WO2024067695A1 - 微流控芯片及检测方法 - Google Patents

微流控芯片及检测方法 Download PDF

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Publication number
WO2024067695A1
WO2024067695A1 PCT/CN2023/122009 CN2023122009W WO2024067695A1 WO 2024067695 A1 WO2024067695 A1 WO 2024067695A1 CN 2023122009 W CN2023122009 W CN 2023122009W WO 2024067695 A1 WO2024067695 A1 WO 2024067695A1
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WIPO (PCT)
Prior art keywords
chamber
liquid
microfluidic chip
plasma
immune reaction
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PCT/CN2023/122009
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English (en)
French (fr)
Inventor
王晶晶
黄斌
何凡
肖育劲
钱纯亘
刘笔锋
Original Assignee
深圳市卓润生物科技有限公司
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Publication of WO2024067695A1 publication Critical patent/WO2024067695A1/zh

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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
    • B01L3/50273—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 means or forces applied to move the fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces

Definitions

  • the present invention relates to the technical field of immune detection, and in particular to a microfluidic chip and a detection method.
  • Chemiluminescent immunoassay is a detection and analysis technology that combines highly sensitive chemiluminescent detection technology with highly specific antigen-antibody immune reaction to detect antigens or antibodies in the test object. It is a new immunoassay technology after radioimmunoassay, enzyme-linked immunosorbent assay, fluorescence immunoassay and time-resolved fluorescence immunoassay.
  • Chemiluminescent immunoassay consists of two main components: the immunoreaction system and the chemiluminescent analysis system.
  • the immunoreaction system is based on the basic principle of antigen-antibody reaction, directly marking the luminescent substance on the antigen or antibody or using the enzyme as the luminescent substrate;
  • the chemiluminescent analysis system uses the chemiluminescent substance to form an excited state through the catalysis of the catalyst and the oxidation of the oxidant. When this unstable excited state molecule returns to the stable ground state, it releases energy and emits photons.
  • the luminescent intensity of the luminescent reaction is measured using a photon signal detector, thereby calculating the content of the substance being measured.
  • the current chemiluminescence immunoassay for clinical whole blood samples is complicated and time-consuming, requiring the addition of capture antibodies, antigens, cleaning fluids, and substrates in batches, and the whole blood samples must be processed in advance to separate the serum before testing.
  • the existing tube-type chemiluminescence reagents use a large amount of reagents. For chemiluminescence with a high reagent cost, reducing the amount of reagents used can significantly reduce the user's testing costs, especially for grassroots medical institutions, where there is a great demand for miniaturized and simplified testing equipment.
  • an embodiment of the present application provides a microfluidic chip.
  • a microfluidic chip comprises a chip body and a liquid capsule component; the chip body has a rotation center, and the chip body is provided with:
  • a whole blood separation structure comprising a plasma chamber, a blood cell chamber and a blood waste liquid chamber, wherein the blood cell chamber and the blood waste liquid chamber are respectively connected to the plasma chamber, the blood waste liquid chamber is close to the inlet of the plasma chamber, and the blood cell chamber is close to the outlet of the plasma chamber;
  • a mixing chamber connected to the plasma chamber
  • a liquid separation channel the liquid separation channel comprises a main channel and a plurality of cup-separating chambers, the main channel has an inlet end and an outlet end, the inlet end is connected to the mixing chamber; the main channel is in an outer spiral shape with the rotation center as the center, and the plurality of cup-separating chambers are directly connected to the main channel;
  • each of the reaction units comprises an immune reaction chamber and a reaction waste liquid chamber connected to the immune reaction chamber, and the plurality of immune reaction chambers are connected to the plurality of cup chambers in a one-to-one correspondence;
  • a substrate reagent chamber, the substrate reagent chamber is communicated with the mixing chamber;
  • the liquid capsule component has a first cavity, a second cavity and a third cavity.
  • the first cavity and the second cavity can be communicated with the mixing cavity respectively, and the third cavity can be communicated with the mixing cavity through the substrate reagent cavity.
  • the above-mentioned microfluidic chip is equipped with a whole blood separation structure, a mixing chamber, a liquid separation channel, multiple reaction units and a substrate reagent chamber on the chip body, and is matched with a liquid capsule component, so that the above-mentioned microfluidic chip can test multiple or multiple immune items with a single sample addition on a limited disk surface, and does not require separate pre-treatment of whole blood samples. It is compatible with whole blood and non-whole blood, and the operation is simple and time-saving.
  • the main flow channel is in the shape of an external spiral line, so that the liquid in the mixing chamber can be distributed to each sub-cup chamber at a lower rotation speed and each sub-cup chamber can be filled, thereby reducing the liquid in the sub-cup chamber to the immune reaction.
  • the requirement for the centrifugal speed in the process of transferring the liquid from the immune reaction chamber to the waste liquid chamber is reduced.
  • FIG1 is a microfluidic chip according to an embodiment of the present invention.
  • FIG2 is an exploded view of the microfluidic chip shown in FIG1 ;
  • FIG3 is a three-dimensional view of a chip body of the microfluidic chip shown in FIG1 ;
  • FIG. 4 is a top view of a chip body of the microfluidic chip shown in FIG. 1 .
  • Separation chamber 114d sacrificial chamber; 115, reaction unit; 115a, immune reaction chamber; 115b, reaction waste chamber; 115c, first microfluidic channel; 115d, second microfluidic channel; 116, substrate reagent chamber; 117, sedimentation tank; 118, first siphon channel; 119, second siphon channel; 120, liquid capsule assembly; 121, first type of liquid capsule; 122, second type of liquid capsule; 123, third type of liquid capsule; 130, cover plate; 140, adhesive layer.
  • the capacity of the chamber refers to the maximum amount of liquid that the chamber can hold;
  • the depth of the components on the chip body refers to the distance from the bottom of the corresponding component to the cover surface, for example, the depth of the collection part refers to the distance from the bottom of the collection part to the cover surface.
  • the bottom surface of each component is a flat surface, that is, the distance from each position on the bottom surface of the component to the cover surface is equal.
  • Microfluidic chips integrate the basic operating units involved in the fields of biology and chemistry, such as reaction, separation, cultivation, sorting and detection, into a very small chip to realize various functions of conventional biological or chemical laboratories.
  • the technology has the characteristics of small injection volume, high integration, easy automation control and high-throughput analysis, making biochemical reaction operations on microfluidic chips more convenient and faster than conventional analytical sample pretreatment.
  • an embodiment of the present application provides a microfluidic chip 10, which includes a chip body 110 and a liquid capsule assembly 120;
  • the chip body 110 has a rotation center, and is provided with a whole blood sampling chamber 111, a non-whole blood sampling chamber 111a, a whole blood separation structure 112, a mixing chamber 113, a liquid separation channel 114, a plurality of reaction units 115 and a substrate reagent chamber 116;
  • the whole blood separation structure 112 has a plasma chamber 112a, a blood cell chamber 112b and a blood waste liquid chamber 112c, the plasma chamber 112a is connected to the whole blood sampling chamber 111, the plasma chamber 112a is connected to the blood cell chamber 112b, the blood waste liquid chamber 112c is connected to the plasma chamber 112a, the blood waste liquid chamber 112c is close to the inlet of the plasma chamber 112a, and the blood cell chamber 112b is close to the outlet of the plasma chamber 112a;
  • the mixing chamber 113 is a plasma
  • the microfluidic chip 10 is provided with a whole blood injection chamber 111, a non-whole blood injection chamber 111a, a whole blood separation structure 112, a mixing chamber 113, a liquid separation channel 114, a plurality of reaction units 115 and a substrate reagent chamber 116 on the chip body 110, and is matched with a liquid capsule component 120, so that the microfluidic chip 10 can test multiple or multiple immune items by adding a sample once, and does not need to pre-treat the whole blood sample separately, and is simple to operate and time-saving.
  • the main flow channel 114a is in the shape of an outer spiral line with the rotation center as the center, and the distance from the main flow channel 114a to the rotation center gradually increases from the inlet end to the outlet end, which makes it possible to distribute the liquid in the mixing chamber 113 to each sub-cup chamber 114b at a lower rotation speed. Therefore, the microfluidic chip 10 can realize a single sample addition test of multiple or multiple immune items on a limited disk surface, and does not need to pre-treat the whole blood sample separately, and is simple to operate, time-saving, and has good accuracy.
  • the substrate reagent chamber 116 may have any shape, and may be composed of any shape and any curved microchannel.
  • the liquid separation channel 114 also includes a sacrificial chamber 114d, which is connected to the main channel 114a and separated from the cup-separating chamber 114b.
  • the sacrificial chamber 114d is closer to the inlet end than the cup-separating chamber 114b.
  • the setting of the sacrificial chamber 114d makes it less likely that the actual liquid filled in the first cup-separating chamber 114b from the inlet end is less than the preset volume, thereby affecting the next reaction.
  • the sacrificial chamber 114d can also effectively reduce the liquid impact force on the first cup-separating chamber 114b, thereby improving the accuracy of the first immune reaction chamber 115a.
  • the main channel 114a is in an outer spiral shape with the rotation center as the center, and the plurality of sub-cup cavities 114b are arranged in parallel at equal angles on the outer periphery of the main channel 114a with the rotation center as the center and are directly connected to the main channel 114a.
  • the diameter of the microfluidic chip 10 is 100 mm to 140 mm.
  • the diameter of the microfluidic chip 10 is 100 mm, 120 mm, 130 mm or 140 mm.
  • the material of the chip body 110 includes but is not limited to glass, PDMS, PMMA, PET or PC.
  • the material of the chip body 110 is an opaque material. Opaque materials as the material of the chip body 110 are more conducive to subsequent detection.
  • the material of the chip body 110 is a colored opaque material.
  • the diameter of the chip body 110 is 100mm to 140mm.
  • the diameter of the chip body 110 is 100mm, 120mm, 130mm or 140mm. Please refer to Figures 3 and 4. Specifically, the whole blood injection chamber 111 and the non-whole blood injection chamber 111a are used to hold the added sample.
  • the capacity of the whole blood injection chamber 111 is above 400 ⁇ L, and the capacity of the non-whole blood injection chamber 111a is above 1 ⁇ L. According to the above setting, the dosage of at least 20 reaction units 115 can be met. Further, the capacity of the whole blood injection chamber 111 is greater than 450 ⁇ L; the capacity of the non-whole blood injection chamber 111a is greater than 50 ⁇ L. Further, the capacity of the whole blood injection chamber 111 is 400 ⁇ L to 600 ⁇ L; the capacity of the non-whole blood injection chamber 111a is 1 ⁇ L to 200 ⁇ L.
  • the whole blood injection chamber 111 is fan-shaped, and the whole blood injection chamber 111 protrudes from the rotation center toward the edge of the chip body 110;
  • the non-whole blood injection chamber 111a is fan-shaped, and the non-whole blood injection chamber 111a protrudes from the rotation center toward the edge of the chip body 110.
