WO2020010913A1 - 一种微流控检测芯片及其制备方法、固定装置和离心式检测装置 - Google Patents

一种微流控检测芯片及其制备方法、固定装置和离心式检测装置 Download PDF

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Publication number
WO2020010913A1
WO2020010913A1 PCT/CN2019/086574 CN2019086574W WO2020010913A1 WO 2020010913 A1 WO2020010913 A1 WO 2020010913A1 CN 2019086574 W CN2019086574 W CN 2019086574W WO 2020010913 A1 WO2020010913 A1 WO 2020010913A1
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chip
detection
sample
area
microfluidic
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PCT/CN2019/086574
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English (en)
French (fr)
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方雪恩
吴静
钱江洪
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上海速创诊断产品有限公司
上海速芯生物科技有限公司
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Priority to US16/632,037 priority Critical patent/US11123731B2/en
Priority to EP19834509.2A priority patent/EP3674713B1/en
Publication of WO2020010913A1 publication Critical patent/WO2020010913A1/zh

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    • 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
    • G01N33/6854—Immunoglobulins
    • 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
    • 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
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    • 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
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    • 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
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    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
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    • 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
    • B01L3/502753—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 bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B27/00—Layered products comprising a layer of synthetic resin
    • B32B27/06—Layered products comprising a layer of synthetic resin as the main or only constituent of a layer, which is next to another layer of the same or of a different material
    • B32B27/08—Layered products comprising a layer of synthetic resin as the main or only constituent of a layer, which is next to another layer of the same or of a different material of synthetic resin
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    • B32B3/26—Layered products comprising a layer with external or internal discontinuities or unevennesses, or a layer of non-planar form; Layered products having particular features of form characterised by a particular shape of the outline of the cross-section of a continuous layer; characterised by a layer with cavities or internal voids ; characterised by an apertured layer
    • B32B3/30—Layered products comprising a layer with external or internal discontinuities or unevennesses, or a layer of non-planar form; Layered products having particular features of form characterised by a particular shape of the outline of the cross-section of a continuous layer; characterised by a layer with cavities or internal voids ; characterised by an apertured layer characterised by a layer formed with recesses or projections, e.g. hollows, grooves, protuberances, ribs
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    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/028—Modular arrangements
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    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0684—Venting, avoiding backpressure, avoid gas bubbles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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    • B01L2200/12—Specific details about manufacturing devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01L2300/06—Auxiliary integrated devices, integrated components
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    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
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    • B32B38/10—Removing layers, or parts of layers, mechanically or chemically

Definitions

  • the disclosure belongs to the field of medical detection, and particularly relates to a microfluidic detection chip for detecting immune indicators and a detection device using the same.
  • Immunochromatographic technology has experienced the development from colloidal gold qualitative and semi-quantitative to fluorescent quantitative since its birth.
  • the main method is to use a combination of nitrocellulose membrane, glass fiber membrane and absorbent paper as a mobile substrate to combine different markers.
  • the coated antibody is fixed on it for double antibody sandwich or competitive reaction. Because of its convenient use, fast detection, the use of filters to separate whole blood, and the low requirements for manufacturing equipment and testing equipment are currently widely used in the field of POCT (real-time diagnosis).
  • the microfluidic detection chip developed in the 1990s refers to a scientific and technical system that processes and controls a liquid with a volume of 10 -9 to 10 -8 liters by using channels of tens to hundreds of microns. Many advantages are low consumption, fast analysis speed, automated analysis process, easy integration, and high throughput.
  • the benefits of the microfluidic detection chip for immunoassay are: substrates such as PMMA and PS have strong adsorption force on proteins, and then use the capillary force of the flow channel or apply driving force to guide the flow of liquid, which can easily realize immune chromatography
  • the reaction combined with the detection device, can conveniently carry out quantitative determination of the test substance in the sample.
  • Patent Document 1 discloses a microfluidic chip for fluorescence immunoassay and a preparation method thereof.
