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WO2025086145A1 - Puce microfluidique et procédé de préparation de puce microfluidique - Google Patents

Puce microfluidique et procédé de préparation de puce microfluidique Download PDF

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Publication number
WO2025086145A1
WO2025086145A1 PCT/CN2023/126471 CN2023126471W WO2025086145A1 WO 2025086145 A1 WO2025086145 A1 WO 2025086145A1 CN 2023126471 W CN2023126471 W CN 2023126471W WO 2025086145 A1 WO2025086145 A1 WO 2025086145A1
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WO
WIPO (PCT)
Prior art keywords
channel
sample
microfluidic chip
reaction
injection
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2023/126471
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English (en)
Chinese (zh)
Inventor
齐欣
赵新
刘娜
李瑞环
王成
王永
兰青阔
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Tianjin Academy of Agricultural Sciences
Original Assignee
Tianjin Academy of Agricultural Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin Academy of Agricultural Sciences filed Critical Tianjin Academy of Agricultural Sciences
Priority to PCT/CN2023/126471 priority Critical patent/WO2025086145A1/fr
Publication of WO2025086145A1 publication Critical patent/WO2025086145A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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

Definitions

  • the present invention relates to the field of microfluidic technology, and in particular to a microfluidic chip and a method for preparing the microfluidic chip.
  • Microfluidic chips are also called Miniaturized Total Analytical Systems or Micro Total Analysis Systems (MTAS). They can control the flow of reaction liquids in micron-scale channels and cavities, thereby achieving reaction closure.
  • Microfluidic chip technology involves knowledge in disciplines and fields such as chemistry, fluid mechanics, biology, medicine, microelectronics, and new materials.
  • molecular detection technology based on polymerase chain reaction (PCR) is widely used in medicine, agriculture, food and other fields.
  • PCR reaction can amplify trace amounts of target DNA in vitro, and has the characteristics of strong specificity, high sensitivity, and relatively simple operation.
  • the widely used PCR technologies mainly include real-time fluorescence quantitative PCR technology, digital PCR technology, isothermal amplification PCR technology, etc.
  • Microfluidic chip technology can concentrate multiple steps of sample detection on a single chip, integrate functional units through the combined design of flow channels, microvalves, cavities, etc., and ultimately achieve miniaturization and automation of chip devices.
  • the samples to be tested can be diverted to multiple independent high-throughput and high-efficiency reaction units at the same time, so that the same sample can be tested in parallel in multiple tasks as needed.
  • traditional microfluidic chips have defects such as low detection throughput and low integration, which makes it difficult to promote the use of microfluidic chips.
  • the object of the present invention is to provide a microfluidic chip, the detection throughput of the microfluidic chip is improved.
  • the present invention provides a microfluidic chip, including a reaction module, wherein the reaction module includes:
  • An injection channel one end of which is provided with an injection port
  • the sample splitting flow channel includes a plurality of sample splitting flow channels, all of which are sequentially connected to the sample inlet flow channel along the direction of fluid movement in the sample inlet flow channel; the length of the flow channel in the sample splitting flow channel connected to the sample inlet flow channel gradually shortens along the direction of fluid movement in the sample inlet flow channel;
  • a reaction pool wherein the sample outlet of each sample distribution channel is respectively provided with the reaction pool.
  • the sample inlet channel includes a first channel and a second channel connected to the first channel, the sample inlet is arranged on the first channel, the sample splitting channel is connected to the second channel, and the second channel is an arc-shaped channel.
  • the sample distribution channel is radially arranged along the length direction of the second channel.
  • the above-mentioned microfluidic chip further includes a substrate, the reaction modules include multiple ones, and all the reaction modules are arranged on the substrate.
  • each reaction module is provided with 32 sample distribution channels.
  • reaction modules are symmetrically distributed on opposite sides of the center line of the substrate.
  • the injection port is located at one end of the injection channel close to the center line of the substrate.
  • the height of the reaction liquid in the reaction pool is lower than the height of the bottom end of the sample distribution channel.
