WO2022205529A1 - Dispositif d'encapsulation de puce microfluidique multicouche, et puce microfluidique multicouche et son application - Google Patents

Dispositif d'encapsulation de puce microfluidique multicouche, et puce microfluidique multicouche et son application Download PDF

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Publication number
WO2022205529A1
WO2022205529A1 PCT/CN2021/088336 CN2021088336W WO2022205529A1 WO 2022205529 A1 WO2022205529 A1 WO 2022205529A1 CN 2021088336 W CN2021088336 W CN 2021088336W WO 2022205529 A1 WO2022205529 A1 WO 2022205529A1
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substrate
fluid channel
normally closed
hole
microfluidic chip
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PCT/CN2021/088336
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English (en)
Chinese (zh)
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张秀莉
罗勇
邓权锋
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苏州大学
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    • 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
    • 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
    • B01L3/502707—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 the manufacture of the container or its components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G01N33/5304—Reaction vessels, e.g. agglutination plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6866—Interferon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/525—Tumor necrosis factor [TNF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5406—IL-4
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5412—IL-6
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5428—IL-10
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/545—IL-1
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/55—IL-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma

Definitions

  • the invention relates to the technical field of microfluidic chips, in particular to a multilayer microfluidic chip packaging device, a multilayer microfluidic chip and applications thereof.
  • Microfluidics is a technology that controls fluids in micron-scale channels.
  • the layers of a multi-layer microfluidic chip applying microfluidic technology need to be tightly fitted.
  • the purpose of the tight fit is to prevent the test solution from leaking.
  • the gaps between the layers bleed.
  • the bonding methods of multi-layer microfluidic chips mainly include the following: 1) Thermal bonding, this method is mainly used for materials such as glass, silicon, rigid plastics, etc., with many steps, complicated operations, and professional skills; 2) Plasma sealing, but this method is limited to a limited number of materials such as PDMS, and has low efficiency, poor repeatability, and is difficult to industrialize production; 3) By means of pressure bonding, the layers are closely attached to each other, which often uses elastic The combination of different materials and hard materials, such as PDMS elastic layer and PMMA board, and the use of screws and nuts to apply pressure to make some deformation of the elastic layer, and achieve the sealing effect by extrusion. The disadvantage of this method is that the pressure is difficult to achieve. Precise control.
  • the microfluidic chip package in the prior art lacks a simple, reliable and standardized substrate bonding design.
  • the technical problem to be solved by the present invention is to overcome the defect that the microfluidic chip package in the prior art lacks a simple, reliable and standardized substrate bonding design.
  • a first aspect of the present invention provides a multi-layer microfluidic chip package device, comprising a multi-layer substrate, and some of the substrates in the multi-layer substrate are provided with fluid channels;
  • the multi-layer substrate includes a first substrate and a second substrate, the first substrate is provided with a convex portion, the second substrate is provided with a concave portion matched with the convex portion, and the convex portion is connected to different positions of the concave portion , to adjust the distance between the first substrate and the second substrate;
  • the multilayer substrate includes a third substrate made of elastic material, the third substrate is arranged between the first substrate and the second substrate, and the thickness of the third substrate is greater than that of the first substrate and the second substrate distance between, so that the third substrate is elastically deformed under the pressing of the first substrate and the second substrate, so that the first substrate and the third substrate, as well as the second substrate and The third substrate is closely attached.
  • the concave portion includes a plurality of teeth, a gear is formed between two adjacent teeth, and the convex portion can be clamped on the gear.
  • the convex portion and/or the concave portion is a hard material capable of being slightly deformed, so that the convex portion can move and switch on the gear position of the concave portion.
  • a porous membrane is further included, and the porous membrane is disposed between the first substrate and the second substrate.
  • a second aspect of the present invention provides a multi-layer microfluidic chip, including the above-mentioned multi-layer microfluidic chip packaging device;
  • a normally closed valve is also included, the normally closed valve is connected to the fluid channel on the substrate, and the normally closed valve communicates with different fluid channels on the substrate in an open state.
  • the substrate is provided with a hole for holding the test solution, the hole is connected to a normally closed valve through a fluid channel, and the normally closed valve controls the transfer of the test solution between the holes through the fluid channel.