  • the widths of the whole blood injection chamber 111 and the non-whole blood injection chamber 111a gradually increase in the clockwise direction. According to such a configuration, it is convenient for the sample to enter the downstream after centrifugation, reduce the centrifugation time, and improve the detection efficiency.
  • the whole blood separation structure 112 is used to process the blood sample to separate serum or plasma from the whole blood for downstream detection.
  • the whole blood separation structure 112 includes a plasma chamber 112a, a blood cell chamber 112b and a blood waste liquid chamber 112c.
  • the whole blood is separated into serum or plasma and blood cells, and the serum or plasma stays in the plasma chamber 112a while the blood cells stay in the blood cell chamber 112b, thereby achieving separation.
  • the blood in the plasma chamber 112a exceeds its capacity, it enters the blood waste liquid chamber 112c, thereby achieving quantification of the blood.
  • the chip body 110 has a cover surface 110a; the plasma chamber 112a has a flow-aiding surface, and the distance from the flow-aiding surface to the cover surface 110a gradually decreases along the direction from the entrance of the plasma chamber 112a to the outlet of the plasma chamber 112a.
  • the plasma chamber 112a is substantially funnel-shaped, and the distance between the side walls of the plasma chamber 112a gradually narrows in the reverse direction from the entrance of the plasma chamber 112a to the exit of the plasma chamber 112a. It is understood that the shape of the plasma chamber 112a is not limited to the above, and the shape of the blood cell chamber 112b is also not limited.
  • the volume ratio of the plasma chamber 112a to the blood cell chamber 112b is 1:(1-5).
  • the volume ratio of the plasma chamber 112a to the blood cell chamber 112b is set to 1:(1-5) to fully separate the plasma from the blood cells, so that there is no other interference such as hemoglobin in the plasma.
  • the volume ratio of the plasma chamber 112a to the blood cell chamber 112b is 1:(2-5). Setting the volume ratio of the plasma chamber 112a to the blood cell chamber 112b to 1:(2-5) can make the whole blood separation structure compatible with normal whole blood samples of almost all age groups and genders.
  • the volume ratio of the plasma chamber 112a to the blood cell chamber 112b is 1:2, 1:3, 1:4 or 1:5.
  • the blood waste liquid chamber 112c is used to hold whole blood that exceeds the carrying capacity of the plasma chamber 112a, that is, to hold excess whole blood. During use, excess whole blood will overflow and enter the blood waste liquid chamber 112c.
  • the entrance of the blood waste liquid chamber 112c is close to the entrance of the plasma chamber 112a. It is understood that in other embodiments, the entrance of the plasma chamber 112a is not limited to this, and can also be other positions, as long as it can receive the whole blood overflowing from the plasma chamber 112a.
  • the blood waste liquid chamber 112c includes a platform portion 112d and a collecting portion 112e, the platform portion 112d is connected to the plasma chamber 112a, and the collecting portion 112e is connected to the platform portion 112d, and the depth of the platform portion 112d is less than the depth of the collecting portion 112e.
  • the whole blood overflowing from the plasma chamber 112a flows into the collecting portion 112e through the platform portion 112d.
  • the chip body 110 also has a blood sample sufficient amount detection chamber 112f for reflecting whether the amount of whole blood is sufficient for subsequent detection.
  • the blood sample sufficient amount detection chamber 112f is connected to the blood waste liquid chamber 112c and is farther away from the rotation center than the blood waste liquid chamber 112c.
  • the blood sample sufficient amount detection chamber 112f is connected to the platform portion 112d, and the blood sample sufficient amount detection chamber 112f and the collecting portion 112e are spaced apart and located at one end of the platform portion 112d away from the rotation center, and the blood sample sufficient amount detection chamber 112f is closer to the blood cell chamber 112b.
  • the blood waste liquid chamber 112c is also provided with a vent hole.
  • the vent hole is provided to facilitate the waste blood to enter the blood waste liquid chamber 112c.
  • the chip body 110 is also provided with a sedimentation tank 117.
  • the sedimentation tank 117 is used to preliminarily filter possible clots in the blood sample, including blood clots, larger fat masses, etc., to prevent clogging of the flow channel in the chip.
  • the sedimentation tank 117 is located between the whole blood sampling chamber 111 and the plasma chamber 112a, and the whole blood sampling chamber 111 is connected to the plasma chamber 112a through the sedimentation tank 117.
  • the mixing chamber 113 is a place where serum or plasma is evenly mixed with the diluent, and is also the only way for the cleaning liquid to enter the liquid separation channel 114, and is also a place where the luminescent substrate and the luminescent substrate diluent are evenly mixed.
  • the mixing chamber 113 is connected to the plasma chamber 112a and the non-whole blood injection chamber 111a. More specifically, the mixing chamber 113 is connected to the plasma chamber 112a through the first siphon channel 118, and the mixing chamber 113 is connected to the non-whole blood injection chamber 111a through the microchannel 120.
  • the width of the first siphon channel 118 is 0.2mm to 1.5mm, and the depth of the first siphon channel 118 is 0.1mm to 1mm.
  • the size of the first siphon flow channel 118 is set as described above to ensure that during the whole blood separation process, the whole blood is prevented from passing through the first siphon flow channel 118; however, after the whole blood separation step is completed, the centrifugal speed is reduced so that the separated plasma can pass through the first siphon flow channel 118. In short, it is ensured that the whole blood does not pass through but the separated plasma passes.
  • the width of the microchannel 120 is The microchannel 120 has a width of 0.2 mm to 1.5 mm and a depth of 0.1 mm to 1 mm.
  • one-third of the area of the first siphon flow channel 118 near the mixing chamber 113 is coated with a hydrophobic reagent, and two-thirds of the area of the first siphon flow channel 118 near the main channel 114ad is coated with a hydrophilic reagent.
  • the above coating treatment can effectively enhance the siphon effect of the first siphon flow channel 118.
  • the coating method can be ultrasonic spraying, micro-spotting, etc. In an optional specific example, the ultrasonic spraying scheme is selected. The position that does not need to be sprayed is made into a mask, covered on the chip body 110, and placed in an ultrasonic spraying instrument.
  • the instrument sprays the reagent in the form of particles to the chip surface not covered by the mask, and the reagent adheres to the surface of the microchannel.
  • the width of the first siphon flow channel 118 is 0.1 to 1.0 mm, and the depth of the first siphon flow channel 118 is 0.1 to 1.0 mm.
  • the mixing chamber 113 is roughly crescent-shaped around the center of rotation. It can be understood that in other embodiments, the shape of the mixing chamber 113 is not limited to the above, and can also be other shapes.
  • the arc-shaped liquid separation channel 114 is used to quantitatively transport the liquid in the mixing chamber 113 to the reaction unit 115.
  • the liquid separation channel 114 includes a main channel 114a and a plurality of spaced-apart cup chambers 114b.
  • the main channel 114a is arc-shaped, and the main channel 114a protrudes from the rotation center to the edge of the chip body 110, and the plurality of cup chambers 114b are distributed at intervals along the circumference of the main channel 114a on the side of the main channel 114a away from the rotation center.
  • the arc-shaped liquid separation channel is conducive to transferring all the liquid to each cup chamber 114b at a low speed.
  • the width of the main channel 114a is 0.5mm to 3mm, and the depth of the main channel 114a is 1.5mm to 3mm.
  • the width of the main channel 114a is 1 to 2 mm, and the depth of the main channel 114a is 0.5 to 3.5 mm.
  • the ratio of the distance from the first cup-dividing cavity 114b to the rotation center to the distance from the last cup-dividing cavity to the rotation center is 1:(1.05-1.2).
  • each cup-dividing cavity 114b can be filled with liquid from the mixing cavity 113 at a lower rotation speed.
  • the ratio of the distance from the first cup-dividing cavity 114b to the rotation center to the distance from the last cup-dividing cavity to the rotation center is 1:1.12.
  • the angle formed by the rotation center and the inlet end and the outlet end is 0° to 359°. It is understandable that the angle formed by the rotation center and the inlet end and the outlet end is not limited to 0° to 359°, and if the disk size of the chip body 110 is appropriate, it can also exceed 359°, such as 400°, 480°, etc.
  • the mixing chamber 113 is connected to the liquid separation channel 114 through the second siphon channel 119, the width of the second siphon channel 119 is 0.2 mm to 1.5 mm, and the depth of the second siphon channel 119 is 0.1 mm to 1 mm.
  • the size of the second siphon channel 119 is set as above so that when performing a high-speed mixing action, the liquid in the mixing chamber 113 is prevented from entering the liquid separation channel 114 through the second siphon channel 119, and when performing a liquid separation action at a reduced speed, the liquid in the mixing chamber 113 can climb over the valve top of the second siphon channel 119 through capillary force and then enter the liquid separation channel 114.
  • the second siphon flow channel 119 is coated with a hydrophilic reagent in the part close to the mixing chamber 113, the middle part of the second siphon flow channel 118 is coated with a hydrophobic reagent, and the second siphon flow channel 118 is coated with a hydrophilic reagent in the part close to the liquid separation flow channel 114.
  • the coating treatment can effectively enhance the siphon effect of the second siphon flow channel 119.
  • the coating method can be selected from ultrasonic spraying, micro-spotting, etc. In an optional specific example, an ultrasonic spraying scheme is selected. The position where no spraying is required is made into a mask, covered on the chip body 110, and placed in an ultrasonic spraying instrument.
  • the instrument sprays the reagent in the form of particles to the chip surface not covered by the mask, and the reagent adheres to the surface of the microchannel.
  • the width of the second siphon flow channel 119 is 0.1-1.0mm, and the depth of the second siphon flow channel 119 is 0.1-1.0mm.
  • the first siphon flow channel 118 and the second siphon flow channel 119 are both located at one end of the mixing chamber 113 close to the plasma chamber 112 a .
  • the liquid separation channel 114 further includes a waste liquid separation chamber 114c located at the outlet end of the main channel 114a.
  • the waste liquid separation chamber 114c is used to contain excess liquid flowing out of the main channel 114a.
  • multiple reaction units 115 are arranged in parallel with equal angles around the rotation center, and are used for multiple or multiple immune reactions.
  • the parallel arrangement of each reaction unit can avoid the problem of cross contamination of reagents.
  • the reaction material (coated capture antibody or capture antigen carrier and label carrier) of each reaction unit 115 is the same.
  • the reaction materials of the multiple reaction units 115 are not completely the same or completely different, and the multiple reaction units 115 are used for multiple immune reactions.
  • each reaction unit 115 includes an immune reaction chamber 115a and a reaction waste liquid chamber 115b connected to the immune reaction chamber 115a, and the immune reaction chamber 115a is pre-loaded with reagents for immune reactions, and each immune reaction chamber 115a is connected to the liquid separation channel 114.
  • the cup chamber 114b, immune reaction chamber 115a and reaction waste liquid chamber 115b of each reaction unit 115 are arranged in sequence in the radial direction of the main channel 114a along the direction away from the rotation center.
  • the reagent for immune response is a solid reagent.
  • the preparation method of the solid reagent includes at least one of the following: reduced pressure evaporation drying, normal pressure drying, freeze drying, vacuum drying and gasification humidification drying.