  • a method for preparing a microfluidic chip, used to prepare the above-mentioned microfluidic chip, comprises the steps of:
  • sampling channel, sample distribution channel and reaction pool of the microfluidic chip were preliminarily drawn using computer 3D drawing software to form an initial model diagram of the microfluidic chip;
  • the injection channel is designed to be arc-shaped
  • the injection volume and the length of the sampling channel of each reaction pool of a single reaction module are obtained by combining the fluid state, rated pressure and the same injection time conditions;
  • the fluid state is, in the three-dimensional simulation process, the flow state of the fluid in the microfluidic chip is calculated according to the Navier-Stokes equation (a) under the state of conservation of kinetic energy (b);
  • ⁇ represents the fluid density
  • u represents the fluid velocity
  • I represents the turbulence variable
  • ⁇ represents the fluid density
  • u represents the fluid velocity
  • the reaction module is processed by etching.
  • the microfluidic chip provided by the present invention includes a reaction module, the reaction module includes an injection channel, a sample splitting channel and a reaction pool, and an injection port is provided at one end of the injection channel; the sample splitting channel includes multiple sample splitting channels, and all the sample splitting channels are connected to the injection channel in sequence along the direction of fluid movement in the injection channel; the length of the flow channel in the sample splitting channel connected to the injection channel along the direction of fluid movement in the injection channel is gradually shortened; and the sample outlet of each sample splitting channel is respectively provided with a reaction pool.
  • the sample splitting flow channel includes a plurality of sample splitting flow channels, and along the direction of fluid movement in the sample inlet flow channel, the length of the flow channel in the sample splitting flow channel connected to the sample inlet flow channel is gradually shortened, so that the liquid in the sample inlet flow channel passes through the sample splitting flow channel almost synchronously.
  • the samples enter the reaction pool through the channel, realizing multiple reactions simultaneously, thereby increasing the detection throughput of the microfluidic chip.
  • FIG1 is a schematic diagram of the structure of a microfluidic chip provided in an embodiment of the present invention.
  • FIG. 2 is a schematic diagram showing a simulation of the injection volume of the microfluidic chip provided in an embodiment of the present invention for different sample splitting channel lengths 20 milliseconds after injection.
  • Figure 1 1-substrate, 2-inlet, 3-injection channel, 4-sample splitting channel, 5-reaction cell.
  • the core of the present invention is to provide a microfluidic chip, the detection flux of the microfluidic chip is improved.
  • the microfluidic chip provided in a specific embodiment of the present invention includes a reaction module, and the reaction module includes an injection channel 3, a sample splitting channel 4 and a reaction pool 5.
  • the injection channel 3 is provided with an injection port 2, and the injection channel 3 can be one or a combination of a straight channel, a curved channel or a broken line channel.
  • the injection channel 3 and the sample splitting channel 4 are based on achieving automatic flow of fluids, and the specific dimensions are determined according to actual needs, and this application does not make specific limitations.
  • the injection channel 3 and the sample splitting channel 4 are specifically capillary structures.
  • primers and corresponding probes used to detect sample target DNA may be contained in the reaction pool 5.
  • the type of primers depends on actual needs and is not specifically limited in this application.
  • one end of the sample inlet channel 3 is provided with a sample inlet 2;
  • the sample splitting channel 4 includes multiple sample splitting channels, and along the direction of fluid movement in the sample inlet channel 3, the length of the flow channel in the sample splitting channel 4 connected to the sample inlet channel 3 is gradually shortened, so that the liquid in the sample inlet channel 3 passes through the sample splitting channel 4 almost synchronously into the reaction pool 5, and multiple reactions are carried out synchronously. In this way, the detection throughput of the microfluidic chip is improved.
  • the sample splitting channel 4 is radially arranged along the length direction of the second channel.
  • the second channel is preferably a linear channel, so that the fluid can flow smoothly to the reaction pool 5.