  • the microfluidic chip includes a chip substrate, and a microfluidic channel is provided on the substrate.
  • the microfluidic channel includes a sample dropping area and a whole blood filter connected in sequence. Area, antibody coating area, reaction area, detection area, quality control area and waste liquid collection area, and the whole blood filtration area is provided with a red blood cell hemofiltration membrane. During the test, driven by centrifugal force, the whole blood sample is passed through the filter area to remove blood cells and then tested.
  • Patent Document 2 discloses a magnetic particle chemiluminescence microfluidic chip for quantitatively detecting brain natriuretic peptides in whole blood, which is composed of a top tape, a chip substrate, and a bottom tape, wherein a filter region on the chip substrate and a magnetic particle labeled BNP antibody coating region
  • the reaction zone, the washing zone, the detection zone, and the fluid release channel are sequentially connected.
  • antibody-modified magnetic particles are used, and the preparation method is relatively complicated.
  • the detection of the microfluidic chip requires the equipment electromagnet to pull the magnetic particles in the chip, which requires high equipment complexity.
  • the filter area contains hemofiltration membranes, and there are also problems such as detection interference and sample waste.
  • Patent Document 3 discloses a fully automatic microfluidic chip fluorescence immunodetection system, which includes a chip card storage disk, a sample tube disk, and a centrifugation reaction disk.
  • the centrifugation reaction disk has one or more chip card slots, and the size of the chip card slot Matching the chip detection card, the chip detection card is sequentially arranged with a sample loading slot, a fluorescent probe slot, a reaction detection slot and a waste liquid slot, and a microchannel connected to the above slot.
  • Patent Document 4 discloses a stacked three-layer chip structure, the second layer is a double-sided adhesive film, and the microstructure is etched on it, which is easy to operate and has high production efficiency.
  • the disadvantage is that the microstructure on the double-sided adhesive film is not described in detail.
  • no other microstructures are designed on the first layer and the third layer.
  • the chip prepared by this method needs to cooperate with micropump microvalves and special detection equipment. Etc., suitable for some scientific research fields, not suitable for the instant detection of biological samples in clinical.
  • Patent Document 1 CN106807461A
  • Patent Document 2 CN105259162A
  • Patent Document 3 CN207036852U
  • Patent Document 4 CN203663854U.
  • the present disclosure designs a self-driven and short
  • the microfluidic detection chip for immune index detection combined with time centrifugation and its preparation method are simple in chip preparation and assembly, low requirements for detection equipment, and can be conveniently applied to the instant detection of biological samples in clinic.
  • the present disclosure also provides a fixing device based on the microfluidic detection chip to achieve the stability and convenience of multiple chip clamping and fixing.
  • the present disclosure also provides a centrifugal detection device based on the microfluidic detection chip.
  • the present disclosure includes the following technical solutions:
  • a microfluidic detection chip the detection chip includes three layers stacked, the upper layer of the chip includes a sample area and a vent hole; the lower layer of the chip is provided with a slope structure or a groove; the middle layer of the chip is a double-sided adhesive layer
  • a sample flow channel is separated on the double-sided adhesive layer by an adhesive region and a non-adhesive region, and the sample flow channel includes a sample tank region, a flow channel detection region, and a waste liquid tank region, wherein the sample tank region and the The sample loading area on the upper layer of the chip corresponds, the waste liquid tank area covers at least the slope structure or groove of the lower layer of the chip, and the flow channel detection area is arc-shaped.
  • the microfluidic detection chip according to [1] is also provided with a gradient structure, the included angle of the gradient structure is 15 ° -45, and one or more of the gradient structures are distributed on the gradient structure Cylindrical raised drainage points.
  • the material of the upper layer and the lower layer of the chip is selected from polystyrene (PS), polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), glass or polycarbonate (PC); preferably, the upper and lower layers of the chip
  • the material is selected from polymethyl methacrylate (PMMA), polystyrene (PS) or polycarbonate (PC);
  • the chip intermediate layer is polyethylene terephthalate (PET) glue or polymethyl Methyl acrylate (PMMA) glue.