  • the reagent for immune response includes a capture antibody or a capture antigen, a labeled antibody, and a substrate component, and the capture antibody or the capture antigen, and the labeled antibody can be set accordingly according to the specific detection substance.
  • the distance between the bottom of the immune reaction chamber 115 b and the lower surface of the chip body 110 is not less than 0.3 mm.
  • the cup chamber 114b is connected to the immune reaction chamber 115a through the first microchannel 115c, and the immune reaction chamber 115a is connected to the reaction waste liquid chamber 115b through the second microchannel 115d.
  • the first microchannel 115c is used to prevent the liquid from entering the immune reaction chamber 115a during the process of the liquid gradually entering the cup chamber 114b, and on the other hand, guide the liquid in the cup chamber 114b to enter the immune reaction chamber 115a after the liquid fills all the cup chambers 114b.
  • the second microchannel 115d is used to prevent the substances in the immune reaction chamber 115a from entering the reaction waste liquid chamber 115b during the immune reaction process, and on the other hand, guide the liquid in the immune reaction chamber 115a to enter the reaction waste liquid chamber 115b when it needs to be discarded.
  • the width of the first microchannel 115c and the second microchannel 115d are independently 0.2mm to 0.7mm, and the depth of the first microchannel 115c and the second microchannel 115d are independently 0.02mm to 0.07mm; the length of the first microchannel 115c is 1.5mm to 2.5mm, and the length of the second microchannel 115d is 3.5mm to 5.5mm. According to the above arrangement, it is more conducive to allow the liquid in the scoring cup chamber 114b to enter the immune reaction chamber 115a at a lower speed without flowing out from the second microchannel 115d. When it is necessary to centrifuge the liquid in the immune reaction chamber 115a into the waste liquid chamber 115b, the liquid can also smoothly enter the waste liquid chamber 115b.
  • the first microchannel 115c and the second microchannel 115d are microchannels that have been hydrophobically treated. Furthermore, the first microchannel 115c and the second microchannel 115d are coated with a hydrophobic reagent, respectively, which can effectively increase the obstruction effect, so that multiple cleanings can be completed smoothly.
  • the coating method can be selected from ultrasonic spraying, micro-spotting, etc. In an optional specific example, an ultrasonic spraying scheme is selected. The position that does not need to be sprayed is made into a mask, covered on the chip body 110, and placed in an ultrasonic spraying instrument.
  • the instrument sprays the reagent in the form of particles to the surface of the chip not covered by the mask, and the reagent adheres to the surface of the microchannel.
  • the hydrophobic treatment of the first microchannel 115c and the second microchannel 115d it is more conducive to the speed difference to achieve the obstruction and liquid guiding effect of the first microchannel 115c and the second microchannel 115d, so as to meet the multiple cleaning and drainage processes of the chip.
  • the immune reaction chamber 115a is cylindrical, and the positive projection of the reaction waste liquid chamber 115b on the cover surface 110a is an isosceles trapezoid. It is understandable that in other embodiments, the shapes of the immune reaction chamber 115a and the reaction waste liquid chamber 115b are not limited to the above, and can also be other shapes.
  • a labeled antibody and a carrier coated with a capture antibody or a capture antigen are provided in the immune reaction chamber 115a.
  • the label of the labeled antibody is not particularly limited, and can be used for chemiluminescent immunoassay, or for immunoassays such as fluorescence and colloidal gold;
  • the carrier coated with the capture antibody or the capture antigen includes but is not limited to magnetic beads, latex particles, and the like.
  • the substrate reagent chamber 116 is used to pre-install the luminescent substrate.
  • the substrate reagent chamber 116 is pre-installed with the luminescent substrate.
  • the substrate reagent chamber 116 is located on a side of the blood waste liquid chamber 112c away from the plasma chamber 112a and close to the whole blood sampling chamber 111, and the substrate reagent chamber 116 is connected to the mixing chamber 113 through a microchannel.
  • the microfluidic chip 10 further includes a liquid capsule component 120 having a first cavity, a second cavity and a third cavity, wherein the first cavity and the second cavity are respectively connected to the mixing cavity, and the third cavity is connected to the mixing cavity via the substrate reagent cavity.
  • the liquid capsule component 120 includes a first type of liquid capsule 121 storing a plasma diluent, a second type of liquid capsule 122 storing a cleaning liquid, and a third type of liquid capsule 123 storing a luminescent substrate diluent.
  • the first type of liquid capsule 121 is located in the first cavity
  • the second type of liquid capsule 122 is located in the second cavity.
  • the third type of liquid capsule 123 is located in the third cavity.
  • the liquid capsule component 120 is located on the cover surface 110a, and is used to provide diluent and cleaning liquid to the chip body 110 to complete the immune response.
  • the liquid capsule component 120 includes a plurality of liquid capsules. Further, the liquid capsule includes a first type of liquid capsule 121 storing a plasma diluent, a second type of liquid capsule 122 storing a cleaning liquid, and a third type of liquid capsule 123 storing a luminescent substrate diluent.
  • the plasma diluent in the first type of liquid capsule 121 and the cleaning liquid in the second type of liquid capsule 122 can flow into the mixing chamber 113, and the luminescent substrate diluent in the third type of liquid capsule 123 can flow into the mixing chamber 113 through the substrate reagent chamber 116.
  • the liquid capsule of the liquid capsule component 120 in the above-mentioned microfluidic chip 10 can be omitted, and additional liquid capsules can be used when used, that is, the corresponding liquid capsules are added to the first cavity, the second cavity and the third cavity when used.
  • the liquid in the liquid capsule can also be directly correspondingly contained in the first cavity, the second cavity and the third cavity, and the corresponding chambers are connected when the corresponding liquid is needed.
  • the microfluidic chip 10 also includes a puncturing member (not shown) corresponding to the liquid capsule.
  • a puncturing member (not shown) corresponding to the liquid capsule.
  • the puncturing member can puncture the liquid capsule corresponding to it so that the liquid stored in the liquid capsule can flow out.
  • the puncturing member is located on the chip body 110 and extends from the chip body 110 in the direction close to the liquid capsule.
  • the puncturing member includes a sharp thorn portion, which is used to puncture the liquid capsule.
  • the shape of the sharp thorn portion is a square cone, a cone and a blade. When not in use (when the liquid capsule does not need to be punctured), there is a gap between the puncturing member and the liquid capsule.
  • the puncturing member can be replaced by other switches that can control the opening and closing of the liquid capsule.
  • the liquid capsule component 120 has a liquid capsule cavity for accommodating the liquid capsule, and the number of the liquid capsule cavity corresponds to the number of the liquid capsules.
  • the microfluidic chip 10 further includes a first piercing member corresponding to the first type of liquid capsule 121, a second piercing member corresponding to the second type of liquid capsule 122, and a third piercing member corresponding to the third type of liquid capsule 123.
  • the first piercing member, the second piercing member, and the third piercing member all have a spike portion, and the shape of the spike portion is a square cone, a cone, and a blade shape.
  • the number of the first piercing member, the second piercing member, and the third piercing member are independently at least 1. Specifically, the number of the second type of liquid capsule 122 corresponds to the number of cleaning times.
  • the number of the second type of liquid capsule 122 is two.
  • multiple second type of liquid capsules 122 are arranged at intervals.
  • the number of cleaning times here refers to the number of times the antigen-antibody complex formed by the capture antigen or capture antibody and the detected substance and the antigen-antibody complex with a label are cleaned.
  • the number of the first type of liquid capsule 121 is one
  • the number of the second type of liquid capsule 122 is three
  • the number of the third type of liquid capsule 123 is one.
  • the diluent of the first type of liquid capsule 121 may be the same as the diluent of the third type of liquid capsule 123 .
  • the volumes of the first type of liquid sac 121 , the second type of liquid sac 122 , and the third type of liquid sac 123 are independently 100 ⁇ L to 1200 ⁇ L.
  • the microfluidic chip 10 also includes a cover plate 130, which is covered on the covering surface 110a.
  • the covering of the cover plate 130 and the chip body 110 blocks the openings of each chamber on the chip toward the covering surface 110a, so that the liquid in the chamber will not overflow from the opening and affect the reaction.
  • the first type of liquid capsule 121, the second type of liquid capsule 122 and the third type of liquid capsule 123 are all located on the side of the cover plate 130 away from the chip body 110, and the cover plate 130 is provided with through holes corresponding to the first type of liquid capsule 121, the second type of liquid capsule 122 and the third type of liquid capsule 123 respectively.
  • the through holes provided on the cover plate 130 are used to allow the liquid in the liquid capsule to flow into the mixing chamber 113.
  • the thickness of the chip body 110 is 2mm to 8mm; the thickness of the cover plate 130 is 0.5mm to 2mm.
  • the material of the cover plate 130 includes but is not limited to glass, PDMS, PMMA, PET or PC.
  • the microfluidic chip 10 further includes an adhesive layer 140 for bonding the cover plate 130 and the chip body 110.
  • the thickness of the adhesive layer 140 is 0.03 mm to 0.2 mm.
  • an embodiment of the present application also provides a method for preparing the above-mentioned microfluidic chip 10, which includes: obtaining a chip body 110 by injection molding or CNC machining; placing a luminescent substrate in a substrate reagent chamber 116, a labeled antibody and different carriers coated with capture antibodies or capture antigens in each of the immune reaction chambers 115a, and then placing an adhesive layer 140 on the chip body 110; covering the cover plate 130 on the adhesive layer 140 and then pressurizing and bonding them; bonding the first type of liquid capsule 121, the second type of liquid capsule 122 and the liquid capsule to corresponding positions of the cover plate 130 to obtain the microfluidic chip 10.
  • connection between the cover plate 130 and the chip body 110 is not limited to the above-mentioned adhesive layer 140 connection, and can also be ultrasonic Wave welding, laser welding, etc.
  • adhesive layer 140 and other sealing technologies can also be used at the same time.
  • the luminescent substrate, labeled antibody and luminescent substrate are all placed in corresponding chambers in the form of freeze-dried pellets.
  • an embodiment of the present application further provides a detection method, which uses the above-mentioned microfluidic chip to perform immune detection. Specifically, it includes the following steps:
  • the whole blood sample is injected into the injection cavity, the whole blood is separated by the whole blood separation structure to obtain the plasma located in the plasma cavity;
  • the diluted plasma reacts with the pre-loaded reagents for immune reaction to form antigen-antibody complexes
  • the luminescent substrate dilution liquid is transferred to each immune reaction chamber through the substrate reagent chamber, the mixing chamber, and the liquid separation channel for reaction, and a chemiluminescent reaction occurs under the catalytic action of the enzyme.