  • the hole includes a hole a, a hole b and a hole c, and the volume of the hole b and/or the hole c is greater than the volume of the hole a;
  • the fluid channel includes a fluid channel a, a fluid channel b and a fluid channel c,
  • the hole a, the hole b, and the hole c are connected to the normally closed valve through the fluid passage a, the fluid passage b, and the fluid passage c, respectively.
  • the fluid channel further includes a fluid channel d, the fluid channel d is connected to a normally closed valve, and the normally closed valve is respectively connected to the fluid channel a and the fluid channel b; the fluid channel further includes a fluid channel e, so The fluid passage e is connected to a normally closed valve, and the normally closed valve is respectively connected to the fluid passage a and the fluid passage c.
  • the holes a are arranged side by side on the substrate in a row-like arrangement, and one of the holes a in each row is divided into a plurality of chambers by a porous membrane in the vertical direction, and one of the chambers is provided with a biochemical reaction vector.
  • a third aspect of the present invention provides an application of the above-mentioned multilayer microfluidic chip in immunodetection.
  • the present invention effectively adjusts and controls the distance between the substrates through the movement switching of the convex portion in the concave portion, so as to realize the tight connection between the substrates.
  • the present invention does not require a pressure generating device, which significantly reduces The volume of the chip makes the chip more beautiful, and it is easy to standardize the production of multi-layer substrates by injection molding and laser engraving.
  • FIG. 1 is a schematic structural diagram of Embodiment 1 of the present invention.
  • FIG. 2 is a schematic diagram 1 of a substrate bonding principle in Embodiment 1 of the present invention.
  • FIG. 3 is a schematic diagram 2 of the substrate bonding principle in the first embodiment of the present invention.
  • FIG. 4 is a first structural schematic diagram of Embodiment 3 of the present invention.
  • FIG. 5 is a second structural schematic diagram of Embodiment 3 of the present invention.
  • FIG. 6 is a schematic diagram of the normally closed valve in the open state in the third embodiment of the present invention.
  • FIG. 7 is a schematic diagram of the normally closed valve in a closed state in the third embodiment of the present invention.
  • FIG. 8 is a schematic structural diagram of a multi-layer microfluidic chip in Embodiments 4-7 of the present invention.
  • Example 9 is a schematic diagram of the standard curve of AMH detection in Example 4 of the present invention.
  • Figure 10 is a schematic diagram of the standard curve for the detection of the inflammatory factor IL-6 in Example 6 of the present invention.
  • microfluidic chip fabrication technology and use method are conventional technologies and methods in the field of biochemical analysis.
  • the first embodiment includes:
  • a multi-layer microfluidic chip package device includes a multi-layer substrate, some of the multi-layer substrates are provided with fluid channels 170 , and the multi-layer substrate includes a first substrate 110 and a second substrate Two substrates 120 , a porous membrane 160 is provided between the first substrate 110 and the second substrate 120 , the first substrate 110 is provided with a convex portion 140 , and the second substrate 120 is provided with a concave portion 150 which is matched with the convex portion 140 .
  • the first substrate 110 and the second substrate 120 are rigid substrates
  • the convex portion 140 is similar to a male header structure
  • the concave portion 150 is similar to a female header structure
  • both the protruding direction of the male header and the recessed direction of the female header are perpendicular to the surface of the substrate , when the package is attached, the male header can be embedded in the female header, so that the distance between the first substrate 110 where the male header is located and the second substrate 120 where the female header is located can be fixed.
  • the first substrate 110 and the second substrate 120 may be rigid substrates.
  • the multilayer substrate includes a third substrate 130 , the third substrate 130 is an elastic substrate, the third substrate 130 is disposed between the first substrate 110 and the second substrate 120 , and the thickness of the third substrate 130 is greater than that of the first substrate 110 The distance from the second substrate 120 so that the third substrate 130 is elastically deformed under the pressing of the first substrate 110 and the second substrate 120, so that the first substrate 110 and the third substrate 130, and the second substrate 120 It is closely attached to the third substrate 130 to realize the sealing of the upper fluid channel 170 .
  • the third substrate 130 may be a composite substrate.
  • the composite substrate is formed by stacking multiple layers of sub-substrates. Some of the sub-substrates are made of elastic material and some are hard.
  • the substrate is made of elastic material, and the sub-substrates directly attached to each hard sub-substrate up and down are all of elastic material.