  • the thickness of the upper layer of the chip and the lower layer of the chip are 1.5-2.5mm, and the thickness of the middle layer of the chip is 0.05-0.5mm.
  • the method for preparing a microfluidic detection chip according to any one of the technical solutions [1] to [6], which at least includes 1) etching a sample fluid channel on the double-sided adhesive layer by a laser, 2) The peeling layer on the side of the double-sided adhesive layer is peeled off, and it is pasted on the surface of the lower layer of the chip; 3) spotting in the sample fluid flow channel, and drying, sticking the upper layer of the chip and pressing.
  • a fixing device for a microfluidic detection chip characterized in that the fixing device is used to simultaneously fix a plurality of microfluidic detection chips according to any one of the technical solutions [1] to [6],
  • the fixing device includes a central fixing plate and a rotating tray.
  • the rotating tray is divided into a plurality of microfluidic detection chips according to any one of the technical solutions [1]-[6] by the central fixing plate above it.
  • the fixing device mainly uses each side of the central fixing plate to fix a plurality of microfluidic detection chips according to any one of the technical solutions [1] to [6] on the rotating tray.
  • a centrifugal detection device which includes at least the following modules:
  • the microfluidic module includes the microfluidic detection chip according to any one of the technical solutions [1] to [6];
  • a fixing module including the fixing device described in [8] or [9];
  • a rotation module connected to the fixing device for driving the fixing device to rotate
  • a detection module is configured to detect a flow channel detection area of the microfluidic detection chip through a light source transmission area of the fixing device, and output a detection result.
  • the disclosure provides a microfluidic detection chip combining self-drive and short-time centrifugation, which has the advantages of: 1) no whole blood filtering step, avoiding interference and sample waste caused by the separation of the whole blood filter membrane, and the whole blood sample directly Participate in the reaction and use the sample more efficiently; 2)
  • the combination of self-driven and short-time centrifugation can well control the fluid flow rate, which is conducive to the chromatographic reaction.
  • the capillary action and centrifugal action are used to drive the sample to flow in the fluid channel. Residual liquid in the detection area of the main flow channel, and a groove is provided in the lower layer of the chip to facilitate collection of waste liquid during centrifugation.
  • This design reduces the non-specific binding and detection background to a certain extent, and improves detection sensitivity; Design and fix more fluorescent microspheres and antibodies to improve detection sensitivity; 4) Chip preparation and assembly are simpler, microfluidic channels are set on the double-sided adhesive layer, and micro-layers are also designed on the first and third layers. Structure, so there is no need to cooperate with micro-pumps and micro-valves, etc., and the requirements for detection equipment are small, and it can be easily applied to a single item of immunology or more Head measurement.
  • the fixing device of the microfluidic detection chip of the present disclosure ensures the centrifugal deviation of the chip under high-speed centrifugation through the limit of the central fixing plate.
  • the curved design on the side fully considers the possibility of taking the chip by hand. Operability and comfort.
  • the centrifugal detection device based on the microfluidic detection chip has the advantages of convenient assembly, simple operation, and high detection sensitivity, and is suitable for large, medium and small medical institutions, emergency departments, community hospitals, family detection and scientific research.
  • FIG. 1 is an external view of a first embodiment of a microfluidic detection chip provided by the present disclosure.
  • FIG. 2 is an upper structure diagram of a first embodiment of a microfluidic detection chip provided by the present disclosure.
  • FIG. 3 is a lower structure diagram of a first embodiment of a microfluidic detection chip provided by the present disclosure.
  • FIG. 4 is a structural diagram of an intermediate layer of a first embodiment of a microfluidic detection chip provided by the present disclosure.
  • FIG. 5 is a schematic diagram of a reagent fixing region of a first embodiment of a microfluidic detection chip provided by the present disclosure.
