WO2022174471A1 - Fully-integrated pathogen nucleic acid test microfluidic chip - Google Patents
Fully-integrated pathogen nucleic acid test microfluidic chip Download PDFInfo
- 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
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- layer
- chamber
- nucleic acid
- reagent storage
- liquid
- Prior art date
Links
- 244000052769 pathogen Species 0.000 title claims abstract description 33
- 230000001717 pathogenic effect Effects 0.000 title claims abstract description 28
- 238000011330 nucleic acid test Methods 0.000 title abstract 4
- 239000007788 liquid Substances 0.000 claims abstract description 116
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 93
- 238000003860 storage Methods 0.000 claims abstract description 81
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 66
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 66
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 66
- 238000006243 chemical reaction Methods 0.000 claims abstract description 50
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 48
- 230000003321 amplification Effects 0.000 claims abstract description 47
- 239000012530 fluid Substances 0.000 claims abstract description 43
- 238000000605 extraction Methods 0.000 claims abstract description 35
- 238000011065 in-situ storage Methods 0.000 claims abstract description 35
- 239000002699 waste material Substances 0.000 claims abstract description 33
- 238000004140 cleaning Methods 0.000 claims abstract description 31
- 239000002390 adhesive tape Substances 0.000 claims abstract description 8
- 230000009471 action Effects 0.000 claims abstract description 4
- 238000001514 detection method Methods 0.000 claims description 58
- 238000002156 mixing Methods 0.000 claims description 12
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 9
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 9
- 239000012295 chemical reaction liquid Substances 0.000 claims description 6
- 239000010410 layer Substances 0.000 description 98
- 239000000243 solution Substances 0.000 description 10
- 238000000034 method Methods 0.000 description 8
- 238000007789 sealing Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 241000711573 Coronaviridae Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000001125 extrusion Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011895 specific detection Methods 0.000 description 3
- 241001112090 Pseudovirus Species 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- 239000000853 adhesive Substances 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 239000012790 adhesive layer Substances 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000012864 cross contamination Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 description 2
- 229940069446 magnesium acetate Drugs 0.000 description 2
- 235000011285 magnesium acetate Nutrition 0.000 description 2
- 239000011654 magnesium acetate Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 238000007397 LAMP assay Methods 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000012197 amplification kit Methods 0.000 description 1
- 230000002457 bidirectional effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000012925 reference material Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- 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.
Abstract
Disclosed is a fully-integrated pathogen nucleic acid test microfluidic chip, comprising a fluid pipeline layer, an elastic adhesive tape layer and a reagent storage layer, wherein the reagent storage layer is configured for respectively storing chambers for collection liquid, cleaning liquid, a reaction reagent and waste liquid, an in-situ extraction amplification chamber having capture filter paper is provided in the fluid pipeline layer, the elastic adhesive tape layer is sandwiched between the fluid pipeline layer and the reagent storage layer, and the collection liquid, the cleaning liquid and the reaction reagent in the reagent storage layer pass through the elastic adhesive tape layer to flow to the fluid pipeline layer under the action of an external force, and flow to the in-situ extraction amplification chamber for reaction through a flow channel of the fluid pipeline layer. The fully-integrated pathogen nucleic acid test microfluidic chip of the present invention can integrate pathogen sample treatment, nucleic acid extraction and in-situ amplification into the same microfluidic chip device, such that complex operations and dependence on a professional laboratory during an actual pathogen nucleic acid test are eliminated.
Description
本发明涉及生物分析检测领域，具体的说，涉及一种全集成病原体核酸检测微流控芯片。The invention relates to the field of biological analysis and detection, in particular to a fully integrated pathogen nucleic acid detection microfluidic chip.
作为体外诊断领域的主要检测方式之一，核酸检测是实现病原体早期、快速、特异检测的最直接、最可靠、最灵敏的方法，其能快速对检测样本中病原体核酸进行检测，为感染病例确诊提供科学检验依据。As one of the main detection methods in the field of in vitro diagnosis, nucleic acid detection is the most direct, reliable and sensitive method to achieve early, rapid and specific detection of pathogens. Provide scientific evidence.
传统的核酸检测方法如Southern blot印迹杂交、琼脂糖凝胶电泳等，具有费时费力，灵敏度低，难以实现高通量检测，甚至还需使用放射性试剂等缺点。核酸扩增的方法如聚合酶链式反应PCR、环介导等温扩增LAMP，重组酶聚合酶扩增RPA等，可极大地提高检测灵敏度，同时还因操作相对简单使其成为现代分子生物学实验常用方法。通过核酸扩增进行病原体检测一般包含样本前处理、核酸提取、扩增、检测四个步骤。传统的在实验室进行病原体检测的方法需要有专业人员在具有专业条件的实验室中完成，需要人工操作来完成试剂的转移和添加，依旧需要完成大量繁琐操作，在操作的过程中还可能会将反应试剂暴露在空气中，极易造成污染，而且还需借助于温度控制仪来提高精确地温度控制，因而限制了传统的实验室核酸检测实现对病原体的快速、灵敏检测。Traditional nucleic acid detection methods, such as Southern blot hybridization, agarose gel electrophoresis, etc., are time-consuming and labor-intensive, have low sensitivity, are difficult to achieve high-throughput detection, and even require the use of radioactive reagents. Nucleic acid amplification methods such as polymerase chain reaction PCR, loop-mediated isothermal amplification of LAMP, recombinase polymerase amplification of RPA, etc., can greatly improve the detection sensitivity, and at the same time, because of the relatively simple operation, it has become a modern molecular biology method. Commonly used experimental methods. Pathogen detection by nucleic acid amplification generally includes four steps: sample pretreatment, nucleic acid extraction, amplification, and detection. The traditional method of pathogen detection in the laboratory needs to be completed by professionals in a laboratory with professional conditions, and manual operation is required to complete the transfer and addition of reagents, and a large number of tedious operations still need to be completed. Exposing the reaction reagents to the air can easily cause contamination, and a temperature controller is needed to improve precise temperature control, which limits the rapid and sensitive detection of pathogens by traditional laboratory nucleic acid detection.