  • the microfluidic chip also includes a substrate 1, and the reaction modules include multiple ones, and all the reaction modules are arranged on the substrate 1 to improve the integration of the reaction modules. Specifically, the number of reaction modules on the same substrate 1 can be 4-10. Each reaction module is provided with 10-40 sample distribution channels 4.
  • the substrate 1 is provided with 8 reaction modules, and each reaction module is provided with 32 sample distribution channels 4.
  • the chip is configured in this way to carry out the detection of 256 targets for one sample, or 32 targets for each of 8 samples.
  • DNA primers and corresponding probes for detecting targets are embedded in each reaction pool 5, and corresponding detection equipment and nucleic acid amplification reaction reagents are provided.
  • the microfluidic chip provided in this application provides a high-throughput detection method for multiple detection fields such as genetically modified component screening, crop strain identification, food inspection, and medical diagnosis.
  • the reaction modules are preferably symmetrically distributed on opposite sides of the center line of the substrate 1.
  • the reaction modules can be evenly distributed in the circumferential direction with the center of the substrate 1 as the center.
  • the injection port 2 is located at one end of the injection channel 3 close to the center line of the substrate 1. The distance traveled by the injection needle between each injection port 2 is reduced, and the injection speed is further improved.
  • the height of the reaction liquid in the reaction pool 5 is lower than the height of the bottom of the sample distribution channel 4. This arrangement prevents the overflow of the reaction liquid during the sample addition process from causing mutual contamination of the detection reaction. Specifically, the volume of the microfluidic chip reaction pool 5 should be larger than the total volume of the detection experiment reaction liquid in actual application.
  • the present application provides a method for preparing a microfluidic chip, which is used to prepare any of the above-mentioned microfluidic chips, comprising the steps of:
  • sampling channel 3, the sample distribution channel 4 and the reaction pool 5 of the microfluidic chip are preliminarily drawn using computer three-dimensional drawing software to form an initial model diagram of the microfluidic chip;
  • the injection channel 3 adopts an arc design
  • the injection volume of each reaction pool 5 and the length of the sample distribution channel 4 of a single reaction module are obtained under the conditions of fluid state, rated pressure and equal injection time;
  • the collective model of the microfluidic chip can be modified and improved according to the simulation calculation results for the preparation of the microfluidic chip.
  • the fluid state is calculated according to the Navier-Stokes equation (a) under the condition of conservation of kinetic energy (b) during the three-dimensional simulation process.
  • ⁇ represents the fluid density
  • u represents the fluid velocity
  • I represents the turbulence variable
  • ⁇ represents the fluid density
  • u represents the fluid velocity
  • the relationship between the injection time, injection pressure and injection volume of the microfluidic chip was simulated by three-dimensional simulation software to determine the injection time and injection pressure of the microfluidic chip.
  • the color on the right represents the volume fraction of the solution in the flow channel. The volume fraction gradually decreases from top to bottom. After the preset injection time, the injection volume at the reaction pool position is almost the same.
  • reaction module is processed by etching, and the etching of the microfluidic chip is completed by using a laser etching method according to the size of each component in the drawing of the microfluidic chip.
  • the proportion of each component in the single target detection system is determined by extracting the DNA of the sample to be detected, and the detection reaction premix is prepared according to the number of detection targets and the proportion of the components in the reaction system. Then, the volume proportion of each component in the single detection target detection system is determined.
  • the injection pressure and injection time are selected, and the sample is dispensed into each reaction pool 5 by using the capillary pressure injection method.
  • the premixed liquid and the pre-embedded target primers and probes mix the reaction system under the action of the injection pressure.
  • the microfluidic chip after sample injection is placed in a device equipped with a temperature control and fluorescence signal detection unit to perform nucleic acid amplification reaction and real-time detection of results. After amplification, the fluorescence signal analysis is used to determine whether the detection target is detected.
  • the present invention is used for the detection and identification of new varieties of biological breeding industrialization and related products, and has the following advantages:
  • the product has high detection throughput, wide application range, high integration and portability.