  • FIG. 6 is a schematic diagram of a first embodiment of a microfluidic detection chip provided by the present disclosure fixed to a fixed device of the present disclosure and detected.
  • FIG. 7 is an external view of a second embodiment of a microfluidic detection chip provided by the present disclosure.
  • FIG. 8 is a top structural diagram of a second embodiment of a microfluidic detection chip provided by the present disclosure.
  • FIG. 9 is a lower-level structure diagram of a second embodiment of a microfluidic detection chip provided by the present disclosure.
  • FIG. 10 is a structural diagram of an intermediate layer of a second embodiment of a microfluidic detection chip provided by the present disclosure.
  • FIG. 11 is a schematic diagram of a reagent fixing area of a second embodiment or a third embodiment of a microfluidic detection chip provided by the present disclosure.
  • FIG. 12 is a schematic diagram of a second embodiment or a third embodiment of a microfluidic detection chip provided in the present disclosure fixed to a fixed device of the present disclosure and detected.
  • FIG. 13 is a schematic assembly view of a fourth embodiment of a fixing device for a microfluidic detection chip provided by the present disclosure.
  • FIG. 14 is a schematic diagram of a positioning chute fit of a fourth embodiment of a fixing device for a microfluidic detection chip provided by the present disclosure.
  • the microfluidic detection chip includes three layers stacked: the upper layer of the chip, the lower layer of the chip, and the middle layer of the chip.
  • the shape of the chip can be oval-like, square, rectangular, polygonal, or circular. Preferably, the chip is oval-like to achieve better grip.
  • the thickness of the upper and lower layers of the chip are both 1.5 to 2.5 mm. If the thickness is too thin, the amount of sample loaded on the chip is too small and it is easy to deform. If the thickness is too thick, the light transmission will be affected, affecting the detection results, and it will not meet the chip small size. Demand.
  • the thickness of the chip intermediate layer is 0.05 to 0.5 mm. The upper layer and the lower layer of the chip are adhered and fixed by the middle layer or the middle layer and the buckle.
  • the material of the upper layer and the lower layer of the chip is selected from the group consisting of polystyrene, polydimethylsiloxane, polymethyl methacrylate, polyethylene terephthalate, glass or polycarbonate;
  • the material of the upper layer and the lower layer of the chip is selected from polymethyl methacrylate, polystyrene or polycarbonate;
  • the middle layer of the chip is polyethylene terephthalate glue or polymethyl methacrylate gum.
  • the upper layer of the chip is mainly used to introduce the sample into the detection area.
  • the upper layer of the chip contains a sample adding area, and a sample adding hole is provided on the sample adding area for adding a sample.
  • the sample adding hole can be designed to be a round shape that can closely match the standard pipette tip of a conventional experiment in biological experiments.
  • the diameter can be between 2mm and 3mm, and the sample added through the sampling hole can flow along the sample flow channel. Due to the combination of gravity and capillary action self-driving and short-term centrifugation adopted in the present disclosure, there is no need to fix a whole blood filtering device such as a whole blood filtration membrane in the sample adding area of the present disclosure, which can reduce sample waste and improve sample utilization efficiency.
  • the shape of the sample application area corresponds to the shape of the sample slot area of the chip middle layer.
  • the sample application area is an irregular fan.
  • the height is 0.2-0.6mm, the distance between drainage points is 1-5mm, and the number of drainage points is 3-10. It plays the role of storing samples and drainage.
  • the upper layer of the chip also includes vent holes, that is, vent holes in the sample addition area and vent holes in the waste liquid pool.
  • the vent hole is a through hole communicating with the atmosphere, preferably a circular through hole to improve the fluidity of the sample solution, and the diameter can be in the range of 0.5 to 2.0 mm.
  • a sample application area vent hole and a waste liquid tank vent hole are provided on the upper layer of the chip.