微流控芯片技术(Microfluidics)是把生物、化学、医学分析过程的样品制备、反应、分离、检测等基本操作单元集成到一个微尺度的芯片上，自动完成分析全过程。由于它在生物、化学、医学等领域的巨大潜力，已经发展成为进行核酸检测的一个重要的载体工具之一。微流控芯片在核酸提取扩增检测上已有诸多引用，但是大多数都依旧停留在实验室阶段。究其因是其需要各种微阀、微泵和辅助设备，增加了芯片应用的复杂度，使得制造难度、成本大大升高，限制了其进一步在临床上的应用，而且难以实现全集成式的病原体核酸检测，因此急需一种全集成、高灵敏度的微流控芯片，在改进核酸检测过程的同时，降低检测时间和芯片制造难度与成本。Microfluidic chip technology (Microfluidics) 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.
发明公开Invention Disclosure
为了解决现有核酸检测技术中存在的不足，提供一种可以快速、高灵敏的集核酸捕获及原位扩增于一体的全集成病原体核酸检测微流控芯片。In order to solve the deficiencies in the existing nucleic acid detection technology, 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.
本发明提供一种全集成病原体核酸检测微流控芯片，包括流体管道层、弹性胶带层和试剂存储层，所述试剂存储层设有用于分别存储采集液、清 洗液、反应试剂和废液的腔室，所述流体管道层内设有带有捕获滤纸的原位提取扩增腔室，所述弹性胶带层夹设在流体管道层和试剂存储层之间，所述试剂存储层中的储采集液、清洗液和反应试剂在外力作用下穿过弹性胶带层流至流体管道层，并通过流体管道层的流道流至原位提取扩增腔室进行反应。所述弹性胶带层为双面带有粘性的材质能够将下方的流体管道层个上方的试剂存储层密封连接在一起。所述弹性胶带层可以为双面胶。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.
进一步，所述流体管道层和试剂存储层的腔室相对应，且在上下对应的两个腔室之间的弹性胶带层设有由上向下连通的单向阀。Further, 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.
进一步，所述试剂存储层上依次设置的采集液腔室、清洗液腔室、混匀腔室和废液池，所述试剂存储层上可拆卸的连接有反应试剂存储套管，所述采集液、清洗液、反应试剂分别密封存储在采集液腔室、清洗液腔室、反应试剂存储套管中。Further, 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.
进一步，所述反应试剂存储套管包括由下至上依次连接的下端旋冒、储液腔室和上端旋冒，所述上端旋冒上连接有推杆，所述胶塞球位于储液腔室的底壁与推杆之间，所述试剂存储层上设有与储液腔室下端相匹配的接口。Further, the 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.
进一步，所述流体管道层上与混匀腔室对应位置设有原位提取扩增腔室中设有捕获滤纸固定位，用于捕获核酸的捕获滤纸连接在捕获滤纸固定位处；所述混匀腔室对应位置设有原位提取扩增腔室之间的弹性胶带层设有去粘性层。Further, 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.
进一步，所述流体管道层上与采集液腔室对应的位置设有采集液池，所述采集液池与采集液腔室之间的弹性胶带层设有由上向下连通的单向阀；所述流体管道层上与清洗液腔室对应的位置设有清洗液池，所述清洗液池与清洗液腔室之间的弹性胶带层设有由上向下连通的单向阀；所述流体管道层上反应试剂存储套管相对应的位置，设有反应液池，所述反应液池与反应试剂存储套管之间的弹性胶带层设有由上向下连通的单向阀。Further, a collection liquid pool is provided on the fluid pipeline layer at a position corresponding to the collection liquid chamber, and the elastic tape layer between the collection liquid pool and the collection liquid chamber is provided with a one-way valve that communicates from top to bottom; A cleaning liquid pool is provided on the fluid pipeline layer at a position corresponding to the cleaning liquid chamber, and an elastic tape layer between the cleaning liquid pool and the cleaning liquid chamber is provided with a one-way valve communicating from top to bottom; the The position corresponding to the reaction reagent storage sleeve on the fluid pipeline layer is provided with a reaction liquid pool, and the elastic tape layer between the reaction liquid pool and the reaction reagent storage sleeve is provided with a one-way valve communicating from top to bottom.
进一步，所述由上向下连通的单向阀为带有通孔的去粘性层，所述通孔流体管道层上的流道或者各个反应池相连通，所述通孔与试剂存储层室的腔室的出液***错设置，当液体流向去粘性层时，去粘性层向下发生形变，是得其通过通孔形成通路；而当液体由下向上流动时，由于通孔与出 液***错设置，去粘性层向上挤压使得通孔封闭，此时单向阀关闭。Further, 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.
进一步，所述带有通孔的去粘性层即为在对应的腔室之间的位置的弹性胶带层去粘性，去粘性后的弹性胶带层上设有通孔。Further, 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.
进一步，所述反应池靠近原位提取扩增腔室的一侧设有镁离子复溶腔室，所述镁离子复溶腔室一侧与反应池相连通，另一端可以通过挤压对应设置的去粘性层与原位提取扩增腔室相连通；所述流体管道层上设有第一流道，所述第一流道的一端分别与清洗液池和采集液池相连接，另一端可以通过挤压对应设置的去粘性层与原位提取扩增腔室相连通；所述原位提取扩增腔室靠近废液池的一侧设有与废液池联通的第二流道，所述第二流道和废液池之间设有由下向上连通的单向阀。Further, a magnesium ion redissolving chamber is provided on the side of the reaction tank close to the in-situ extraction and amplification chamber, one side of the magnesium ion redissolving chamber is communicated with the reaction tank, and the other end can be correspondingly set by extrusion. 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.