  • the sample injection channel 3, the sample distribution channel 4 and the reaction pool on the substrate 1 are sealed by a sealing film.
  • the processed microfluidic chip has strong sealing performance, which can reduce the impact of manual operation and environment on the detection results, and the detection is more accurate.
  • the microfluidic chip provided in this application can be used for polymerase chain reaction technology, mainly referring to real-time fluorescence PCR technology, the purpose of which is to use in vitro amplification to obtain sufficient results in a short period of time in vitro.
  • the DNA primer for detecting the sample target is a DNA sequence designed and synthesized based on the specific sequence of the target DNA to be detected and combined with the characteristics of the polymerase chain reaction.
  • the corresponding probe is designed and synthesized based on the fragment amplified by the primer DNA sequence and is fluorescently labeled to facilitate the characterization of subsequent test results.
  • the present invention coats the target primer DNA and the fluorescently labeled DNA probe in the reaction pool 5 of the microfluidic chip by freeze coating and freezes them.
  • the source of the DNA primer sequence of the detection target is mainly the related sequences in the qualitative PCR method of herbicide-resistant, insect-resistant and herbicide-resistant corn transformants, herbicide-resistant, insect-resistant and quality-improved soybean transformants, insect-resistant, insect-resistant and herbicide-resistant rice transformants, insect-resistant and herbicide-resistant cotton transformants and their derivative varieties involved in the national standards of the People's Republic of China, as well as the common transgenic and product component detection genes bar, pat, CaMV35S promoter, FMV 35S promoter, NOS promoter, NOS terminator and CaMV35S, etc., totaling 256 detection targets.
  • Genomic DNA from crops and related products is extracted by cell lysis, DNA separation and precipitation. It is diluted as a detection template, and a premix is prepared according to the proportion of each component in the reaction solution and the number of test samples.
  • the reaction solution is filled into the reaction pool 5 by capillary pressure, and the reaction system is mixed under the action of pressure injection.
  • the microfluidic chip is etched, mainly including an injection port 2, an injection channel 3, a sample splitting channel 4, and a reaction pool 5, with a total of 8 reaction modules, which can meet the detection of 8 samples, 32 targets per sample, or 256 targets per sample.
  • the identification primers and probes are designed, and the typing and identification of species SNP sites are carried out based on the fluorescent probe method, and the amplification primers and probes are embedded in the reaction pool 5.
  • the detection of SNP sites in the genome of crops such as corn, wheat, soybean, rice, and cotton is carried out.
  • Application method Use a DNA extraction kit to extract the genomic DNA of the sample to be tested, and dilute the DNA to an appropriate concentration as a test template. Prepare a premix according to the proportion of each component in the reaction solution and the number of test samples, use capillary pressure to fill the reaction solution into the reaction pool 5, and mix the reaction system under the action of pressure injection.
  • the microfluidic chip with sample loading is placed in a heating and fluorescence signal detection device, and the reaction temperature, reaction time and fluorescence signal acquisition time are set. After the amplification is completed, the fluorescence signal analysis is used to determine whether relevant genetically modified components are detected.
  • the microfluidic chip is etched, mainly including an injection port 2, an injection channel 3, a sample splitting channel 4, and a reaction pool 5, with a total of 8 reaction modules, which can meet the detection of 8 samples, 32 targets per sample, or 256 targets per sample.
  • Primers and probes are designed according to the nucleic acid sequence characteristics of pathogenic bacteria and embedded in the reaction pool 5.
  • the genomic DNA of the sample to be tested is extracted by cell lysis, DNA separation and precipitation, and diluted as a test template.
  • the premix is prepared according to the proportion of each component in the reaction solution and the number of test samples.
  • the reaction solution is filled into the reaction pool 5 using capillary pressure, and the reaction system is mixed under the action of pressure injection.
  • the microfluidic chip with sample loading is placed in a heating and fluorescence signal detection device, and the reaction temperature, reaction time and fluorescence signal acquisition time are set. After the amplification is completed, the fluorescence signal analysis is used to determine whether relevant genetically modified components are detected.