  • card posts are provided on the upper layer of the chip.
  • the number of card posts can be set according to actual needs, and 2-4 can be provided. For better assembly adhesion, it is further preferable to set 4 card posts.
  • a slope structure or a groove is provided on the lower layer of the chip to guide and store the waste liquid.
  • the included angle of the gradient structure is 15 ° -45 °, and several cylindrical convex drainage points are distributed in the gradient structure for drainage of waste liquid, and the diameter of the drainage points is 0.2-0.6mm.
  • the height is 0.2-0.6mm, the spacing of drainage points is 1-5mm, and the number of drainage points is 3-10.
  • a groove is provided on the lower layer of the chip to more conveniently collect the residual waste liquid thrown out of the flow channel detection area during centrifugation, and the groove corresponds to the lower half of the waste liquid tank area of the chip middle layer, Shaped, depth is 1-2mm.
  • the lower layer of the chip further includes a drainage groove at a position corresponding to the sample loading area of the upper layer of the chip.
  • the drainage groove is a channel with a width of 0.2-0.6mm and a depth of 0.1-0.5mm, in order to guide the sample into the flow channel Detection area.
  • the lower layer of the chip may also include a certain number of card post holes, which correspond to the card posts of the upper layer of the chip to strengthen the adhesiveness of the three layers of the chip.
  • the middle layer of the chip is a double-sided adhesive layer, and a sample flow channel is separated by an adhesive region and an adhesive-free region on the double-sided adhesive layer.
  • the sample flow channel includes a sample tank region, a flow channel detection region, and a waste liquid tank region.
  • the sample slot area corresponds to the sample addition area on the upper layer of the chip, and the shape of the sample slot area may be the same as the sample addition area on the chip.
  • the main role of the waste liquid tank area is to store waste liquid, which is equivalent to a waste liquid pool.
  • the shape of the waste liquid tank area at least completely covers the slope of the lower layer of the chip.
  • the waste liquid tank area may be the same as or larger than the size of the slope structure or groove of the lower layer of the chip.
  • the flow channel detection area is arc-shaped, and the bending radius of the flow channel detection area is 25-35mm (radius centered on the centrifugal axis of the rotating tray of the fixed device as the center of the circle), the arc is in the range of 1.8-2.2rad, and the width is 2 -4mm.
  • the surface of the fluid channel of the sample is fixed with fluorescent microsphere-antibody markers and antibodies for immunological detection, and increasing the number of fluorescent microsphere-antibody markers and antibodies can further improve detection sensitivity.
  • the chip middle layer may also include a certain number of card post holes, corresponding to the card post on the chip to reinforce the adhesion of the three layers of the chip.
  • the method for preparing a microfluidic detection chip according to the present disclosure includes at least:
  • a laser engraving machine can be used to etch the double-sided adhesive layer.
  • traditional microfluidic detection chips use a lithography machine to etch the channel structure on the chip layer such as PDMS or PMMA.
  • the lithography machine is expensive and the light source is contaminated during the lithography process.
  • the upper etching channel structure can improve production efficiency, reduce production costs and reduce environmental pollution during preparation;
  • an antibody-coated fluorescent microsphere spotting area is set at the intersection of the flow channel detection area and the sample slot area, and the second spotting area of the detection antibody and the first detection antibody are set in order along the direction of sample flow.
  • Spotting area and quality control antibody spotting area The quality control antibody spotting area is set up to reduce variation between batches and / or batches of products and improve product quality.
  • microfluidic detection chip of the present disclosure When the microfluidic detection chip of the present disclosure is used, 100-300 ⁇ l of fresh blood can be directly dripped into the sample well, and the sample flows in the sample flow channel under the action of gravity and capillary action, and is first captured with the coating.
  • the fluorescent microspheres of the antibody are mixed and then react with the detection antibody and the polyclonal antibody to generate a fluorescent signal.
  • 10-15 minutes after the sample is added to the sample well the sample can react and flow into the waste liquid tank.