进一步，所述由下向上连通的单向阀为带有通孔的去粘性层，所述通孔与废液池相连通，所述第二流道的出液口与通孔相对于去粘性层交错设置，当第二流道的液体流向废液池时，去粘性层向上发生形变是得第二流道与通孔相连通，液体流向废液池；而当液体由上向下流动时，由于通孔与第二流道的出液***错设置，去粘性层向下挤压使得通孔封闭，此时单向阀关闭。Further, 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. When the liquid in the second flow channel flows to the waste liquid pool, 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.
进一步，所述废液池上设有废液池排气孔，所述试剂存储层的外壁连接有气膜，所述气模与试剂存储层的外壁形成一密闭的容纳空间，所述容纳空间与废液池排气孔相连通。Further, 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, and 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.
进一步，所述试剂存储层设有把手。Further, the reagent storage layer is provided with a handle.
本发明还提供一种全集成病原体核酸检测***，包括上述的全集成病原体核酸检测微流控芯片。The present invention also provides a fully integrated pathogen nucleic acid detection system, including the above-mentioned fully integrated pathogen nucleic acid detection microfluidic chip.
本发明的全集成病原体核酸检测微流控芯片可将病原体样本处理、核酸提取、原位扩增集成于同一微流控芯片装置中，因而摆脱了实际进行病原体核酸检测时的繁琐操作以及对专业实验室的依赖。使用本发明提供的一种全集成病原体核酸检测微流控芯片，可真正实现全集成式、快速、高灵敏的病原体核酸检测。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.
图1为本发明的全集成病原体核酸检测微流控芯片的结构示意图；1 is a schematic structural diagram of a fully integrated pathogen nucleic acid detection microfluidic chip of the present invention;
图2为本发明的全集成病原体核酸检测微流控芯片的正视图；2 is a front view of a fully integrated pathogen nucleic acid detection microfluidic chip of the present invention;
图3为本发明的全集成病原体核酸检测微流控芯片的后视图；3 is a rear view of the fully integrated pathogen nucleic acid detection microfluidic chip of the present invention;
图4为本发明的全集成病原体核酸检测微流控芯片的试剂存储套管的结构示意图；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;
图5为具体实施例1中的检测结果图。FIG. 5 is a graph of the detection results in the specific embodiment 1. FIG.
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.
本发明提供一种全集成病原体核酸检测微流控芯片，如图1所示包括流体管道层1、弹性胶带层2和试剂存储层3，所述流体管道层包括流体管道，可对试剂进行微量操作；复溶腔室，可供冻干试剂进行复溶；核酸捕获滤纸固定位，可固定滤纸以进行核酸捕获；反应腔室，可供核酸扩增腔室以对滤纸捕获后的核酸进行原位扩增。所述弹性胶带层，选用双面胶基材，对流体管道层和试剂存储层进行粘接，以封装芯片。通过局部去粘性处理还可配合其弹性特点起弹性阀作用，以配合流体管道层以对试剂进行定向微量操作。所述试剂存储层包括，试剂存储腔室和废液池，以进行试剂存储和试剂处理后的废液；反应密封阀，密封反应腔室；混匀腔室，可对进行中的反应进行试剂混合，增强反应效果。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. For the elastic tape layer, a double-sided adhesive base material is used to bond the fluid pipeline layer and the reagent storage layer to encapsulate the chip. Through the local de-sticking treatment, it can also function as an elastic valve in combination with its elastic characteristics, so as to cooperate with the fluid pipeline layer to perform directional micro-manipulation of the reagent. The reagent storage layer includes a reagent storage chamber and a waste liquid pool for reagent storage and waste liquid after reagent treatment; a reaction sealing valve to seal the reaction chamber; a mixing chamber for reagents in the ongoing reaction. Blend for enhanced response.
如图2所述，所述试剂存储层包括依次设置的采集液腔室32、清洗液腔室33、反应试剂存储套管34、混匀腔室37和废液池36；所述反应试剂存储套管34可以为设置在试剂存储层3上的通孔也可以为与试剂存储层的底部可拆卸的连接独立装置；As shown in FIG. 2 , the reagent storage layer includes a collection liquid chamber 32 , a cleaning liquid chamber 33 , a reaction reagent storage sleeve 34 , a mixing chamber 37 and a waste liquid pool 36 arranged in sequence; the reaction reagent storage The sleeve 34 can be a through hole provided on the reagent storage layer 3 or an independent device detachably connected to the bottom of the reagent storage layer;
所述流体管道层1上与混匀腔室37对应位置设有原位提取扩增腔室17中设有捕获滤纸固定位18，用于捕获核酸的核酸捕获滤纸19连接在捕获滤纸固定位18处；所述混匀腔室37对应位置设有原位提取扩增腔室17之间的弹性胶带层2设有去粘性层；所述流体管道层1上与采集液腔室32对应的位置设有采集液池12，所述采集液池12与采集液腔室32之间的弹性胶带层2设有去粘性层，所述去粘性层上设有由上向下连通的单向阀；所述流体管道层1上与清洗液腔室33对应的位置设有清洗液池13，所述 清洗液池13与清洗液腔室33之间的弹性胶带层2设有去粘性层，所述去粘性层上设有由上向下连通的单向阀；所述流体管道层1上反应试剂存储套管34相对应的位置，设有反应液池14，所述反应液池14与反应试剂存储套管34之间的弹性胶带层2设有去粘性层，所述去粘性层上设有由上向下连通的单向阀；所述反应池14靠近原位提取扩增腔室4的一侧设有镁离子复溶腔室15，所述镁离子复溶腔室15一侧与反应池14相连通，另一端可以通过挤压对应设置的去粘性层与原位提取扩增腔室17相连通。第一流道11，所述第一流道11的一端分别与清洗液池和采集液池相连接，另一端可以通过挤压对应设置的去粘性层与原位提取扩增腔室17相连通。所述原位提取扩增腔室17靠近废液池的一侧设有与废液池联通的第二流道16，所述第二流道16和废液池36之间设有去粘性层，所述去粘性层上设有由下向上连通的单向阀。