  • all the elastic sub-substrates in the composite substrate are elastically deformed under the pressing of the first substrate 110 and the second substrate 120, so that the elastic sub-substrates in the composite substrate are elastically deformed.
  • the upper and lower sub-substrates or the first substrate 110 or the second substrate 120 which are directly attached to themselves are closely attached.
  • the third substrate 130 may also be composed of a single elastic sub-substrate, which is not limited in the present invention.
  • the female head is designed as a tooth-like structure, which specifically includes a plurality of teeth 152 , and a gear 151 is located between two adjacent teeth 152 .
  • the male head is designed as a T-shaped structure, which specifically includes a protrusion adapted to the gear position 151 of the female head, and the protrusion can be clamped on the gear position 151 to realize the bonding of the first substrate 110 and the second substrate 120 position.
  • the material of the male head and/or the female head is a hard material that can be slightly deformed, such as a hard plastic that can be slightly deformed, such as polycarbonate (PC), polycarbonate and acrylonitrile-butadiene - blends of styrene terpolymers (PC/ABS), polyamide (PA), polyether ether ketone (PEEK), polyketone (POK) and polyoxymethylene (POM), etc.), such as male head convex From now on, it can be moved and switched on the gear position 151 of the female header, so as to adjust the distance between the first substrate 110 where the male header is located and the second substrate 120 where the female header is located.
  • a hard plastic that can be slightly deformed
  • PC/ABS polyamide
  • PA polyether ether ketone
  • POK polyketone
  • POM polyoxymethylene
  • the protrusion of the male head exerts a force on the teeth 152 when moving, so that the teeth 152 shrink and deform in a direction away from the protrusion, so that the protrusion slides from the previous gear 151 to the next gear 151, After the force on the teeth 152 disappears, the teeth 152 are reset, so that the protrusions of the male head are located on the gear position 151 .
  • the above embodiment effectively adjusts and controls the distance between the substrates through the cooperation of the female head and the male head, so as to realize the tight connection between the substrates.
  • the present embodiment does not need a pressure generating device, which significantly reduces the The volume of the chip is reduced, making the chip more beautiful, and it is easy to standardize the production of multi-layer substrates by injection molding and laser engraving.
  • the second embodiment of a multilayer microfluidic chip packaging device provided by the present invention will be introduced below.
  • the second embodiment is implemented based on the above-mentioned first embodiment, and is expanded on the basis of the first embodiment to a certain extent.
  • the tips of the teeth can be designed in a circular arc shape, so that when the protrusions slide, the circular arc teeth 152 can reduce the wear on the protrusions.
  • a further improvement is that the teeth 152 are also provided with a snap groove, and the two snap grooves are smoothly connected to the arc-shaped tip portion. Specifically, the protrusion of the male head exerts a force on the teeth 152 when moving, so that the protrusions are separated from the grooves on the teeth 152 and slide to the tip of the teeth 152, and the teeth 152 shrink in the direction away from the protrusions.
  • the above-mentioned second embodiment is an improvement made on the basis of the first embodiment in order to improve the stability of the male head clamping position.
  • the present invention can also have other improvements, and the present invention is not limited by this.
  • the third embodiment includes:
  • the multi-layer microfluidic chip of this embodiment includes the multi-layer microfluidic chip package device of the above-mentioned embodiment and a normally closed valve.
  • the normally closed valve is connected to the fluid channel 170 on the substrate and is normally closed. The valve communicates with the different fluid channels 170 on the substrate in an open state.
  • the multi-layer microfluidic chip package device is formed by stacking the 4-layer rigid substrate and the 3-layer elastic substrate in the first embodiment using the three sets of male and female header structures in the first embodiment, The specific content thereof has been described in detail in the foregoing embodiment, and is not repeated in this embodiment.
  • the four layers of rigid substrates are, from top to bottom, a first rigid substrate, a second rigid substrate, a third rigid substrate and a fourth rigid substrate, wherein the fourth rigid substrate is provided with a fluid channel , the fluid channel is connected to the normally closed valve.
  • the processing of the normally closed valve involves the close adhesion of the elastic film and the chip substrate.