  • the microfluidic detection chip is placed on the detection device and fixed. In the present disclosure, multiple chips can be fixed on the same fixing device, a centrifugal force is applied for 1-3 minutes, and the rotation speed is in the range of 2000-6000rpm. The purpose is to dry the flow. Residual waste liquid in the test area. Then read the fluorescence intensity of the spot of the control antibody spot and the spot of the antibody spot in the detection area, and then fit the calibration curve by the detection standard to calculate the
  • the present disclosure Since the microfluidic detection chip of the present disclosure needs to work under high-speed centrifugation, in order to solve the problem of clamping and fixing of the microfluidic detection chip under centrifugal force, the present disclosure also designs a microfluidic detection chip that cooperates with the present disclosure. Used fixtures.
  • the fixing device is used for simultaneously fixing a plurality of microfluidic detection chips according to the present disclosure.
  • the fixing device includes a central fixing plate and a rotating tray.
  • the rotating tray is divided into the central fixing plate above the rotating tray.
  • a plurality of regions matching the shape of the microfluidic detection chip according to the present disclosure, and the fixing device mainly uses a plurality of sides of the central fixing plate to respectively fix a plurality of microfluidic detection chips according to the present disclosure on the
  • the rotating tray has a light source transmission area, and the light source transmission area is coaxial with the flow channel detection area of the microfluidic detection chip according to the present disclosure.
  • the light transmission area is arc-shaped.
  • the center fixing plate and the rotating tray can be fixed with screws.
  • the present disclosure is provided with a positioning chute on the side of the central fixing plate, the positioning chute is uniformly arranged along the side of the central fixing plate, and the side of the microfluidic detection chip of the disclosure
  • the chute forms a clamping structure with the positioning chute.
  • each microfluidic detection chip cooperates with two positioning chute to prevent circumferential movement between the microfluidic detection chip and the central fixed disk.
  • a recessed structure corresponding to the microfluidic detection chip may be formed on the side of the central fixed plate with an elastic ball plunger.
  • Flexible limit assembly corresponding to each microfluidic detection chip, generally two flexible ball plungers can be set.
  • the fixing device can simultaneously fix 3 microfluidic detection chips of the present disclosure at a time, and the central fixing plate has 3 curved side surfaces, and each curved side surface is provided.
  • the two sides of the outer side of the rotating tray can be set to be arc-shaped to match the arcuate structure on both sides of the microfluidic detection chip. The operability and comfort of the chip.
  • a foot post hole may be provided correspondingly in the rotating tray, which is beneficial to the firmness of the chip on the tray.
  • the present disclosure also provides a centrifugal detection device based on the microfluidic detection chip of the present disclosure, which includes at least the following modules:
  • a microfluidic module including the microfluidic detection chip described in the present disclosure
  • a fixing module including the fixing device according to the present disclosure for fixing a plurality of microfluidic detection chips
  • a rotation module is connected to the fixing device and is used to drive the rotation of the fixing device to achieve high-speed centrifugation of the microfluidic detection chip.
  • the rotation module includes a stepper motor, which may be a high-speed A micro stepping motor; and a detection module, configured to detect a flow channel detection area of the microfluidic detection chip through a light source through hole of the fixing device, and output a detection result.
  • the detection module includes:
  • a light propagation unit configured to propagate the first light to the light source transmission region; when the rotation module drives the flow channel reaction region of the microfluidic detection chip to rotate to the light source transmission region Above the passing zone, when the first light illuminates a sample in the flow channel reaction zone through the light source transmission zone, the light propagation unit propagates a second light emitted by the sample excited by the first light;
  • a detection unit configured to detect the second light and generate a first signal according to the second light, the first signal is an electrical signal, and the first signal is used to analyze the sample, and Using the first signal as the detection result.
  • the centrifugal detection device further includes a temperature control module and an analysis module.
  • the temperature control module is used to control the temperature of the flow channel detection area.