In-situ extraction and amplification chamber 17 is provided with a capture filter paper fixing position 18 in the position corresponding to the mixing chamber 37 on the fluid pipeline layer 1, and the nucleic acid capture filter paper 19 for capturing nucleic acids is connected to the capture filter paper fixing position 18 The corresponding position of the mixing chamber 37 is provided with an in-situ extraction and amplification chamber 17. The elastic tape layer 2 between the amplification chambers 17 is provided with a detackifying layer; the position corresponding to the collection liquid chamber 32 on the fluid pipeline layer 1 A collection liquid pool 12 is provided, and the elastic tape layer 2 between the collection liquid pool 12 and the collection liquid chamber 32 is provided with a detackifying layer, and the detackifying layer is provided with a one-way valve communicating from top to bottom; The fluid pipeline layer 1 is provided with a cleaning liquid pool 13 at a position corresponding to the cleaning liquid chamber 33 , and the elastic tape layer 2 between the cleaning liquid pool 13 and the cleaning liquid chamber 33 is provided with a detackifying layer. There is a one-way valve connected from top to bottom on the de-viscosity layer; the position corresponding to the reaction reagent storage sleeve 34 on the fluid pipeline layer 1 is provided with a reaction liquid pool 14, and the reaction liquid pool 14 is connected with the reaction reagent. The elastic tape layer 2 between the storage sleeves 34 is provided with a detackifying layer, and the detackifying layer is provided with a one-way valve communicating from top to bottom; the reaction pool 14 is close to the in-situ extraction and amplification chamber 4. 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.
进一步，所述由上向下连通的单向阀为带有通孔的去粘性层，所述通孔流体管道层上的流道或者各个反应池相连通，所述通孔与试剂存储层室的腔室的出液***错设置，当液体流向去粘性层时，去粘性层向下发生形变，是得其通过通孔形成通路；而当液体由下向上流动时，由于通孔与出液***错设置，去粘性层向上挤压使得通孔封闭，此时单向阀关闭。Further, 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.
所述由下向上连通的单向阀为带有通孔的去粘性层，所述通孔与废液池相连通，所述第二流道的出液口与通孔相对于去粘性层交错设置，当第二流道的液体流向废液池时，去粘性层向上发生形变是得第二流道与通孔相连通，液体流向废液池；而当液体由上向下流动时，由于通孔与第二流道的出液***错设置，去粘性层向下挤压使得通孔封闭，此时单向阀关闭。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. Setting, when the liquid in the second flow channel flows to the waste liquid pool, the de-viscosity layer is deformed 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, due to The through hole and the liquid outlet of the second flow channel are arranged alternately, and the detackifying layer is pressed downward to close the through hole, and at this time, the one-way valve is closed.
所述试剂存储层还包括密封阀室35，当反应试剂注入原位提取扩增腔室开始进行反应时，密封阀室35的内部腔室最低端会塞上一个直径略大于密封阀内部圆孔直径的胶塞，胶塞靠挤压作用挤压原位提取扩增腔室两端的双面胶带，以此封住反应腔室两端进行密封。The reagent storage layer also includes 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.
所述反应试剂存储套管34包括由下至上依次连接的下端旋冒341、储液腔室342、和上端旋冒343，所述上端旋冒343上连接有推杆344，所述储液腔室342内设有胶塞球345，所述胶塞球位于推杆344与储液腔室342底壁之间所述试剂存储层3上设有与储液腔室342下端相匹配的接口，使用时，可以去除下端旋冒341后将储液腔室342的下端直接与接口相连接。所述储液腔室342的下端直接与接口之间密封连接。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. In use, 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.
所述废液池36上设有废液池排气孔361，所述试剂存储层的外壁连接有气膜38，所述气模38与试剂存储层的外壁形成一密闭的容纳空间，所述容纳空间与废液池排气孔361相连通。The waste liquid pool 36 is provided with a waste liquid pool exhaust hole 361, the outer wall of the reagent storage layer is connected with a gas film 38, and the gas mold 38 and the outer wall of the reagent storage layer form a closed accommodating space. The accommodating space communicates with the exhaust hole 361 of the waste liquid pool.
所述试剂存储层设有把手31。The reagent storage layer is provided with a handle 31 .
具体实施例1Specific Example 1
以通过重组酶聚合酶扩增反应RPA检测空气生物气溶胶中新冠病毒为例，该芯片具体实施方式如下：Taking the detection of the new coronavirus in the air bioaerosol by the recombinase polymerase amplification reaction RPA as an example, the specific implementation of the chip is as follows:
存储试剂：试剂存储层与流体管道层通过弹性胶带层封装后即可进行试剂预存，以备后续实验；此时采集液腔室存储试剂可为2mL的病毒裂解液、清洗液腔室试剂可为1mLDEPC水(DNase、RNase free)(货号：R0022，生产商：碧云天)，反应试剂套管内的存储试剂可为DNA恒温快速扩增试剂盒(荧光型)(货号：WLE8202KIT，生产商：安普未来)，镁离子复溶腔室的表面有晶体状2.5μL280nM醋酸镁冻干后试剂成分。Reagent storage: After the reagent storage layer and the fluid pipeline layer are encapsulated by the elastic tape layer, the reagents can be pre-stored for subsequent experiments; at this time, the storage reagents in the collection liquid chamber can be 2 mL of virus lysate, and the cleaning liquid chamber reagents can be 1mL DEPC water (DNase, RNase free) (Item No.: R0022, Manufacturer: Biyuntian), the storage reagent in the reaction reagent sleeve can be DNA Constant Temperature Rapid Amplification Kit (Fluorescent Type) (Item No.: WLE8202KIT, Manufacturer: Anpu future), the surface of the magnesium ion reconstitution chamber has crystal-like 2.5 μL 280nM magnesium acetate lyophilized reagent components.