  • the commonly used method is the dovetail clamping, which not only increases the volume of the chip, but also affects the appearance of the entire chip. Uncontrollable, affecting the repeatability and reliability of the entire analysis. Therefore, there are not many practical applications of normally closed valves, and their advantages in fluid control cannot be brought into play. Therefore, the present invention provides a simple and reliable normally closed valve that can be standardized and processed. Please refer to FIG. 6 and FIG. 7 for its specific structure.
  • the middle layer elastic film 220 is provided with the male headers described in the above embodiments
  • the lower layer rigid substrate 230 is provided with the female headers described in the above embodiments
  • the middle layer elastic film 220 is sucked in The pressure channel of the lower layer and the fluid channel 170 of the upper rigid substrate 210 become connected, and fluid flow can be performed, as shown in FIG. 7 for details.
  • the substrate of the chip is provided with a hole for holding the test solution, the hole is connected to the normally closed valve through the fluid channel 170 , and the normally closed valve controls the transfer of the test solution between the holes through the fluid channel 170 .
  • the shape of the hole on the chip substrate can be cylindrical, cylindrical, conical, a large column is connected to a small column, a large column is connected to a small column and other shapes; the holes can be light-transmitting or opaque; The volume is 1 to 1000 microliters.
  • the test solution contained in the well can be an enzyme-labeled secondary antibody, a fluorescently-labeled secondary antibody, a Raman-labeled secondary antibody, a washing solution, a substrate, a stop solution, an oxygen donor, a chemiluminescence test solution, and the like.
  • the holes include holes a311, holes b312 and holes c313, wherein the holes a311 are arranged side by side on the substrate in a row-like arrangement, and one of the holes a311 in each row is perforated in the vertical direction
  • the membrane 160 is divided into a plurality of chambers 330, wherein one chamber 330 is provided with a biochemical reaction carrier 340, and the biochemical reaction carrier 340 can be porous materials such as magnetic beads, microbeads, nanoparticles, polyurethane foam, paper and MOF.
  • the volume of the hole b312 and the hole c313 is greater than the volume of the hole a311.
  • the fluid channel 170 includes a fluid channel 170a, a fluid channel 170b, a fluid channel 170c, a fluid channel 170d and a fluid channel 170e, and the hole a311, the hole b312 and the hole c313 are respectively connected to the normally closed valve through the fluid channel 170a, the fluid channel 170b and the fluid channel 170c,
  • the fluid passage 170d is connected to normally closed valves respectively connecting the fluid passage 170a and the fluid passage 170b
  • the fluid passage 170e is connected to the normally closed valves respectively connecting the fluid passage 170a and the fluid passage 170c.
  • the normally closed valve controls whether the fluid channel 170 is connected or not, so as to control the transfer of the test solution between the holes.
  • This embodiment provides the application of a multi-layer microfluidic chip in detecting AMH.
  • the specific structure of the chip, the mark of the hole and the mark of the normally closed valve are shown in Figure 8.
  • the chip consists of 5 layers of hard substrates and 4 layers of elastic The substrates are superimposed, the material of the rigid substrate is PC/ABS, and the material of the elastic substrate is PDMS, which uses the four sets of male and female head locking structures in the above embodiment.
  • the pore size of the porous membrane 160 is 10 microns, the immune carrier is glass beads of 100 microns, and the surface of the glass beads is modified with a primary antibody for AMH.
  • Oxygen donor is placed, stop solution is placed in well 020; AMH enzyme-labeled secondary antibody is placed in wells A1-A8, patient serum samples are placed in wells B1-B3, and AMH of different concentrations is placed in wells B4-B8
  • the standard solution, the volume of each well is 140 microliters, and the transfer of the test solution between wells on the chip is controlled by the normally closed valve.
  • the method for detecting AMH using a multi-layer microfluidic chip includes the following steps:
  • the cleaning liquid in the hole 017 is evenly distributed to B1-B8. After cleaning for a period of time, by controlling the normally closed valves 010, 011 and 013, the cleaning liquid in the holes B1-B8 is all into separate wells 11, 21, 31 and 41, this cleaning step is repeated three times;
  • the enzyme-labeled secondary antibody solution in wells A1-A8 flows into wells B1-B8, and after incubation for a period of time, the enzyme-labeled secondary antibody binds to AMH on the glass bead wall;
  • the cleaning liquid in the hole 017 is evenly distributed to B1-B8. After cleaning for a period of time, by controlling the normally closed valves 010, 011 and 013, the cleaning liquid in the holes B1-B8 is all into separate wells 11, 21, 31 and 41, this cleaning step is repeated three times;
  • Step 7 Enzyme catalyzes the substrate
  • the substrate and oxygen supply in wells 018 and 019 are evenly distributed into wells B1-B8, and after incubation for a period of time, the enzyme on the surface of the glass beads catalyzes the substrate solution , so as to develop color;
  • Step 9 Detection
  • This embodiment realizes the automatic quantitative detection of AMH content in three patient samples, and the results are obtained within 2 hours. Because of the large sample volume and the use of microbeads for signal amplification, the accuracy of the results is higher than that of traditional 96-well plates, and lower cost.