  • the centrifugal speed is 1000rpm to 5000rpm, and the centrifugal time is 90s to 150s;
  • the step of distributing the diluted plasma in the mixing chamber to each immune reaction chamber through the liquid separation channel includes: using a centrifugal speed of 100rpm to 1200rpm for 1s to 60s to distribute the diluted plasma to each sub-cup chamber; and using a centrifugal speed of 1000rpm to 2000rpm for 1s to 60s to distribute the plasma in each sub-cup chamber to each immune reaction chamber;
  • the centrifugal speed is 1000rpm ⁇ 2000rpm, and the centrifugal time is 1s ⁇ 60s;
  • the centrifugal speed is 100rpm ⁇ 1200rpm, and the centrifugal time is 1s ⁇ 60s; in the step of introducing the cleaning solution into each immune reaction chamber through the mixing chamber, the centrifugal speed is 100rpm ⁇ 1200rpm, and the centrifugal time is 1s ⁇ 60s; in the step of introducing the cleaning solution into
  • the above detection method comprises the following steps:
  • S19 Collect information from each immune reaction chamber, analyze data, and output results.
  • step S2 the centrifugal speed 1 is 100-1200 rpm, and the centrifugal time is 1-60 s.
  • step S3 the centrifugal speed 2 is 100-1400 rpm, and the centrifugal time is 1-60 s.
  • step S4 the centrifugal speed 3 is 1000-5000 rpm, and the centrifugal time is 90-150 s.
  • step S5 the centrifugal speed 4 is 100-1200 rpm, and the centrifugal time is 1-60 s.
  • step S6 the centrifugal speed 5 is 100-4000 rpm, and the centrifugal time is 1-60 s.
  • step S7 step S10, step S13, step S15 and step S17
  • the centrifugal speed 6 is 100-1200 rpm
  • the centrifugal time is 1-60 s.
  • step S8 the centrifugal speed 7 is 1000-2000 rpm, and the centrifugal time is 1-60 s.
  • step S9 and step S12 the centrifugal speed 8 is 1000-2500 rpm, and the centrifugal time is 1-60 s.
  • step S11 the centrifugal speed 9 is 100-1200 rpm, and the centrifugal time is 1-180 s.
  • Adding a non-whole blood sample for example, serum, plasma, etc.
  • the diluted sample solution reacts with the pre-installed reagent for immune reaction to form an antigen-antibody complex
  • the luminescent substrate dilution solution is transferred to each immune reaction chamber through the substrate reagent chamber, the mixing chamber, and the liquid separation channel for reaction, and a chemiluminescent reaction occurs under the catalysis of the enzyme;
  • the centrifugal speed is 100rpm to 1200rpm, and the centrifugal time is 1s to 60s;
  • the step of distributing the diluted sample liquid in the mixing chamber to each immune reaction chamber through the liquid separation channel includes: using a centrifugal speed of 100rpm to 1200rpm for 1s to 60s to distribute the diluted sample liquid to each sub-cup chamber; and using a centrifugal speed of 1000rpm to 2000rpm for 1s to 60s to distribute the diluted sample liquid in each sub-cup chamber to each immune reaction chamber;
  • the centrifugal speed is 1000rpm ⁇ 2000rpm, and the centrifugal time is 1s ⁇ 60s; in the step of introducing the cleaning solution into each immune reaction chamber through the mixing chamber, the centrifugal speed is 100rpm ⁇ 1200rpm, and the centrifugal time is
  • the above detection method comprises the following steps:
  • S19 Collect information from each immune reaction chamber, analyze data, and output results.
  • step S2 the centrifugal speed 1 is 100-1200 rpm, and the centrifugal time is 1-60 s.
  • step S3 the centrifugal speed 2 is 100-1400 rpm, and the centrifugal time is 1-60 s.
  • step S4 the centrifugal speed 3 is 1000-5000 rpm, and the centrifugal time is 90-150 s.
  • step S5 the centrifugal speed 4 is 100-1200 rpm, and the centrifugal time is 1-60 s.
  • step S6 the centrifugal speed 5 is 100-4000 rpm, and the centrifugal time is 1-60 s.
  • step S7 step S10, step S13, step S15 and step S17
  • the centrifugal speed 6 is 100-1200 rpm
  • the centrifugal time is 1-60 s.
  • step S8 the centrifugal speed 7 is 1000-2000 rpm, and the centrifugal time is 1-60 s.
  • step S9 and step S12 the centrifugal speed 8 is 1000-2500 rpm, and the centrifugal time is 1-60 s.
  • step S11 the centrifugal speed 9 is 100-1200 rpm, and the centrifugal time is 1-180 s.
  • the rotation radius is 60 mm.

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Abstract

一种微流控芯片(10)及检测方法，微流控芯片(10)包括芯片主体(110)及液囊组件(120)；芯片主体(110)具有旋转中心，芯片主体(110)上设置有全血分离结构(112)、混匀腔(113)、分液流道(114)、多个反应单元(115)及底物试剂腔(116)。上述微流控芯片在有限的盘面上集成了多个反应，使用操作简单且准确性高。

Description

微流控芯片及检测方法
本申请要求于2022年09月27日提交中国专利局、申请号为2022111793514、发明名称为“微流控芯片及检测方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及免疫检测技术领域，具体涉及一种微流控芯片及检测方法。
背景技术
化学发光免疫分析是将高灵敏度的化学发光检测技术与高特异性的抗原抗体免疫反应相结合，用于检测被测物中抗原或抗体的检测分析技术，是继放射免疫分析、酶联免疫分析、荧光免疫分析和时间分辨荧光免疫分析之后的一项新的免疫分析技术。
化学发光免疫分析包括两个主要组成部分：免疫反应系统和化学发光分析系统。免疫反应系统是根据抗原抗体反应的基本原理，将发光物质直接标记在抗原或抗体上或将酶用于发光底物；化学发光分析系统是利用化学发光物质经催化剂的催化和氧化剂的氧化形成激发态，当这种不稳定的激发态分子返回到稳定的基态时，释放能量发射出光子，利用光子信号检测仪测定发光反应的发光强度，从而计算出被测物质含量。