样品采集：通过生物气溶胶采样器(规格：便携式生物气溶胶采样器WA-400，生产商：北京鼎蓝科技有限公司)对公共空间环境中的病原体进行采集30分钟，此时检测芯片裂解液腔室通过管阀直接与该生物气溶胶采样器出样口相接通，即可将空气中的新冠病毒颗粒直接采集至采集液腔室，此实验中选用新型冠状病毒(2019-nCoV)假病毒核糖核酸标准物质代替(货号：NIM-RM5203，生产商：中国计量科学研究院)；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. In this experiment, the new coronavirus (2019-nCoV) fake Viral RNA reference material instead (Item No.: NIM-RM5203, manufacturer: China Institute of Metrology);
病原体裂解：将检测芯片装载至全集成病原体核酸分析仪上，一次可装载8位检测芯片，让病原体在采集液腔室进行裂解，释放核酸；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;
核酸捕获：开始进样，对采集液腔室施压，试剂流过采集液腔室下端的单向阀流向采集液池并通过第一流道流向原位提取扩增腔室17中的核酸捕获滤纸，流过核酸捕获滤纸时，核酸捕获滤纸对试剂中的核酸进行捕获、富集；Nucleic acid capture: start the sample injection, pressurize the collection liquid chamber, the reagent flows through the one-way valve at the lower end of the collection liquid chamber to the collection liquid pool and flows to the nucleic acid capture filter paper in the in-situ extraction and amplification chamber 17 through the first flow channel , when passing through the nucleic acid capture filter paper, the nucleic acid capture filter paper captures and enriches the nucleic acid in the reagent;
滤纸清洗：对清洗液腔室施压，使清洗液腔室中的清洗液试剂流过清洗液腔室下端单向阀，经过第一流到层流向核酸捕获滤纸，进行滤纸上的核酸清洗；Filter paper cleaning: Pressure is applied to the cleaning fluid chamber, so that the cleaning fluid reagent in the cleaning fluid chamber flows through the one-way valve at the lower end of the cleaning fluid chamber, and flows to the nucleic acid capture filter paper through the first flow to the laminar flow to clean the nucleic acid on the filter paper;
核酸原位扩增：对反应试剂存储套管加压，使反应试剂存储套管的反应体系试剂经过反应池和镁离子复溶腔室，将镁离子复溶腔室中的固态状醋酸镁进行复溶，随后流至原位提取扩增腔室，接着，密封原位提取扩增腔室两端的密封阀，加热膜开始加热，反应开始。反应时，混匀腔室在仪 器推杆的作用下进行双向混匀，增强反应效果；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. During the reaction, the mixing chamber performs bidirectional mixing under the action of the instrument push rod to enhance the reaction effect;
扩增结果检测：配合全集成核酸检测仪实时进行最高四色荧光扫描检测，并输出荧光扩增曲线，给出检测结果。Amplification result detection: with the fully integrated nucleic acid detector, the highest four-color fluorescence scanning detection is performed in real time, and the fluorescence amplification curve is output to give the detection result.
以20copies/mL的假病毒样品为例，按照上述方法进行检测，检测结果如附图5所示，图5中，左侧为20copies/mL的假病毒样品，右侧为空白对照。从图5中的结果可以看出，本发明的芯片可配合全集成核酸检测仪将待测样本的“样本前处理、核酸提取、扩增、检测”于一体，操作简便，可避免传统实验室所需各种繁琐操作；芯片全封闭式设计，试剂预混装，无需手工配液，安全可靠，与环境无交叉污染；芯片配合壳聚糖修饰的特异性滤纸进行核酸捕获富集，可进行原位扩增，检测灵敏度可达20copies/mL，尤其适用于空气气溶胶中、物品表面上的低copy病原体待检样本，这对新冠疫情而言，可大大助力疫情防控；搭配全集成核酸分析仪，可一次性集成多个芯片进行高通量检测；试剂存储腔室存储试剂种类、试剂存储腔室的腔室数量弹性可调节，可适应多种不同条件下，对多种不同病原体进行特异性检测。Taking the pseudovirus sample of 20copies/mL as an example, the detection is carried out according to the above method, and the detection result is shown in Figure 5. In Figure 5, the left side is the pseudovirus sample of 20copies/mL, and the right side is the blank control. It can be seen from the results in Fig. 5 that 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. For the new crown epidemic, it can greatly help epidemic prevention and control; with fully integrated nucleic acid 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.
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 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
本发明的芯片可配合全集成核酸检测仪将待测样本的“样本前处理、核酸提取、扩增、检测”于一体，操作简便，可避免传统实验室所需各种繁琐操作；芯片全封闭式设计，试剂预混装，无需手工配液，安全可靠，与环境无交叉污染；芯片配合壳聚糖修饰的特异性滤纸进行核酸捕获富集，可进行原位扩增，检测灵敏度可达20copies/mL，尤其适用于空气气溶胶中、物品表面上的低copy病原体待检样本；搭配全集成核酸分析仪，可一次性集成多个芯片进行高通量检测；试剂存储腔室存储试剂种类、试剂存储腔室的腔室数量弹性可调节，可适应多种不同条件下，对多种不同病原体进行特异性检测。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; with 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.
