the specific implementation of the chip is as follows:
  • Sample collection The pathogens in the public space environment were collected for 30 minutes by a bioaerosol sampler (specification: portable bioaerosol sampler WA-400, manufacturer: Beijing Dinglan Technology Co., Ltd.), and the chip lysate was detected at this time.
  • the chamber is directly connected to the sample outlet of the bioaerosol sampler through a tube valve, and the new coronavirus particles in the air can be directly collected into the collection liquid chamber.
  • the new coronavirus (2019-nCoV) fake Viral RNA reference material instead (Item No.: NIM-RM5203, manufacturer: China Institute of Metrology);
  • Pathogen lysis Load the detection chip on the fully integrated pathogen nucleic acid analyzer, and 8-bit detection chips can be loaded at a time, so that the pathogen can be lysed in the collection liquid chamber to release nucleic acid;
  • Nucleic acid in-situ amplification pressurize the reaction reagent storage sleeve, so that the reaction system reagents in the reaction reagent storage sleeve pass through the reaction pool and the magnesium ion redissolving chamber, and the solid magnesium acetate in the magnesium ion redissolving chamber is carried out. Reconstituted, and then flowed to the in-situ extraction and amplification chamber, then, the sealing valves at both ends of the in-situ extraction and amplification chamber are sealed, the heating membrane starts to heat, and the reaction starts.
  • the mixing chamber performs bidirectional mixing under the action of the instrument push rod to enhance the reaction effect;
  • the chip of the present invention can cooperate with a fully integrated nucleic acid detector to integrate "sample pretreatment, nucleic acid extraction, amplification, and detection" of the sample to be tested, and the operation is simple and can avoid the need for traditional laboratory Various tedious operations are required; the chip is fully enclosed design, the reagents are pre-mixed, and no manual solution is required, which is safe and reliable, and has no cross-contamination with the environment; the chip is combined with chitosan-modified specific filter paper for nucleic acid capture and enrichment. In situ amplification, the detection sensitivity can reach 20copies/mL, especially suitable for low-copy pathogen samples in air aerosols and on the surface of items to be tested.
  • the analyzer can integrate multiple chips at one time for high-throughput detection; the reagent storage chamber stores the types of reagents, and the number of chambers in the reagent storage chamber can be adjusted flexibly, which can adapt to a variety of different conditions for a variety of different pathogens. specific detection.
  • the chip of the present invention can cooperate with a fully integrated nucleic acid detector to integrate "sample pretreatment, nucleic acid extraction, amplification, and detection" of the sample to be tested, and the operation is simple and can avoid various tedious operations required by traditional laboratories;
  • the chip is fully enclosed It is safe and reliable, and has no cross-contamination with the environment;
  • the chip is combined with chitosan-modified specific filter paper for nucleic acid capture and enrichment, and in situ amplification can be performed, and the detection sensitivity can reach 20copies /mL, especially suitable for low-copy pathogen samples to be tested in aerosols and on the surface of objects;
  • a fully integrated nucleic acid analyzer multiple chips can be integrated at one time for high-throughput detection;
  • the reagent storage chamber stores the types of reagents, The number of chambers in the reagent storage chamber can be adjusted elastically, which can be adapted to perform specific detection of a variety of different pathogens under a variety of different conditions.

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  • Chemical & Material Sciences (AREA)
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  • Health & Medical Sciences (AREA)
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  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

La présente invention concerne une puce microfluidique entièrement intégrée pour le test d'acide nucléique des agents pathogènes, comprenant une couche de canalisation de fluide, une couche de bande adhésive élastique et une couche de stockage de réactifs, la couche de stockage de réactifs étant conçue pour stocker respectivement des chambres pour un liquide de collecte, un liquide de nettoyage, un réactif de réaction et un liquide résiduel, une chambre d'amplification d'extraction in situ comportant un papier filtre de capture étant prévue dans la couche de canalisation de fluide, la couche de bande adhésive élastique est intercalée entre la couche de canalisation de fluide et la couche de stockage de réactif, et le liquide de collecte, le liquide de nettoyage et le réactif de réaction dans la couche de stockage de réactif passent à travers la couche de bande adhésive élastique pour s'écouler vers la couche de canalisation de fluide sous l'action d'une force externe, et s'écoulent vers la chambre d'amplification d'extraction in situ pour réaction à travers un canal d'écoulement de la couche de canalisation de fluide. La puce microfluidique entièrement intégrée pour le test d'acide nucléique des agents pathogènes de la présente invention peut intégrer le traitement de l'échantillon d'agent pathogène, l'extraction de l'acide nucléique et l'amplification in situ dans le même dispositif de puce microfluidique, de sorte que les opérations complexes et la dépendance à l'égard d'un laboratoire professionnel pendant un test d'acide nucléique d'agent pathogène réel sont éliminées.
PCT/CN2021/078003 2021-02-19 2021-02-26 Puce microfluidique entièrement intégrée pour le test d'acide nucléique des agents pathogènes WO2022174471A1 (fr)

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CN202110188881.4A CN112940922A (zh) 2021-02-19 2021-02-19 一种全集成病原体核酸检测微流控芯片
CN202110188881.4 2021-02-19

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CN114164088B (zh) * 2021-12-10 2024-03-29 西安交通大学 一种居家核酸检测的芯片结构、卡盒及方法
CN114100722B (zh) * 2021-12-27 2024-06-04 北京梓晶生物科技有限公司 便携式全自动核酸恒温扩增装置
CN114570449B (zh) * 2022-04-26 2022-08-16 广州国家实验室 液体转移装置及多路并联的液体转移装置
WO2023231158A1 (fr) * 2022-05-30 2023-12-07 广州达安基因股份有限公司 Dispositif de test d'acide nucléique portatif comportant une puce microfluidique, et son procédé d'utilisation

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