然而，目前的对于临床全血样本的化学发光免疫分析步骤繁杂且耗时，需陆续分次加入捕捉抗体、抗原、清洗液、底物，并且全血样本需提前对样本进行处理以分离出血清后才能进行检测。并且现有的管式化学发光的试剂用量比较大，对于试剂成本较高的化学发光来说减少试剂使用量可以显著降低用户的检测成本，特别是对于基层的医疗机构，小型化、简单化的检验设备需求较大。
发明内容
基于此，本申请一实施例提供一种微流控芯片。
一种微流控芯片，包括芯片主体以及液囊组件；所述芯片主体具有旋转中心，所述芯片主体上设置有：
全血分离结构，具有血浆腔、血球腔和血液废液腔，所述血球腔和所述血液废液腔分别与所述血浆腔连通，所述血液废液腔靠近所述血浆腔的入口，所述血球腔靠近所述血浆腔的出口；
混匀腔，与所述血浆腔连通；
分液流道，所述分液流道包括主流道、多个分杯腔，所述主流道具有入口端和出口端，所述入口端与所述混匀腔连通；所述主流道以所述旋转中心为中心呈外螺旋线状，多个所述分杯腔与所述主流道直接连通；
多个反应单元，多个所述反应单元间隔排布，各个所述反应单元均包括免疫反应腔和与所述免疫反应腔连通的反应废液腔，多个所述免疫反应腔与多个所述分杯腔一一对应连通；以及
底物试剂腔，所述底物试剂腔与所述混匀腔连通；
所述液囊组件具有第一腔、第二腔和第三腔，所述第一腔和所述第二腔能够分别与所述混匀腔连通，所述第三腔能够经所述底物试剂腔与所述混匀腔连通。
上述微流控芯片通过在芯片主体上设置全血分离结构、混匀腔、分液流道、多个反应单元和底物试剂腔，并搭配液囊组件，使得上述微流控芯片在有限的盘面上可以实现一次加样就可测试多个或多种免疫项目，且不需要单独对全血液样本进行前处理，兼容全血及非全血，操作简捷，耗时少。此外，主流道呈外螺旋线状，而使得以较低的转速就可以将混匀腔中的液体分配到各个分杯腔中且各个分杯腔都能被充满，从而降低了分杯腔中的液体到免疫反应 腔的过程中对离心转速的要求，从而也降低了免疫反应腔中的液体到废液腔的过程中对离心转速的要求。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图，其中：
图1为一是实施例的微流控芯片；
图2为图1所示的微流控芯片的爆炸图；
图3为图1所示的微流控芯片的芯片主体的立体图；
图4为图1所示的微流控芯片的芯片主体的俯视图。
附图标记：
10、微流控芯片；110、芯片主体；110a、盖合面；111、全血进样腔；111a、非全血
进样腔；112、全血分离结构；112a、血浆腔；112b、血球腔；112c、血液废液腔；112d、平台部；112e、收集部；112f、血样足量检测腔；113、混匀腔；114、分液流道；114a、主流道；114b、分杯腔；114c、分流废液腔；114d、牺牲腔；115、反应单元；115a、免疫反应腔；115b、反应废液腔；115c、第一微流道；115d、第二微流道；116、底物试剂腔；117、沉降池；118、第一虹吸流道；119、第二虹吸流道；120、液囊组件；121、第一类液囊；122、第二类液囊；123、第三类液囊；130、盖板；140、粘合层。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使本发明公开内容更加透彻全面。
需要说明的是，当元件被表述“固定于”另一个元件，它可以直接在另一个元件上、或者其间可以存在一个或多个居中的元件。当一个元件被表述“连接”另一个元件，它可以是直接连接到另一个元件、或者其间可以存在一个或多个居中的元件。
当使用术语“垂直的”、“水平的”、“左”、“右”、“上”、“下”、“内”、“外”、“底部”等指示方位或位置关系时，是为基于附图所示的方位或位置关系，仅为了便于描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本申请的限制。此外，术语“第一”、“第二”等仅用于描述目的，而不能理解为指示或暗示相对重要性。术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。本文中，“可选地”表示举例说明。术语“多个”是指至少两个；术语“多种”是指至少两种。
需要说明的是，在本文中，腔室的容量是指腔室能够承装液体的最大量；芯片主体上的部件的深度均是指部件对应的底到盖合面的距离，例如，收集部的深度是指收集部的底部到盖合面的距离。另外，在本文中，如未特殊说明，各个部件的底面均是平整面，也即是部件底面上的各个位置到盖合面的距离相等。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
微流控芯片把生物和化学领域中所涉及的反应、分离、培养、分选检测等基本操作单元集成到一块很小的芯片上，用以实现常规生物或化学实验室的各种功能。由于微流控芯片技 术具有进样量小、集成度高、易实现自动化控制和高通量分析的特点，使得在微流控芯片上进行生化反应操作较常规的分析样品前处理更方便、快速。
请参阅图1～图3，本申请一实施方式提供了一种微流控芯片10，该微流控芯片10包括芯片主体110和液囊组件120；芯片主体110具有旋转中心，芯片主体110上设置有全血进样腔111、非全血进样腔111a、全血分离结构112、混匀腔113、分液流道114、多个反应单元115和底物试剂腔116；全血分离结构112具有血浆腔112a、血球腔112b和血液废液腔112c，血浆腔112a与全血进样腔111连通，血浆腔112a与血球腔112b连通，血液废液腔112c与血浆腔112a连通，血液废液腔112c靠近血浆腔112a的入口，血球腔112b靠近血浆腔112a的出口；混匀腔113与血浆腔112a连通；分液流道114包括主流道114a、多个分杯腔114b，主流道114a具有入口端和出口端，入口端与混匀腔113连通，主流道114a呈弧状，多个分杯腔114b在主流道114a的周向上间隔排布并与主流道114a连通，主流道114a到旋转中心的距离自入口端到出口端逐渐增大；多个反应单元115沿旋转周向间隔分布，各个反应单元115均包括免疫反应腔115a和与免疫反应腔115a连通的反应废液腔115b，免疫反应腔115a预装有用于免疫反应的试剂(例如标记抗体和包被有捕获抗体或捕获抗原的载体)，各个免疫反应腔115a均与分液流道114；预装有发光底物的底物试剂腔116与混匀腔113连通。
上述微流控芯片10通过在芯片主体110上设置全血进样腔111、非全血进样腔111a、全血分离结构112、混匀腔113、分液流道114、多个反应单元115和底物试剂腔116，并搭配液囊组件120，使得上述微流控芯片10一次加样就可测试多个或多种免疫项目，且不需要单独对全血液样本进行前处理，操作简捷，耗时少。此外，主流道114a以旋转中心为中心呈外螺旋线状，主流道114a到旋转中心的距离自入口端到出口端逐渐增大，这使得以较低的转速就可以将混匀腔113中的液体分配到各个分杯腔114b中。所以，上述微流控芯片10在有限的盘面上可以实现一次加样测试多个或多种免疫项目，且不需要单独对全血液样本进行前处理，操作简捷，耗时少，且准确性好。
进一步地，底物试剂腔116的形状任意，可以是任意形状与任意弯曲的微流道共同组成。
进一步地，分液流道114还包括牺牲腔114d，牺牲腔114d与主流道114a连通并与分杯腔114b间隔，牺牲腔114d较分杯腔114b更靠近入口端；牺牲腔114d的设置使得从入口端数的第一个分杯腔114b，不容易出现实际填充的液体少于预设体积，而影响下一步反应；牺牲腔114d同时可以有效减轻第一个分杯腔114b承受的液体冲击力，从而提高第一个免疫反应腔115a的准确性。
进一步地，主流道114a以旋转中心为中心呈外螺旋线状，多个分杯腔114b在主流道114a的外周上、以旋转中心为中心等角度并联式间隔排布并与主流道114a直接连通。
在一些实施例中，微流控芯片10的直径为100mm～140mm。可选地，微流控芯片10的直径为100mm、120mm、130mm或140mm。
可选地，芯片主体110的材料包括但不限于玻璃、PDMS、PMMA、PET或PC。在一些实施例中，芯片主体110的材料为不透明材料。不透明材料作为芯片主体110的材料更利于后续检测。可选地，芯片主体110的材料为带颜色的不透明材料。在一些实施例中，芯片主体110的直径为100mm～140mm。可选地，芯片主体110的直径为100mm、120mm、130mm或140mm。请参阅图3和图4，具体地，全血进样腔111、非全血进样腔111a用于承装添加的样品。在本实施方式中，全血进样腔111的容量在400μL以上，非全血进样腔111a的容量在1μL以上。按照上述设置可以满足至少20个反应单元115的用量。进一步地，全血进样腔111的容量在450μL以上；非全血进样腔111a的容量在50μL以上。更进一步地，全血进样腔111的容量为400μL～600μL；非全血进样腔111a的容量在1μL～200μL。可选地，全血进样腔111呈扇环状，全血进样腔111自旋转中心向芯片主体110的边缘凸出； 非全血进样腔111a呈扇环状，非全血进样腔111a自旋转中心向芯片主体110的边缘凸出。在图示的实施例中，全血进样腔111、非全血进样腔111a的宽度沿顺时针方向逐渐增大。按照如此设置便于样品经离心进入下游，减少离心时间，提高检测效率。
具体地，全血分离结构112用于对血液样本进行处理，以从全血中分离出血清或血浆用于下游检测。全血分离结构112包括血浆腔112a、血球腔112b和血液废液腔112c。在离心时，全血被分离成血清或血浆和血细胞，血清或血浆停留在血浆腔112a中而血细胞留在血球腔112b中，从而实现分离。在血浆腔112a中的血液超过其容量时，进入血液废液腔112c，从而实现对血液的定量。进一步地，芯片主体110具有盖合面110a；血浆腔112a具有助流面，助流面到盖合面110a的距离沿血浆腔112a的入口到血浆腔112a的出口方向逐渐减小。通过助流面的设置，使得在离心时血浆腔112a中的血细胞更容易进入血球腔112b而使得全血分离更彻底，利于后续检测。可选地，血浆腔112a大致呈漏斗状，血浆腔112a的侧壁之间的距离在沿血浆腔112a的入口至血浆腔112a的出口反向上逐渐收窄。可以理解的是，血浆腔112a的形状不限于上述，血球腔112b的形状也不限。
在本实施方式中，血浆腔112a与血球腔112b的容量之比为1:(1～5)。将血浆腔112a与血球腔112b的容量之比设置为1:(1～5)，将血浆与血细胞充分分离，使得血浆中无血红蛋白等其他干扰物。进一步地，血浆腔112a与血球腔112b的容量之比为1:(2～5)。将血浆腔112a与血球腔112b的容量之比设置为1:(2～5)可以使得全血分离结构能够兼容几乎所有年龄段及性别的正常全血样本。在一个可选地具体示例中，血浆腔112a与血球腔112b的容量之比为1:2、1:3、1:4或1:5。
具体地，血液废液腔112c用于承装超过血浆腔112a承载量的全血，即用于承装多余的全血。在使用过程中，多余的全血会溢出而进入血液废液腔112c。在图示的实施例中，血液废液腔112c的入口靠近血浆腔112a的入口。可以理解的是，在其他实施例中，血浆腔112a的入口不限于此，还可以是其他位置，只要能接收从血浆腔112a溢出的全血即可。进一步地，血液废液腔112c包括平台部112d和收集部112e，平台部112d与血浆腔112a连通，收集部112e与平台部112d连通，平台部112d的深度小于收集部112e的深度。在使用时，从血浆腔112a溢出的全血经平台部112d流入收集部112e。通过设计平台部112d和收集部112e，可使得进入血液废液腔112c的全血不容易逆行回血浆腔112a。更进一步地，芯片主体110还具有用于反应全血的量是否足够用于后续检测的血样足量检测腔112f，血样足量检测腔112f与血液废液腔112c连通且较血液废液腔112c更远离旋转中心。在图示的实施例中，血样足量检测腔112f与平台部112d连通，血样足量检测腔112f与收集部112e间隔地位于平台部112d远离旋转中心的一端，血样足量检测腔112f更靠近血球腔112b。