Claims (12)
- 一种核酸检测微流控芯片，其特征在于，包括流体管道层、弹性胶带层和试剂存储层，所述试剂存储层用于存储液体的腔室，所述流体管道层内设有带有捕获滤纸的原位提取扩增腔室，所述弹性胶带层夹设在流体管道层和试剂存储层之间，所述试剂存储层中的液体在外力作用下穿过弹性胶带层流至流体管道层，并通过流体管道层的流道流至原位提取扩增腔室进行反应。A nucleic acid detection microfluidic chip, characterized in that it includes a fluid pipeline layer, an elastic adhesive tape layer and a reagent storage layer, the reagent storage layer is used for a chamber for storing liquid, and the fluid pipeline layer is provided with a capture In-situ extraction and amplification chamber of filter paper, the elastic tape layer is sandwiched between the fluid pipeline layer and the reagent storage layer, and the liquid in the reagent storage layer flows through the elastic tape layer to the fluid pipeline layer under the action of external force , and flow to the in-situ extraction and amplification chamber through the flow channel of the fluid pipeline layer for reaction.
- 根据权利要求2所述的核酸检测微流控芯片，其特征在于，所述流体管道层和试剂存储层的腔室相对应，且在上下对应的两个腔室之间的弹性胶带层设有由上向下连通的单向阀。The nucleic acid detection microfluidic chip according to claim 2, wherein the fluid pipeline 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 One-way valve connected from top to bottom.
- 根据权利要求2所述的核酸检测微流控芯片，其特征在于，所述试剂存储层包括依次设置的采集液腔室、清洗液腔室、反应试剂存储套管、混匀腔室和废液池；所述采集液、清洗液、反应试剂分别密封存储在采集液腔室、清洗液腔室、反应试剂存储套管中。The nucleic acid detection microfluidic chip according to claim 2, wherein the reagent storage layer comprises a collection liquid chamber, a cleaning liquid chamber, a reaction reagent storage sleeve, a mixing chamber and a waste liquid arranged in sequence pool; the collection liquid, cleaning liquid, and reaction reagent are sealed and stored in the collection liquid chamber, the cleaning liquid chamber, and the reaction reagent storage sleeve, respectively.
- 根据权利要求3所述的核酸检测微流控芯片，其特征在于，所述反应试剂存储套管包括由下至上依次连接的下端旋冒、储液腔室和上端旋冒，所述上端旋冒上连接有推杆，所述胶塞球位于储液腔室的底壁与推杆之间，所述试剂存储层上设有与储液腔室下端相匹配的接口。The nucleic acid detection microfluidic chip according to claim 3, wherein the reaction reagent storage sleeve comprises a lower end spinner, a liquid storage chamber and an upper end spinner sequentially connected from bottom to top, and the upper end spinner A push rod is connected to the top, the rubber stopper ball is located between the bottom wall of the liquid storage chamber and the push rod, and the reagent storage layer is provided with an interface matching the lower end of the liquid storage chamber.
- 根据权利要求4所述的核酸检测微流控芯片，其特征在于，所述流体管道层上与混匀腔室对应位置设有原位提取扩增腔室中设有捕获滤纸固定位，用于捕获核酸的捕获滤纸连接在捕获滤纸固定位处；所述混匀腔室对应位置设有原位提取扩增腔室之间的弹性胶带层设有去粘性层。The nucleic acid detection microfluidic chip according to claim 4, wherein the fluid pipeline layer is provided with an in-situ extraction and amplification chamber at a position corresponding to the mixing chamber, and a capture filter paper fixing position is provided in the chamber for The capture filter paper for capturing nucleic acid is connected at the fixed position of the capture filter paper; the corresponding position of the mixing chamber is provided with a detackifying layer on the elastic tape layer between the in-situ extraction and amplification chambers.
- 根据权利要求5所述的核酸检测微流控芯片，其特征在于，所述流体管道层上与采集液腔室对应的位置设有采集液池，所述采集液池与采集液腔室之间的弹性胶带层设有由上向下连通的单向阀；所述流体管道层上与清洗液腔室对应的位置设有清洗液池，所述清洗液池与清洗液腔室之间的弹性胶带层设有由上向下连通的单向阀；所述流体管道层上反应试剂存储套管相对应的位置，设有反应液池，所述反应液池与反应试剂存储套管之间的弹性胶带层设有由上向下连通的单向阀。The nucleic acid detection microfluidic chip according to claim 5, wherein a collection liquid pool is provided on the fluid pipeline layer at a position corresponding to the collection liquid chamber, and the collection liquid pool is located between the collection liquid chamber and the collection liquid chamber. The elastic tape layer is provided with a one-way valve that communicates from top to bottom; the position corresponding to the cleaning liquid chamber on the fluid pipeline layer is provided with a cleaning liquid pool, and the elasticity between the cleaning liquid pool and the cleaning liquid chamber The tape layer is provided with a one-way valve that communicates from top to bottom; the position corresponding to the reaction reagent storage sleeve on the fluid pipeline layer is provided with a reaction liquid pool, and the space between the reaction liquid pool and the reaction reagent storage sleeve is provided. The elastic tape layer is provided with a one-way valve communicating from top to bottom.
- 根据权利要求6所述的核酸检测微流控芯片，其特征在于，所述由上向下连通的单向阀为带有通孔的去粘性层，所述通孔流体管道层上的流道或者各个反应池相连通，所述通孔与试剂存储层室的腔室的出液***错设置。The nucleic acid detection microfluidic chip according to claim 6, wherein the one-way valve connected from top to bottom is a detackifying layer with through holes, and the flow channels on the through-hole fluid pipeline layer are Or each reaction cell is in communication, and the through holes are arranged in a staggered manner with the liquid outlet of the chamber of the reagent storage layer chamber.