  • the fifth embodiment of the application of a multilayer microfluidic chip provided by the present invention in immune detection will be introduced below.
  • the fifth embodiment is implemented based on the fourth embodiment above, and is expanded to a certain extent on the basis of the fourth embodiment. .
  • the design of the microfluidic chip in this embodiment is the same as that in the fourth embodiment, and the operation is also the same as that of the fourth embodiment.
  • the difference is that the holes B1-B8 contain 8 patient samples, so the microfluidic chip can realize 8 patient samples. Automated quantitative detection of AMH content.
  • This embodiment provides the application of a multi-layer microfluidic chip in the detection of inflammatory factors.
  • the specific structure of the chip, the mark of the hole and the mark of the normally closed valve are shown in Figure 8.
  • the chip consists of a 5-layer hard substrate and a 4-layer
  • the elastic substrates are superimposed, the material of the rigid substrate is PC, and the material of the elastic substrate is PDMS.
  • the pore size of the porous membrane 160 is 10 microns
  • the immune carrier is 200 micron silica particles
  • the surface of the silica particles is modified with a primary antibody corresponding to the inflammatory factor IL6, the cleaning solution is contained in well 017, the substrate is contained in well 018, and the The oxygen supply body is contained, and the stop solution is contained in the hole 020.
  • Wells A1-A8 contain IL6 enzyme-labeled secondary antibodies
  • wells B1-B3 contain clinical samples
  • wells B4-B8 contain different concentrations of IL6 standard.
  • the volume of each well is 140 microliters, and the transfer of the test solution between wells on the chip is controlled by a normally closed valve.
  • the method for detecting immune factors using a multi-layer microfluidic chip includes the following steps:
  • the cleaning liquid in the hole 017 is evenly distributed to B1-B8. After cleaning for a period of time, by controlling the normally closed valves 010, 011 and 013, the cleaning liquid in the holes B1-B8 is all into separate wells 11, 21, 31 and 41, this cleaning step is repeated three times;
  • the enzyme-labeled secondary antibody solution in wells A1-A8 flows into wells B1-B8, and after incubation for a period of time, the enzyme-labeled secondary antibody binds to IL6 on the surface of the silica particles;
  • the cleaning liquid in the hole 017 is evenly distributed to B1-B8. After cleaning for a period of time, by controlling the normally closed valves 010, 011 and 013, the cleaning liquid in the holes B1-B8 is all into separate wells 11, 21, 31 and 41, this cleaning step is repeated three times;
  • Step 7 Enzyme catalyzes the substrate
  • the substrate and oxygen supply in wells 018 and 019 are evenly distributed to wells B1-B8, after incubation for a period of time, the enzyme on the surface of the silica gel particles catalyzes the substrate solution, to develop color;
  • Step 9 Detection
  • This example realizes the automatic quantitative detection of IL6 content in three patient samples, and the results are obtained within 2 hours. Because of the large sample volume and the use of silica gel particles for signal amplification, the accuracy of the results is higher than that of traditional 96-well plates. lower cost.
  • the seventh embodiment of the application of a multi-layer microfluidic chip provided by the present invention in immune detection will be introduced below.
  • the seventh embodiment is implemented based on the above-mentioned third embodiment, and is expanded to a certain extent on the basis of the third embodiment. .
  • This embodiment provides the application of the multilayer microfluidic chip in the detection of inflammatory factor storm, and the specific structure of the chip, the marking of the hole and the marking of the normally closed valve are shown in FIG. 8 .
  • the chip is composed of 5 layers of hard substrates and 4 layers of elastic substrates.