在一些实施例中，血液废液腔112c上还设置有透气孔。通过透气孔的设置便于废弃血液进入血液废液腔112c中。进一步地，芯片主体110还设置有沉降池117。具体地，沉降池117用于初步过滤血样中的可能凝块，包括凝血块、较大脂肪团等，以防止堵塞芯片中流道。沉降池117位于全血进样腔111和血浆腔112a之间，全血进样腔111与血浆腔112a通过沉降池117连通。
具体地，混匀腔113是血清或浆与稀释液混合均匀的场所，也是清洗液进入分液流道114的必经之路，还是发光底物与发光底物稀释液混合均匀的场所。混匀腔113与血浆腔112a、非全血进样腔111a连通。更具体地，混匀腔113与血浆腔112a通过第一虹吸流道118连通，混匀腔113与非全血进样腔111a通过微流道120连通。在本实施方式中，第一虹吸流道118的宽度为0.2mm～1.5mm，第一虹吸流道118的深度为0.1mm～1mm。将第一虹吸流道118的尺寸按照上述设置可以确保在全血分离过程中，阻挡全血不会通过第一虹吸流道118；但在结束全血分离步骤后，通过降低离心转速，使得分离出的血浆可以通过第一虹吸流道118，简而言之，确保全血不通过而分离得出的血浆通过。在本实施方式中，微流道120的宽度为 0.2mm～1.5mm，微流道120的深度为0.1mm～1mm。
在一些实施例中，第一虹吸流道118的靠近混匀腔113的三分之一的区域涂布有疏水试剂，第一虹吸流道118的靠近主流道114ad三分之二的区域涂布有亲水试剂。上述涂层处理可以有效增强第一虹吸流道118的虹吸效果。涂布方式可以选择超声喷涂、微量点样器点样等。在一个可选地具体示例中，选择的是超声喷涂方案。将不需要喷涂的位置做成掩膜，覆盖在芯片主体110之上，置于超声喷涂仪器中，仪器将试剂以微粒的形式喷涂到未被掩膜覆盖的芯片表面，试剂附着微流道的表面。在一个可选地具体示例中，第一虹吸流道118的宽度为0.1～1.0mm，第一虹吸流道118的深度为0.1～1.0mm。在图示的实施例中，混匀腔113绕旋转中心大致呈月牙形。可以理解的是，在其他实施例中，混匀腔113的形状不限于上述，还可以是其他形状。
具体地，弧形的分液流道114用于将混匀腔113中的液体定量地输送至反应单元115。分液流道114包括主流道114a和多个间隔设置的分杯腔114b。在使用时，随着离心，从混匀腔113中流出的液体顺着离心方向进入主流道114a并逐步填满每个分杯腔114b。主流道114a呈弧状，主流道114a自旋转中心向芯片主体110的边缘凸起，多个分杯腔114b在主流道114a的远离旋转中心的一侧沿主流道114a的周向间隔分布。弧形的分液流道有利于在低转速下将液体全部转移到每个分杯腔114b中。在本实施方式中，主流道114a的宽度为0.5mm～3mm，主流道114a的深度为1.5mm～3mm。在一个可选地具体示例中，主流道114a的宽度为1～2mm，主流道114a的深度为0.5～3.5mm。
在自主流道114a的入口端到出口端的方向上，第一个分杯腔114b到旋转中心的距离与最后一个分杯腔到旋转中心的距离之比为1:(1.05～1.2)。按照如此设置，可以使得在有较多分杯腔114b(例如分杯腔114b的数量为25个以上)时，也能在较低的转速下，每个分杯腔114b都被充满来自混匀腔113的液体。在图示的实施例中，第一个分杯腔114b到旋转中心的距离与最后一个分杯腔到旋转中心的距离之比为1:1.12。
在一些实施例中，以旋转中心为顶点，旋转中心与入口端和出口端所形成的夹角的大小为0°～359°。可以理解的是，旋转中心与入口端和出口端所形成的夹角的不限于0°～359°，如果芯片主体110的盘面尺寸合适，还可以超过359°，例如400°、480°等。
在本实施方式中，混匀腔113与分液流道114通过第二虹吸流道119连通，第二虹吸流道119的宽度为0.2mm～1.5mm，第二虹吸流道119的深度为0.1mm～1mm。将第二虹吸流道119的尺寸按照上述设置可以使得在执行高速混匀动作时，阻挡混匀腔113内的液体不会经第二虹吸流道119进入到分液流道114中，而在降低转速执行分液动作时，混匀腔113内的液体可以通过毛细力爬过第二虹吸流道119的阀顶，进而进入到分液流道114中。在一些实施例中，第二虹吸流道119在靠近混匀腔113的部分涂布有亲水试剂，第二虹吸流道118的中部涂布有疏水试剂，第二虹吸流道118在靠近分液流道114的部分涂布有亲水试剂，涂层处理可以有效增强第二虹吸流道119的虹吸效果。涂布方式可以选择超声喷涂、微量点样器点样等。在一个可选地具体示例中，选择的是超声喷涂方案。将不需要喷涂的位置做成掩膜，覆盖在芯片主体110之上，置于超声喷涂仪器中，仪器将试剂以微粒的形式喷涂到未被掩膜覆盖的芯片表面，试剂附着微流道的表面。在一个可选地具体示例中，第二虹吸流道119的宽度为0.1-1.0mm，第二虹吸流道119的深度为0.1-1.0mm。在图示的实施例中，第一虹吸流道118和第二虹吸流道119均位于混匀腔113靠近血浆腔112a的一端。
在图示的实施例中，分液流道114还包括位于主流道114a的出口端的分流废液腔114c。分流废液腔114c用于承装多余的从主流道114a流出的液体。
具体地，多个反应单元115以旋转中心为中心等角度间隔并联式间隔分布，用于多个或多种免疫反应。各个反应单元并联式的设置可以避免试剂交叉污染的问题。在一些实施例中，每个反应单元115的反应物质(包被的捕获抗体或捕获抗原的载体和与标记载体)相同，此 时多个反应单元115用于批量的免疫反应。在另一些实施例中，多个反应单元115的反应物质不完全相同或完全不同，此时多个反应单元115用于多种免疫反应。更具体地，每个反应单元115包括免疫反应腔115a和与免疫反应腔115a连通的反应废液腔115b，免疫反应腔115a预装有用于免疫反应的试剂，各个免疫反应腔115a均与分液流道114连通。每个反应单元115的分杯腔114b、免疫反应腔115a和反应废液腔115b在主流道114a的径向上沿远离旋转中心的方向依次排布。
在一些实施例中，用于免疫反应的试剂为固态试剂。可选地，固态试剂的制备方法包括以下的至少一种：减压蒸发干燥、常压干燥、冷冻干燥、真空干燥和气化加湿干燥。可选地，用于免疫反应的试剂包括捕获抗体或捕获抗原、和标记抗体、和底物组分，捕获抗体或捕获抗原、和标记抗体可以根据具体的检测物质进行对应设置。
在一些实施例中，免疫反应腔115b的腔底与芯片主体110的下表面的距离不小于0.3mm。
进一步地，分杯腔114b与免疫反应腔115a通过第一微流道115c连通，免疫反应腔115a与反应废液腔115b通过第二微流道115d连通。第一微流道115c一方面用于在液体逐步进入分杯腔114b的过程中阻碍液体进入免疫反应腔115a，另一方面在液体填满所有分杯腔114b后引导分杯腔114b中的液体进入免疫反应腔115a。第二微流道115d一方面用于阻挡在免疫反应过程中免疫反应腔115a中的物质进入反应废液腔115b，另一方面在需要将免疫反应腔115a中的液体丢弃时引导其进入反应废液腔115b。
在本实施方式中，第一微流道115c和第二微流道115d的宽度分别独立地为0.2mm～0.7mm，第一微流道115c和第二微流道115d的深度分别独立地为0.02mm～0.07mm；第一微流道115c的长度为1.5mm～2.5mm，第二微流道115d的长度为3.5mm～5.5mm。按照上述设置，更利于在较低的转速下可以使得分杯腔114b中的液体进入免疫反应腔115a而不从第二微流道115d流出，在需要将免疫反应腔115a中的液体离心入废液腔115b时，液体也能顺利进入废液腔115b。在一些实施例中，第一微流道115c和第二微流道115d均为经过疏水处理的微流道。进一步地，第一微流道115c和第二微流道115d分别涂布疏水试剂，可以有效的增加阻碍作用，从而使得多次清洗能够顺利完成。涂布方式可以选择超声喷涂、微量点样器点样等。在一个可选地具体示例中，选择的是超声喷涂方案。将不需要喷涂的位置做成掩膜，覆盖在芯片主体110之上，置于超声喷涂仪器中，仪器将试剂以微粒的形式喷涂到未被掩膜覆盖的芯片表面，试剂附着微流道的表面。本实施例中，经过对第一微流道115c和第二微流道115d的疏水处理，更利于转速差实现第一微流道115c和第二微流道115d的阻碍及液体引导作用，满足芯片的多次清洗排液流程。
在图示的实施例中，免疫反应腔115a呈圆柱状，反应废液腔115b在盖合面110a上的正投影呈等腰梯形。可以理解的是，在其他实施例中，免疫反应腔115a和反应废液腔115b的形状不限与上述，还可以是其他形状。
可选地，免疫反应腔115a中设置有标记抗体和包被有捕获抗体或捕获抗原的载体。可以理解的是，标记抗体的标记物没有特别限制，可以用于化学发光免疫分析，也可以用于荧光、胶体金等免疫检测；包被有捕获抗体或捕获抗原的载体包括但不限于磁珠、乳胶微粒等等。
具体地，底物试剂腔116用于预装发光底物。在一些实施例中，底物试剂腔116预置有发光底物。在图示的实施例中，底物试剂腔116位于血液废液腔112c远离血浆腔112a的一侧，并靠近全血进样腔111，底物试剂腔116通过微流道与混匀腔113连通。
在一些实施例中，上述微流控芯片10还包括液囊组件120具有第一腔、第二腔和第三腔，第一腔和第二腔分别与混匀腔连通，第三腔经底物试剂腔与混匀腔连通。液囊组件120包括储存有血浆稀释液的第一类液囊121、储存有清洗液的第二类液囊122和储存有发光底物稀释液的第三类液囊123，第一类液囊121位于第一腔中，第二类液囊122中位于第二腔 中，第三类液囊123位于第三腔中。
具体地，液囊组件120位于盖合面110a上，用于向芯片主体110提供稀释液和清洗液以完成免疫反应。液囊组件120包括多个液囊。进一步地，液囊包括储存有血浆稀释液的第一类液囊121、储存有清洗液的第二类液囊122和储存有发光底物稀释液的第三类液囊123，第一类液囊121中的血浆稀释液和第二类液囊122中的清洗液均能够流入混匀腔113，第三类液囊123中的发光底物稀释液能够经底物试剂腔116流入混匀腔113。可以理解的是，在一些实施例中，上述微流控芯片10中的液囊组件120的液囊可以省略，在使用时搭配额外的液囊即可，即在使用时才向第一腔、第二腔和第三腔中加入对应的液囊。另外，还可以将液囊中的液体直接对应地承装在第一腔、第二腔和第三腔中，在需要对应液体时，连通相应的腔室。
在一些实施例中，微流控芯片10还包括与液囊对应刺破件(图未示)。通过外力作用，刺破件能够将与其对应的液囊刺破而使得储存于该液囊中的液体可以流出。在一些实施例中，刺破件位于芯片主体110上并自芯片主体110向靠近液囊的方向延伸。刺破件包括尖刺部，尖刺部用于刺破液囊。可选地，尖刺部的形状为方锥形、圆锥形和刀片形。在未使用时(不需要刺破液囊时)，刺破件与液囊之间有间隙。可以理解的是，在另一些实施例中，刺破件可以用其他能够控制液囊开启和关闭的开关替代。
可选地，液囊组件120具有容纳液囊的液囊腔，液囊腔与液囊的个数对应。