  • the analysis module is configured to receive the detection result, analyze the sample according to the detection result, and output the analysis result.
  • the centrifugal detection device of the present disclosure can realize single-item or multi-item determination of immunology with higher precision and detection sensitivity than traditional paper substrate immunochromatographic methods.
  • paper substrate immunochromatographic products usually have an analytical sensitivity of 0.2ng / mL, and this method can reach 0.05ng / mL.
  • the intra- and inter-assay precision of the immunoassay reagent prepared by this method is less than 10%, while the traditional method is 15% -20%.
  • This embodiment provides a microfluidic detection chip for detecting the content of procalcitonin in whole blood.
  • FIG. 1 shows an external view of a microfluidic detection chip of this embodiment. It can be known from FIGS. 1-4 that the shape of the microfluidic detection chip 1 is oval-like, and the maximum size of the length, width, and height is 55mm ⁇ 35mm ⁇ 4mm.
  • the chip 1 includes three layers stacked: an upper layer 100 of the chip, a lower layer 200 of the chip, and an intermediate layer 300 of the chip.
  • the material of the upper layer 100 of the chip is PMMA, and the shape is oval-like.
  • the thickness of the upper layer 100 is 2.0 mm.
  • the structure of the upper layer 100 of the chip shown in FIG. 2 shows that the upper layer 100 of the chip includes a sample application area 101, and a sample application hole 102 is provided in the sample application area 101 for adding a sample.
  • the diameter of the sample application hole 102 is 4 mm.
  • the shape of the sample application area 101 is an irregular fan shape.
  • the upper layer 100 of the chip further includes two air vents, namely, the air vent 103 of the sample application area and the air vent 104 of the waste liquid pool shown in FIG. 1.
  • the vent hole is a circular through hole communicating with the atmosphere to improve the fluidity of the sample solution, and the diameter is 1.0 mm.
  • the chip upper layer 100 is provided with four card posts 105.
  • the material of the lower chip layer 200 is PMMA, and the shape matches the upper layer of the chip.
  • the thickness of the lower chip layer 200 is 2.0 mm.
  • the structure of the lower layer 200 of the chip shown in FIG. 3 includes a groove 201 for collecting the residual waste liquid during centrifugation, which is irregular in shape, and the maximum length, width, and depth are 32 mm ⁇ 3.3 mm ⁇ 1.5 mm.
  • the lower layer of the chip further includes a card post hole 202 on the lower layer of the chip.
  • the middle layer of the chip is a double-sided adhesive layer, the material is PET adhesive, and the thickness is 0.05mm.
  • the structure of the chip intermediate layer 300 shown in FIG. 4, the sample flow channel (ie, the non-adhesive region 302) is separated by an adhesive region 301 (dark region) and an adhesive-free region 302 (white region) on the double-sided adhesive layer.
  • the sample flow channel can be further subdivided into three areas: a sample tank area 303, a flow channel detection area 304, and a waste liquid tank area 305.
  • the sample tank area 303 has the same shape as the sample addition area 101 on the upper layer of the chip.
  • the waste liquid tank area 305 completely covers the recess 201 of the lower layer of the chip, and its area is larger than the area of the recess.
  • the width of the flow channel detection area is 3 mm and the length is 30 mm.
  • the bending radius of the flow channel detection area is 32 mm and the radian is 2.09 rad.
  • the chip intermediate layer further includes a card post hole 306 in the chip intermediate layer.
  • the detection chip When preparing the detection chip, first remove the adhesive film on the side of the chip intermediate layer 300 and stick it on the lower layer 200. As shown in FIG. 5, 0.05 ng of the quality control antibody spot area 5 in the exposed sample fluid channel is added. For goat anti-mouse polyclonal antibody, add 0.02 ng of procalcitonin detection antibody to the first spot 6 of the detection antibody, and add 0.02 ng of procalcitonin capture antibody to the fluorescent microsphere spot area of the coated antibody. Fluorescent microspheres with a solid content of 0.01%. After drying at 37 ° C for 4 hours, the upper cover of the chip was stuck and pressed.