- 根据权利要求7所述的核酸检测微流控芯片，其特征在于，所述反应池靠近原位提取扩增腔室的一侧设有镁离子复溶腔室，所述镁离子复溶腔室一侧与反应池相连通，另一端可以通过挤压对应设置的去粘性层与原位提取扩增腔室相连通；所述流体管道层上设有第一流道，所述第一流道的一端分别与清洗液池和采集液池相连接，另一端可以通过挤压对应设置的去粘性层与原位提取扩增腔室相连通；所述原位提取扩增腔室靠近废液池的一侧设有与废液池联通的第二流道，所述第二流道和废液池之间设有设有去粘性层，所述去粘性层身上设有弹性阀，所述弹性阀为由下向上连通的单向阀。The nucleic acid detection microfluidic chip according to claim 7, wherein a magnesium ion reconstitution chamber is provided on the side of the reaction pool close to the in-situ extraction and amplification chamber, and the magnesium ion reconstitution chamber One side is communicated with the reaction tank, and the other end can be communicated with the in-situ extraction and amplification chamber by squeezing the correspondingly arranged de-viscosity layer; the fluid pipeline layer is provided with a first flow channel, and one end of the first flow channel They are respectively connected with the cleaning liquid pool and the collection liquid pool, and the other end can be communicated with the in-situ extraction and amplification chamber by squeezing the correspondingly arranged detackifying layer; the in-situ extraction and amplification chamber is close to one of the waste liquid pools. The side is provided with a second flow channel that communicates with the waste liquid pool, a de-viscosity layer is arranged between the second flow channel and the waste liquid pool, and an elastic valve is provided on the body of the de-viscosity layer. One-way valve connecting from bottom to top.
- 根据权利要求8所述的核酸检测微流控芯片，其特征在于，所述由下向上连通的单向阀为带有通孔的去粘性层，所述通孔与废液池相连通，所述第二流道的出液口与通孔相对于去粘性层交错设置，当第二流道的液体流向废液池时，去粘性层向上发生形变是得第二流道与通孔相连通，液体流向废液池；而当液体由上向下流动时，由于通孔与第二流道的出液***错设置，去粘性层向下挤压使得通孔封闭，此时单向阀关闭。The nucleic acid detection microfluidic chip according to claim 8, wherein the one-way valve that communicates from bottom to top is a detackifying layer with a through hole, and the through hole is communicated with the waste liquid pool, so The liquid outlet and the through hole of the second flow channel are staggered relative to the de-viscosity layer. When the liquid in the second flow channel flows to the waste liquid pool, the de-viscosity layer is deformed upward so that the second flow channel is connected with the through hole. , 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 staggered, the de-viscosity layer is squeezed downward to close the through hole, and the one-way valve is closed at this time. .
- 根据权利要求9所述的核酸检测微流控芯片，其特征在于，所述废液池上设有废液池排气孔，所述试剂存储层的外壁连接有气膜，所述气模与试剂存储层的外壁形成一密闭的容纳空间，所述容纳空间与废液池排气孔相连通。The nucleic acid detection microfluidic chip according to claim 9, wherein 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, and the gas mold is connected to the reagent The outer wall of the storage layer forms a closed accommodating space, and the accommodating space is communicated with the exhaust hole of the waste liquid pool.
- 根据权利要求9所述的核酸检测微流控芯片，其特征在于，所述试剂存储层设有把手。The nucleic acid detection microfluidic chip according to claim 9, wherein the reagent storage layer is provided with a handle.
- 一种全集成病原体核酸检测***，其特征在于，包括权利要求1-11任一所述的核酸检测微流控芯片。A fully integrated pathogen nucleic acid detection system, characterized in that it comprises the nucleic acid detection microfluidic chip according to any one of claims 1-11.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110188881.4 | 2021-02-19 | ||
| CN202110188881.4A CN112940922A (en) | 2021-02-19 | 2021-02-19 | Full-integrated pathogen nucleic acid detection micro-fluidic chip |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2022174471A1 true WO2022174471A1 (en) | 2022-08-25 |
Family
ID=76244290
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2021/078003 WO2022174471A1 (en) | 2021-02-19 | 2021-02-26 | Fully-integrated pathogen nucleic acid test microfluidic chip |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN112940922A (en) |
| WO (1) | WO2022174471A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115814864A (en) * | 2021-09-17 | 2023-03-21 | 上海微创惟微诊断技术有限公司 | In vitro diagnostic and analytical device and reagent cartridge |
| CN114164088B (en) * | 2021-12-10 | 2024-03-29 | 西安交通大学 | Chip structure, card box and method for detecting household nucleic acid |
| CN114100722A (en) * | 2021-12-27 | 2022-03-01 | 北京梓晶生物科技有限公司 | Portable full-automatic nucleic acid constant temperature amplification device |
| CN114570449B (en) * | 2022-04-26 | 2022-08-16 | 广州国家实验室 | Liquid transfer device and multi-path parallel liquid transfer device |
| WO2023231158A1 (en) * | 2022-05-30 | 2023-12-07 | 广州达安基因股份有限公司 | Handheld nucleic acid testing device having micro-fluidic chip, and use method therefor |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102253022A (en) * | 2011-05-04 | 2011-11-23 | 博奥生物有限公司 | Digital signal processing method for detecting microfluidic chip and applied detection device |
| CN106085842A (en) * | 2016-06-16 | 2016-11-09 | 清华大学 | A kind of high flux micro-fluidic chip nucleic acid amplification assays detecting system |
| CN107502544A (en) * | 2017-09-20 | 2017-12-22 | 北京丰特云基科技发展有限公司 | A kind of micro-fluidic chip detecting and controlling system |
| CN107541452A (en) * | 2017-09-20 | 2018-01-05 | 清华大学 | A kind of micro-fluidic chip and chip assembly |