  • the material of the rigid substrate is PC
  • the material of the elastic substrate is PDMS.
  • the pore size of the porous membrane 160 is 10 microns
  • the immune carrier is 200 micron silica particles
  • the surface of the silica particles is modified with 8 kinds of inflammatory factors (IFN- ⁇ , TNF- ⁇ , IL-2, IL-6, IL-1 ⁇ , IL-
  • well 017 contains cleaning solution
  • well 018 contains substrate
  • well 019 contains oxygen supply
  • well 020 contains stop solution.
  • Wells B1-B8 also contain the same clinical sample
  • wells A1-A8 contain enzyme-labeled secondary antibodies corresponding to 8 inflammatory factors.
  • the method for detecting immune factor storm using a multi-layer microfluidic chip includes the following steps:
  • the cleaning liquid in the hole 017 is evenly distributed to B1-B8. After cleaning for a period of time, by controlling the normally closed valves 010, 011 and 013, the cleaning liquid in the holes B1-B8 is all into separate wells 11, 21, 31 and 41, this cleaning step is repeated three times;
  • the enzyme-labeled secondary antibody solution in wells A1-A8 flows into wells B1-B8, and after incubation for a period of time, the enzyme-labeled secondary antibody will bind to the inflammatory factors on the surface of the silica particles;
  • the cleaning liquid in the hole 017 is evenly distributed to B1-B8. After cleaning for a period of time, by controlling the normally closed valves 010, 011 and 013, the cleaning liquid in the holes B1-B8 is all into separate wells 11, 21, 31 and 41, this cleaning step is repeated three times;
  • Step 7 Enzyme catalyzes the substrate
  • the substrate and oxygen supply in wells 018 and 019 are evenly distributed to wells B1-B8, after incubation for a period of time, the enzyme on the surface of the silica gel particles catalyzes the substrate solution, to develop color;
  • Step 9 Detection
  • the reaction solution in the holes B1-B8 flows into the holes C1-C8, and the detection is carried out by a color development method.
  • This embodiment can realize the bedside automatic detection of inflammatory factor storm (8 inflammatory factors) in critically ill patients, and the results can be obtained within 2 hours. Because of the large sample volume and the use of silica gel particles for signal amplification, the accuracy of the results is higher than that of traditional 96-well wells The detection accuracy of the board is lower, and the cost is lower.

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Abstract

La présente invention concerne un dispositif d'encapsulation de puce microfluidique multicouche. Le dispositif comprend un premier substrat et un second substrat, le premier substrat étant pourvu d'une saillie, et le second substrat étant pourvu d'un évidement qui est relié à la saillie d'une manière adaptée, la saillie étant reliée à différentes positions de l'évidement de façon à ajuster la distance entre le premier substrat et le second substrat ; et un substrat multicouche comprenant un troisième substrat fait d'un matériau élastique, le troisième substrat étant disposé entre le premier substrat et le deuxième substrat, et l'épaisseur du troisième substrat est supérieure à la distance entre le premier substrat et le deuxième substrat, de sorte que le troisième substrat génère une déformation élastique sous l'effet de la pression du premier substrat et du deuxième substrat. Dans la présente invention, la distance entre les substrats est efficacement réglée et commandée au moyen de la commutation de mouvement d'une saillie dans un évidement, de telle sorte qu'une connexion serrée entre les substrats est réalisée. Par rapport à l'état de la technique, la présente invention ne nécessite pas d'appareil de génération de pression, réduisant ainsi de manière significative la taille d'une puce, de telle sorte que la puce est plus belle ; et la production standardisée d'un substrat multicouche peut être facilement réalisée au moyen d'un procédé tel que le moulage par injection et la gravure au laser.
PCT/CN2021/088336 2021-03-31 2021-04-20 Dispositif d'encapsulation de puce microfluidique multicouche, et puce microfluidique multicouche et son application WO2022205529A1 (fr)

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WO2016084533A1 (fr) * 2014-11-28 2016-06-02 デクセリアルズ株式会社 Disque maître pour la fabrication de canal de micro-écoulement, produit de transfert, et procédé de fabrication de disque maître pour la fabrication d'un canal de micro-écoulement
CN107312713A (zh) * 2017-07-28 2017-11-03 中科芯瑞(苏州)生物科技有限公司 一种微流控芯片及其应用
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