在一些实施例中可，微流控芯片10还包括与第一类液囊121对应的第一刺破件、与第二类液囊122对应的第二刺破件和第三类液囊123对应的第三刺破件。第一刺破件、第二刺破件和第三刺破件均具有尖刺部，尖刺部的形状为方锥形、圆锥形和刀片形。第一刺破件、第二刺破件和第三刺破件的数量分别独立地为至少1个。具体地，第二类液囊122的个数与清洗的次数相对应。例如，在清洗次数为两次时，第二类液囊122的个数为两个。当然，多个第二类液囊122间隔设置。需要说明的是，此处的清洗次数是指清洗捕获抗原或捕获抗体与被检测物质形成的抗原抗体复合物和带有标记的抗原抗体复合物的次数。在图示的实施例中，第一类液囊121的个数为一个，第二类液囊122的个数为三个，第三类液囊123的个数为一个。可选地，第一类液囊121的稀释液可以与第三类液囊123的稀释液相同。
在一些实施例中，第一类液囊121、第二类液囊122和第三类液囊123的容积分别独立地为100μL～1200μL。
进一步地，微流控芯片10还包括盖板130，盖板130盖合于盖合面110a上。盖板130与芯片主体110的盖合使得芯片上的各个腔室的朝向盖合面110a的开口被封堵，进而腔室中的液体不会从该开口溢出而影响反应。第一类液囊121、第二类液囊122和第三类液囊123均位于盖板130的远离芯片主体110的一侧，盖板130上设有与第一类液囊121、第二类液囊122和第三类液囊123分别对应的通孔。盖板130上设置的通孔用于使得液囊中的液体能够流入混匀腔113。在本实施方式中，芯片主体110的厚度为2mm～8mm；盖板130的厚度为0.5mm～2mm。
可选地，盖板130的材料包括但不限于玻璃、PDMS、PMMA、PET或PC。
更进一步地，微流控芯片10还包括用于粘合盖板130与芯片主体110之间的粘合层140。在本实施方式中，粘合层140的厚度为0.03mm～0.2mm。
此外，本申请一实施方式还提供了一种上述微流控芯片10的制备方法，该方法包括：注塑或数控加工得到芯片主体110；将发光底物置于底物试剂腔116、标记抗体和不同的包被有捕获抗体或捕获抗原的载体置于各个所述免疫反应腔115a中后，将粘合层140放置与芯片主体110上；将盖板130盖合到粘合层140上后加压粘合；将第一类液囊121、第二类液囊122和液囊粘于盖板130的对应处，制得微流控芯片10。
可选地，盖板130与芯片主体110的连接不限于上述的粘合层140连接，还可以是超声 波焊接、激光焊接等。当然，也可以同时采用粘合层140和其他封接技术。
可选地，发光底物、标记抗体和发光底物均以冻干球的形式置于相应的腔室中。
此外，本申请一实施方式还提供一种检测方法，该方法使用上述微流控芯片进行免疫检测。具体地，包括下述步骤：
(一)加入全血样本
将全血样本注入进样腔后，利用全血分离结构分离全血，得到位于血浆腔的血浆；
将血浆腔中的血浆转移到混匀腔；
将血浆稀释液引入混匀腔，以稀释混匀腔中的血浆；
将混匀腔中稀释后的血浆通过分液流道分配至各个免疫反应腔；
在各个免疫反应腔中，稀释后的血浆与预装的用于免疫反应的试剂反应，以形成抗原抗体复合物；
在各个免疫反应腔反应结束后，将各个免疫反应腔中的非特异性物质转移到反应废液腔；
将清洗液通过混匀腔引入各个免疫反应腔，以清洗反应结束后各个免疫反应腔中形成的抗原抗体复合物；
在清洗结束后，将发光底物稀释液经底物试剂腔、混匀腔、分液流道转移至各个免疫反应腔中进行反应，在酶的催化作用下发生化学发光反应。
进一步地，在利用全血分离结构分离全血步骤中，离心的转速为1000rpm～5000rpm，离心时间为90s～150s；在将混匀腔中稀释后的血浆通过分液流道分配至各个免疫反应腔的步骤包括：采用100rpm～1200rpm的离心转速离心1s～60s将稀释后的血浆分配至各个分杯腔中；及采用1000rpm～2000rpm的离心转速离心1s～60s将各个分杯腔中的血浆分配至各个免疫反应腔；在将各个免疫反应腔中的除抗原抗体复合物外的物质转移到反应废液腔的步骤中，离心转速为1000rpm～2000rpm，离心时间为1s～60s；在将清洗液通过混匀腔引入各个免疫反应腔的步骤中，离心转速为100rpm～1200rpm，离心时间为1s～60s；在将发光底物稀释液经底物试剂腔、混匀腔、分液流道转移至各个免疫反应腔的步骤中，离心转速为100～1200rpm，离心时间为1s～60s。
更进一步地，上述检测方法包括如下步骤：
S1：将全血样本注入上述任一实施例的微流控芯片的进样腔中；
S2：以离心转速1将全血样本通过微流道转移至沉降池中；
S3：以离心转速2将沉降池中全血样本转移至血浆腔及血球腔中，多余的全血会通过平台部进入到收集部和血样足量检测腔中。
S4：以离心转速3将血浆腔及血球腔中的全血进行离心，以将血浆与血细胞分离。
S5：以离心转速4将血浆腔中的血浆经由第一虹吸流道进入混匀腔中后,微流控芯片停止转动，外力将第一类液囊顶破，第一类液囊里的用于与血浆混合的稀释液A流入混匀腔。
S6：以混匀离心转速5将血浆与稀释液A充分混匀。
S7：在离心转速6的条件下，将混匀腔中的混合液经第二虹吸流道进入流道和分杯腔中。
S8：在离心转速7的条件下，分杯腔中的混合液经第一微流道进入免疫反应腔，混合液复融预装在免疫反应腔中的冻干球并充分反应后，磁铁上升吸住混合液中的磁颗粒(磁珠)。
S9：以离心转速8将免疫反应腔中的液体经第二微流道进入反应废液腔中。
S10：磁铁下降，微流控芯片停止转动，用外力将其中一个第二类液囊顶破，以将第二类液囊中的清洗液释放，清洗液经微流道进入混匀腔，以离心转速6将混匀腔里的液体经第二虹吸微流道进入主流道和分杯中。
S11：以离心转速7将分杯腔中的液体经第一微流道进入免疫反应腔中后，以离心转速9对磁珠颗粒进行充分清洗，然后磁铁上升吸住混合液中的磁颗粒。
S12：以离心转速8将免疫反应腔中的液体经第二微流道进入反应废液腔中。
S13：磁铁下降，微流控芯片停止转动，外力将另一个第二类液囊顶破以将清洗液释放入混匀腔后，以离心转速6将混匀腔里的清洗液经第二虹吸流道进入主流道和分杯腔中。
S14：重复执行上述步骤S11～13。
S15：外力将另一个第二类液囊顶破以将清洗液释放入混匀腔后，以离心转速6将混匀腔里的清洗液经第二虹吸流道进入主流道和分杯腔中。
S16：重复执行上述步骤S11～13。
S17：外力将第三类液囊顶破以将稀释液释放进入底物试剂腔，复融预装在底物试剂腔的发光底物并进入混匀腔，以离心转速6将混匀腔里的液体经第二虹吸流道进入主流道和分杯腔中。
S18：重复执行上述步骤S11～13。
S19：采集各免疫反应腔的信息，分析数据，输出结果。
在步骤S2中，离心转速1为100～1200rpm，离心时间为1-60s。
在步骤S3中，离心转速2为100～1400rpm，离心时间为1-60s。
在步骤S4中，离心转速3为1000～5000rpm，离心时间为90～150s。
在步骤S5中，离心转速4为100～1200rpm，离心时间为1-60s。
在步骤S6中，离心转速5为100～4000rpm，离心时间为1-60s。
在步骤S7、步骤S10、步骤S13、步骤S15和步骤S17中，离心转速6为100～1200rpm，离心时间为1-60s。
在步骤S8中，离心转速7为1000～2000rpm，离心时间为1-60s。
在步骤S9和步骤S12中，离心转速8为1000～2500rpm，离心时间为1-60s。
在步骤S11中，离心转速9为100-1200rpm，离心时间为1-180s。
(二)加入非全血样本，例如，血清、血浆等其中一种。
将血清或者血浆注入非全血进样腔；
将血清或者血浆转移到混匀腔；
将稀释液引入混匀腔，以稀释混匀腔中的样本；
将混匀腔中稀释后的样本液通过分液流道分配至各个免疫反应腔；
在各个免疫反应腔中，稀释后的样本液与预装的用于免疫反应的试剂反应，以形成抗原抗体复合物；
在各个免疫反应腔反应结束后，将各个免疫反应腔中非特异性物质转移到反应废液腔；
将清洗液通过混匀腔引入各个免疫反应腔，以清洗反应结束后各个免疫反应腔中形成的抗原抗体复合物；
在清洗结束后，将发光底物稀释液经底物试剂腔、混匀腔、分液流道转移至各个免疫反应腔中进行反应，在酶的催化作用下发生化学发光反应；
进一步地，在非全血进样腔进入混匀腔步骤中，离心的转速为100rpm～1200rpm，离心时间为1s～60s；在将混匀腔中稀释后的样本液通过分液流道分配至各个免疫反应腔的步骤包括：采用100rpm～1200rpm的离心转速离心1s～60s将稀释后的样本液分配至各个分杯腔中；及采用1000rpm～2000rpm的离心转速离心1s～60s将各个分杯腔中的稀释后的样本液分配至各个免疫反应腔；在将各个免疫反应腔中的除抗原抗体复合物外的物质转移到反应废液腔的步骤中，离心转速为1000rpm～2000rpm，离心时间为1s～60s；在将清洗液通过混匀腔引入各个免疫反应腔的步骤中，离心转速为100rpm～1200rpm，离心时间为1s～60s；在将发光底物稀释液经底物试剂腔、混匀腔、分液流道转移至各个免疫反应腔的步骤中，离心转速为100～1200rpm，离心时间为1s～60s。
更进一步地，上述检测方法包括如下步骤：
S1：将非全血样本注入上述任一实施例的微流控芯片的非全血进样腔中；
S5：以离心转速4将非全血进样腔中样本经微流道转移进入混匀腔中后,微流控芯片停止转动，外力将第一类液囊顶破，第一类液囊里的用于与血浆混合的稀释液A流入混匀腔。
S6：以混匀离心转速5将血浆与稀释液A充分混匀。
S7：在离心转速6的条件下，将混匀腔中的混合液经第二虹吸流道进入流道和分杯腔中。
S8：在离心转速7的条件下，分杯腔中的混合液经第一微流道进入免疫反应腔，混合液复融预装在免疫反应腔中的冻干球并充分反应后，磁铁上升吸住混合液中的磁颗粒(磁珠)。
S9：以离心转速8将免疫反应腔中的液体经第二微流道进入反应废液腔中。
S10：磁铁下降，微流控芯片停止转动，用外力将其中一个第二类液囊顶破，以将第二类液囊中的清洗液释放，清洗液经微流道进入混匀腔，以离心转速6将混匀腔里的液体经第二虹吸微流道进入主流道和分杯中。
S11：以离心转速7将分杯腔中的液体经第一微流道进入免疫反应腔中后，以离心转速9对磁珠颗粒进行充分清洗，然后磁铁上升吸住混合液中的磁颗粒。
S12：以离心转速8将免疫反应腔中的液体经第二微流道进入反应废液腔中。
S13：磁铁下降，微流控芯片停止转动，外力将另一个第二类液囊顶破以将清洗液释放入混匀腔后，以离心转速6将混匀腔里的清洗液经第二虹吸流道进入主流道和分杯腔中。
S14：重复执行上述步骤S11～13。
S15：外力将另一个第二类液囊顶破以将清洗液释放入混匀腔后，以离心转速6将混匀腔里的清洗液经第二虹吸流道进入主流道和分杯腔中。
S16：重复执行上述步骤S11～13。
S17：外力将第三类液囊顶破以将稀释液释放进入底物试剂腔，复融预装在底物试剂腔的发光底物并进入混匀腔，以离心转速6将混匀腔里的液体经第二虹吸流道进入主流道和分杯腔中。
S18：重复执行上述步骤S11～13。
S19：采集各免疫反应腔的信息，分析数据，输出结果。
在步骤S2中，离心转速1为100～1200rpm，离心时间为1-60s。
在步骤S3中，离心转速2为100～1400rpm，离心时间为1-60s。
在步骤S4中，离心转速3为1000～5000rpm，离心时间为90～150s。
在步骤S5中，离心转速4为100～1200rpm，离心时间为1-60s。
在步骤S6中，离心转速5为100～4000rpm，离心时间为1-60s。
在步骤S7、步骤S10、步骤S13、步骤S15和步骤S17中，离心转速6为100～1200rpm，离心时间为1-60s。
在步骤S8中，离心转速7为1000～2000rpm，离心时间为1-60s。
在步骤S9和步骤S12中，离心转速8为1000～2500rpm，离心时间为1-60s。
在步骤S11中，离心转速9为100-1200rpm，离心时间为1-180s。