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  • Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Est divulguée une puce microfluidique et un procédé de préparation d'une puce microfluidique. La puce microfluidique comprend des modules de réaction ; chaque module de réaction comprend un canal d'écoulement de chargement d'échantillon, des canaux d'écoulement de séparation d'échantillon, et un bassin de réaction ; une entrée d'échantillon est formée à une extrémité de chacun des canaux d'écoulement de chargement d'échantillon ; une pluralité de canaux d'écoulement de séparation d'échantillon sont présents, et tous les canaux d'écoulement de séparation d'échantillon sont raccordés de manière séquentielle aux canaux d'écoulement de chargement d'échantillon dans la direction du mouvement de fluide à l'intérieur des canaux d'écoulement de chargement d'échantillon ; dans la direction du mouvement de fluide à l'intérieur des canaux d'écoulement de chargement d'échantillon, les longueurs de canaux internes des canaux d'écoulement de séparation d'échantillon raccordés aux canaux d'écoulement de chargement d'échantillon raccourcissent progressivement ; et une sortie d'échantillon de chaque canal d'écoulement de séparation d'échantillon est pourvue d'un bassin de réaction. Dans la puce microfluidique selon l'invention, une extrémité de chacun des canaux d'écoulement de chargement d'échantillon est pourvue d'une entrée d'échantillon, une pluralité de canaux de séparation d'échantillon sont présents, et dans la direction du mouvement de fluide à l'intérieur des canaux d'écoulement de chargement d'échantillon, les longueurs de canaux internes des canaux d'écoulement de séparation d'échantillon raccordés aux canaux d'écoulement de chargement d'échantillon raccourcissent progressivement, de sorte qu'un liquide dans les canaux d'écoulement de chargement d'échantillon pénètre presque de manière synchrone dans les bassins de réaction par l'intermédiaire des canaux d'écoulement de séparation d'échantillon, ce qui permet de réaliser de multiples réactions réalisées de manière synchrone. Ceci améliore en outre le débit de détection de la puce microfluidique.
PCT/CN2023/126471 2023-10-25 2023-10-25 Puce microfluidique et procédé de préparation de puce microfluidique Pending WO2025086145A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080073506A1 (en) * 2006-08-24 2008-03-27 Lazar Iuliana M Microfluidic devices and methods facilitating high-throughput, on-chip detection and separation techniques
WO2013160408A2 (fr) * 2012-04-25 2013-10-31 Scope Fluidics Sp. Z O.O. Dispositif microfluidique
US20140044610A1 (en) * 2012-08-13 2014-02-13 Canon Kabushiki Kaisha Microfluidic device
CN105289763A (zh) * 2015-09-24 2016-02-03 基蛋生物科技股份有限公司 一种定量分流的多指标检测微流控芯片
CN107855142A (zh) * 2017-11-01 2018-03-30 深圳市第二人民医院 一种基于微流控技术的检测芯片及检测设备
CN209912962U (zh) * 2019-03-26 2020-01-07 天能电池集团股份有限公司 一种燃料电池用的双极板

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080073506A1 (en) * 2006-08-24 2008-03-27 Lazar Iuliana M Microfluidic devices and methods facilitating high-throughput, on-chip detection and separation techniques
WO2013160408A2 (fr) * 2012-04-25 2013-10-31 Scope Fluidics Sp. Z O.O. Dispositif microfluidique
US20140044610A1 (en) * 2012-08-13 2014-02-13 Canon Kabushiki Kaisha Microfluidic device
CN105289763A (zh) * 2015-09-24 2016-02-03 基蛋生物科技股份有限公司 一种定量分流的多指标检测微流控芯片
CN107855142A (zh) * 2017-11-01 2018-03-30 深圳市第二人民医院 一种基于微流控技术的检测芯片及检测设备
CN209912962U (zh) * 2019-03-26 2020-01-07 天能电池集团股份有限公司 一种燃料电池用的双极板

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