| CN109852530A (en) * | 2019-03-29 | 2019-06-07 | 中国科学院上海微***与信息技术研究所 | A kind of micro-fluidic chip and its device and method integrating circulating tumor cell capture, cracking and detection of nucleic acids |
| CN110452802A (en) * | 2019-08-07 | 2019-11-15 | 深圳市刚竹医疗科技有限公司 | It is complete to extract molecular diagnosis micro-fluidic chip and microfluidic system |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108085314B (en) * | 2016-11-21 | 2021-11-09 | 杭州梓晶生物有限公司 | Aminated filter paper/membrane for nucleic acid extraction and purification and preparation method and application thereof |
| CN207552287U (en) * | 2017-09-20 | 2018-06-29 | 清华大学 | A kind of micro-fluidic chip and chip assembly |
| CN108642148B (en) * | 2018-07-09 | 2024-01-30 | 南京岚煜生物科技有限公司 | Nucleic acid amplification detection micro-fluidic chip and detection method thereof |
| CN110846214A (en) * | 2018-08-01 | 2020-02-28 | 上海真测生物科技有限公司 | Miniature multi-index nucleic acid analysis system and operation method thereof |
| CN110841730B (en) * | 2019-10-21 | 2021-04-27 | 清华大学 | Micro-fluidic chip and tumor DNA detection chip |
| CN111647498B (en) * | 2020-05-25 | 2022-03-22 | 杭州梓晶生物有限公司 | Integrated self-service nucleic acid detection device and use method thereof |
| CN111644213B (en) * | 2020-05-25 | 2021-08-17 | 清华大学 | Fluid control device and fluid control method |
| CN112940928A (en) * | 2021-02-19 | 2021-06-11 | 清华大学 | Fully-integrated pathogen nucleic acid analyzer |
-
2021
- 2021-02-19 CN CN202110188881.4A patent/CN112940922A/en active Pending
- 2021-02-26 WO PCT/CN2021/078003 patent/WO2022174471A1/en active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102253022A (en) * | 2011-05-04 | 2011-11-23 | 博奥生物有限公司 | Digital signal processing method for detecting microfluidic chip and applied detection device |
| CN106085842A (en) * | 2016-06-16 | 2016-11-09 | 清华大学 | A kind of high flux micro-fluidic chip nucleic acid amplification assays detecting system |
| CN107502544A (en) * | 2017-09-20 | 2017-12-22 | 北京丰特云基科技发展有限公司 | A kind of micro-fluidic chip detecting and controlling system |
| CN107541452A (en) * | 2017-09-20 | 2018-01-05 | 清华大学 | A kind of micro-fluidic chip and chip assembly |
| CN109852530A (en) * | 2019-03-29 | 2019-06-07 | 中国科学院上海微***与信息技术研究所 | A kind of micro-fluidic chip and its device and method integrating circulating tumor cell capture, cracking and detection of nucleic acids |
| CN110452802A (en) * | 2019-08-07 | 2019-11-15 | 深圳市刚竹医疗科技有限公司 | It is complete to extract molecular diagnosis micro-fluidic chip and microfluidic system |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112940922A (en) | 2021-06-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2022174471A1 (en) | Fully-integrated pathogen nucleic acid test microfluidic chip | |
| CN110331089B (en) | Full-automatic nucleic acid extraction amplification detection micro-fluidic chip box and application thereof | |
| JP6838127B2 (en) | Test cartridge with integrated transfer module | |
| JP5049274B2 (en) | Cartridge for automated medical diagnosis | |
| CN204727866U (en) | A kind ofly collect the micro fluidic device that nucleic acid amplification and microarray are detected on one | |
| CN101990516B (en) | Multiplex sample preparation system and the use in integrated analysis system thereof | |
| US10752936B2 (en) | Device for analyzing a biological sample | |
| US20110244466A1 (en) | Nucleic acid testing device and method | |
| CN107723210B (en) | Novel micro-fluidic chip device for nucleic acid detection | |
| CN108300640B (en) | Micro-fluidic chip for automatic extraction and detection of nucleic acid | |
| CN206375901U (en) | A kind of integrated closed microfluidic nucleic acid detection means | |
| CN207933420U (en) | A kind of micro-fluidic chip of genetic test | |
| CN111909835A (en) | Closed micro-fluidic nucleic acid detection card box | |
| WO2021254518A1 (en) | Sample treatment and testing apparatus | |
| CN107619775B (en) | Portable nucleic acid detection platform suitable for PCR chromatography | |
| CN113528625A (en) | Microfluidic nucleic acid detection method and application | |
| WO2022174472A1 (en) | Fully-integrated multi-index nucleic acid test microfluidic chip | |
| CN110452806B (en) | Full-automatic micro-fluidic nucleic acid detection chip based on loop-mediated isothermal amplification, kit and method thereof | |
| CN215288792U (en) | Nucleic acid diagnosis micro-fluidic chip | |
| CN115820412A (en) | Microfluidic card box and method for specifically detecting nucleic acid from complex sample | |
| US8808643B1 (en) | Fluidics platform and method for sample preparation and analysis | |
| CN115612593A (en) | Full-integrated nucleic acid detection micro-fluidic chip and nucleic acid detector with same | |
| CN113717827A (en) | Fully-integrated nucleic acid detection micro-fluidic chip and use method thereof | |
| Kwon et al. | Fully automated molecular diagnosis by a novel cartridge-based platform | |
| CN219239665U (en) | Virus detection micro-fluidic chip and virus detection kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21926188 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 21926188 Country of ref document: EP Kind code of ref document: A1 |