需要说明的是，在本实施方式中，旋转半径为60mm。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，便于具体和详细地理解本发明的技术方案，但并不能因此而理解为对发明专利保护范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。应当理解的是，在本领域技术人员在本发明提供的技术方案的基础上，通过合乎逻辑的分析、推理或有限的试验得到的技术方案，均在本发明所附权利要求的保护范围内。因此，本发明专利的保护范围应以所附权利要求的内容为准，说明书及附图可以用 于解释权利要求的内容。

Claims (32)

  1. 一种微流控芯片，包括芯片主体以及液囊组件；所述芯片主体具有旋转中心，所述芯片主体上设置有：
    全血分离结构，具有血浆腔、血球腔和血液废液腔，所述血球腔和所述血液废液腔分别与所述血浆腔连通，所述血液废液腔靠近所述血浆腔的入口，所述血球腔靠近所述血浆腔的出口；
    混匀腔，与所述血浆腔连通；
    分液流道，所述分液流道包括主流道、多个分杯腔，所述主流道具有入口端和出口端，所述入口端与所述混匀腔连通；所述主流道以所述旋转中心为中心呈外螺旋线状，多个所述分杯腔与所述主流道直接连通；
    多个反应单元，多个所述反应单元间隔排布，各个所述反应单元均包括免疫反应腔和与所述免疫反应腔连通的反应废液腔，多个所述免疫反应腔与多个所述分杯腔一一对应连通；以及
    底物试剂腔，所述底物试剂腔与所述混匀腔连通；
    所述液囊组件具有第一腔、第二腔和第三腔，所述第一腔和所述第二腔能够分别与所述混匀腔连通，所述第三腔能够经所述底物试剂腔与所述混匀腔连通。
  2. 根据权利要求1所述的微流控芯片，其特征在于，多个所述分杯腔在所述主流道的外周上以旋转中心为中心等角度间隔并联式排布。
  3. 根据权利要求1所述的微流控芯片，其特征在于，多个所述反应单元以所述旋转中心为中心等角度并联式间隔排布，所述分杯腔、所述免疫反应腔和所述反应废液腔在所述主流道的径向上沿远离所述旋转中心的方向依次排布。
  4. 根据权利要求1所述的微流控芯片，其特征在于，所述液囊组件包括用于储存血浆稀释液的第一类液囊、用于储存清洗液的第二类液囊和用于储存发光底物稀释液的第三类液囊，所述第一类液囊位于所述第一腔中，所述第二类液囊位于所述第二腔中，所述第三类液囊位于所述第三腔中。
  5. 根据权利要求4所述的微流控芯片，其特征在于，所述微流控芯片还包括与所述第一类液囊对应的第一刺破件、与所述第二类液囊对应的第二刺破件和与所述第三类液囊对应的第三刺破件。
  6. 根据权利要求5所述的微流控芯片，其特征在于，所述第一刺破件位于所述芯片主体上并朝向所述第一类液囊，所述第二刺破件位于所述芯片主体上并朝向所述第二类液囊，所述第三刺破件位于所述芯片主体上并朝向所述第三类液囊。
  7. 根据权利要求6所述的微流控芯片，其特征在于，所述第一刺破件、所述第二刺破件和所述第三刺破件均具有尖刺部，所述尖刺部的形状为方锥形、圆锥形和刀片形。
  8. 根据权利要求5所述的微流控芯片，其特征在于，所述第一刺破件、所述第二刺破件和所述第三刺破件的数量分别独立地为0个或至少一个。
  9. 根据权利要求4所述的微流控芯片，其特征在于，所述第二类液囊的数量至少为两个。
  10. 根据权利要求4所述的微流控芯片，其特征在于，所述第一类液囊的数量至少为一个。
  11. 根据权利要求4所述的微流控芯片，其特征在于，所述第三类液囊的数量至少为一个。
  12. 根据权利要求4所述的微流控芯片，其特征在于，所述第一类液囊、所述第二类液囊和所述第三类液囊的容积分别独立地为100μL～1200μL。
  13. 根据权利要求1～12任一项所述的微流控芯片，其特征在于，所述芯片主体的材料为不透明材料。
  14. 根据权利要求1～12任一项所述的微流控芯片，其特征在于，在自所述入口端到所述出口端的方向上，所述第一个分杯腔到所述旋转中心的距离与最后一个分杯腔到所述旋转中心的距离之比为1：(1.05～1.2)。
  15. 根据权利要求1～12任一项所述的微流控芯片，其特征在于，各个所述免疫反应腔预装有用于免疫反应的试剂，各个所述免疫反应腔中的用于免疫反应的试剂为固态试剂。
  16. 根据权利要求15所述的微流控芯片，其特征在于，所述固态试剂的制备方法包括以 下的至少一种：减压蒸发干燥、常压干燥、冷冻干燥、真空干燥和气化加湿干燥。
  17. 根据权利要求1～12、16任一项所述的微流控芯片，其特征在于，所述芯片主体还具有血样足量检测腔，所述血样足量检测腔与所述血液废液腔连通且较所述血液废液腔更远离所述旋转中心。
  18. 根据权利要求1～12、16任一项所述的微流控芯片，其特征在于，所述芯片主体还具有试剂足量检测腔，所述试剂足量检测腔与所述分液流道末端连通，所述试剂足量检测腔还与所述反应废液腔连通，所述试剂足量检测腔为非规则形状以防止液体完全排干。
  19. 根据权利要求1～12、16任一项所述的微流控芯片，其特征在于，所述芯片主体还设置有进样腔以及沉降池，所述血浆腔与所述进样腔连通，所述沉降池位于所述血浆腔与所述进样腔之间，所述沉降池通过流道与所述进样腔连通，所述沉降池通过流道与所述血浆腔连通。
  20. 根据权利要求1～12、16任一项所述的微流控芯片，其特征在于，所述混匀腔与所述血浆腔通过第一虹吸流道连通，所述混匀腔与所述分液流道通过所述第二虹吸流道连通，所述第一虹吸流道和所述第二虹吸流道的宽度分别独立地为0.2mm～1.5mm，所述第一虹吸流道和所述第二虹吸流道的深度分别独立地为0.1mm～1mm。
  21. 根据权利要求1～12、16任一项所述的微流控芯片，其特征在于，所述混匀腔与所述免疫反应腔通过第一微流道连通，所述免疫反应腔与所述反应废液腔通过第二微流道连通，所述第一微流道和所述第二微流道的宽度分别独立地为0.2mm～0.7mm，所述第一微流道和所述第二微流道的深度分别独立地为0.01mm～0.1mm，所述第一微流道的长度为1.5mm～2.5mm，所述第二微流道的长度为2.5mm～5.5mm。
  22. 根据权利要求21所述的微流控芯片，其特征在于，所述第一微流道和所述第二微流道均为经过疏水处理的微流道。
  23. 根据权利要求4～12、16和22中任一项所述的微流控芯片，其特征在于，所述芯片主体具有盖合面，所述微流控芯片还包括盖板，所述盖板盖合于所述盖合面上，所述第一类液囊、所述第二类液囊和所述第三类液囊均位于所述盖板的远离所述芯片主体的一侧，所述盖板上设有与所述第一类液囊、所述第二类液囊和所述第三类液囊分别对应的通孔。
  24. 根据权利要求23所述的微流控芯片，其特征在于，所述微流控芯片还包括用于粘合所述盖板与所述芯片主体的粘合层；所述粘合层的厚度为0.03mm～0.2mm。
  25. 根据权利要求1～12、16、22和24中任一项所述的微流控芯片，其特征在于，所述微流控芯片还满足以下条件中的至少一个：
    (1)所述血浆腔与所述血球腔的容量之比为1:(1～5)；
    (2)所述主流道的宽度为0.5mm～3mm，所述主流道的深度为1.5mm～3mm；
    (3)所述芯片主体的厚度为2mm～8mm；
    (4)以所述旋转中心为顶点，所述旋转中心与所述入口端和所述出口端所形成的夹角的大小为0°～359°；
    (5)所述微流控芯片的直径为100mm～140mm；
    (6)所述免疫反应腔的腔底与所述芯片主体的下表面的距离不小于0.3mm。
  26. 根据权利要求1～12、16、22和24中任一项所述的微流控芯片，其特征在于，所述芯片主体上还设置有非全血进样腔，所述非全血进样腔用于添加血清或血浆；所述非全血进样腔与所述混匀腔连通。
  27. 一种检测方法，所述检测方法的步骤包括：
    将全血样本注入全血分离结构分离全血，得到位于血浆腔的血浆；
    将血浆腔中的血浆转移到混匀腔；
    将血浆稀释液引入混匀腔，以稀释混匀腔中的血浆；
    将混匀腔中稀释后的血浆通过分液流道分配至各个免疫反应腔；
    在各个免疫反应腔中，稀释后的血浆与预装的用于免疫反应的试剂反应，以形成抗原抗体复合物；
    在各个免疫反应腔反应结束后，将各个免疫反应腔中的除抗原抗体复合物外的物质转移到反应废液腔；
    将清洗液通过混匀腔引入各个免疫反应腔，以清洗反应结束后各个免疫反应腔中形成的抗原抗体复合物；
    在清洗结束后，将发光底物稀释液经底物试剂腔、混匀腔、分液流道转移至各个免疫反应腔中进行反应，在酶的催化作用下发生化学发光反应。
  28. 根据权利要求27所述的检测方法，其特征在于，在利用全血分离结构分离全血步骤中，离心的转速为1000rpm～5000rpm，离心时间为90s～150s。
  29. 根据权利要求27所述的检测方法，其特征在于，在将混匀腔中稀释后的血浆通过分液流道分配至各个免疫反应腔的步骤包括：采用100rpm～1200rpm的离心转速离心1s～60s将稀释后的血浆分配至各个分杯腔中；及采用1000rpm～2000rpm的离心转速离心1s～60s将各个分杯腔中的血浆分配至各个免疫反应腔。
  30. 根据权利要求27～29任意一项所述的检测方法，其特征在于，在将各个免疫反应腔中的除抗原抗体复合物外的物质转移到反应废液腔的步骤中，离心转速为1000rpm～2000rpm，离心时间为1s～60s。
  31. 根据权利要求27～29任意一项所述的检测方法，其特征在于，在将清洗液通过混匀腔引入各个免疫反应腔的步骤中，离心转速为100rpm～1200rpm，离心时间为1s～60s。
  32. 根据权利要求27～29任意一项所述的检测方法，其特征在于，在将发光底物稀释液经底物试剂腔、混匀腔、分液流道转移至各个免疫反应腔的步骤中，离心转速为100rpm～1200rpm，离心时间为1s～60s。
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CN111495451A (zh) * 2020-04-30 2020-08-07 微纳芯（苏州）科技有限公司 生化指标检测集成芯片及其检测的方法和应用
KR20220046751A (ko) * 2020-10-07 2022-04-15 바이오뱅크 주식회사 생화학검사와 혈액검사와 면역검사 및 유전자분석을 개별 또는 동시에 수행하는 멀티시스템 및 이에 사용되는 멀티디스크
CN112169853A (zh) * 2020-12-01 2021-01-05 南京岚煜生物科技有限公司 一种多功能微流控检测芯片
CN114849797A (zh) * 2021-01-20 2022-08-05 南京岚煜生物科技有限公司 一种基于相变材料封闭试剂的微流控芯片
CN112763701A (zh) * 2021-02-25 2021-05-07 深圳市亚辉龙生物科技股份有限公司 微流控检测芯片及微流控检测方法
CN113699023A (zh) * 2021-08-27 2021-11-26 中元汇吉生物技术股份有限公司 微流控芯片、混匀装置、分子诊断设备及样本检测方法
CN115055287A (zh) * 2022-08-12 2022-09-16 普迈德（北京）科技有限公司 一种离心式微流控芯片、送盘装置以及血浆分离取样方法
CN115254220A (zh) * 2022-09-27 2022-11-01 深圳市卓润生物科技有限公司 微流控芯片及检测方法
CN115591593A (zh) * 2022-10-09 2023-01-13 深圳市卓润生物科技有限公司（Cn） 检测过敏原的微流控芯片及方法

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