  • the microfluidic detection chip 1 is placed on a fixing device, as shown in FIG. 6, three microfluidic detection chips can be fixed on the fixing device 2, and a centrifugal force of 5000 rpm is applied to the centrifugal axis 3 for 1 minute, and then read Take the detection area and the fluorescence intensity of the antibody spot area.
  • the content of procalcitonin in the sample can be calculated. This method can be used to detect the procalcitonin in whole blood. It only takes 10-15 minutes from the sample loading to the reading.
  • the analysis sensitivity It can reach 0.05ng / mL, and the detection range is 0.05ng / mL-100ng / mL, which is close to the detection performance of chemiluminescence method.
  • This embodiment provides another structure of a microfluidic detection chip for detecting the content of procalcitonin in whole blood.
  • FIG. 7 shows an external view of the microfluidic detection chip of this embodiment.
  • the shape of the microfluidic detection chip is oval-like, and the maximum size of the length, width, and height is 55mm ⁇ 35mm ⁇ 4mm.
  • Chip 1 includes three stacked layers: the upper layer 100, the lower layer 200, and the chip. Middle layer 300.
  • the material of the upper layer 100 of the chip is PMMA, the shape is oval-like, and the thickness of the upper layer 100 is 2.0 mm.
  • the structure of the upper layer 100 of the chip shown in FIG. 8 shows that the upper layer 100 of the chip includes a sample application area 101, and a sample application hole 102 is provided in the sample application area 101 for adding a sample.
  • the diameter of the sample application hole 102 is 3 mm.
  • the sample adding area 101 has a slope with an angle of 25 °, and a plurality of cylindrical convex drainage points 106 having a diameter of 0.4 mm and a height of 0.4 mm are distributed thereon, and the spacing between the drainage points is 4 mm, which functions to drain the sample.
  • the shape of the sample application area 101 is an irregular fan shape.
  • the upper layer 100 of the chip further includes two air vents, namely, the air vent 103 of the sample application area and the air vent 104 of the waste liquid tank shown in FIG. 8.
  • the vent hole is a circular through hole communicating with the atmosphere to improve the fluidity of the sample solution, and the diameter is 1.0 mm.
  • the material of the lower chip layer 200 is PMMA, and the shape matches the upper layer of the chip.
  • the thickness of the lower chip layer 200 is 2.0 mm.
  • the structure of the lower layer 200 of the chip shown in FIG. 9 includes a clip structure 203 with a slope of 20 °, which is shaped, with a maximum length of 41mm and a width of 8mm. There are several 0.4mm in diameter and 0.4mm in height on the gradient structure 203. Cylindrical convex drainage points 106 with a distance of 4 mm between the drainage points play a role in draining the sample.
  • the lower chip layer 200 is provided with a drainage groove 204 at a position corresponding to the sample adding area 101 of the upper chip layer 100.
  • the drainage groove has a width of 0.4 mm and a depth of 0.1 mm. The purpose is to guide the sample into the sample fluid channel.
  • the middle layer of the chip is a double-sided adhesive layer that bonds the upper layer of the chip and the lower layer of the chip and forms a fluid channel.
  • the material is PET glue and the thickness is 0.05mm.
  • FIG. 10 shows the structure of the chip intermediate layer 300.
  • the chip intermediate layer is a double-sided adhesive layer, and a sample flow channel is separated by an adhesive region 301 and an adhesive-free region 302 on the double-sided adhesive layer.
  • the sample flow channel includes The sample tank region 303, the flow channel detection region 304, and the waste liquid tank region 305, wherein the sample tank region 303 corresponds to the sample loading region 101 on the upper layer of the chip, and the waste liquid tank region 305 is on the chip lower layer.
  • the waste liquid tank area 305 can completely cover the gradient structure 203 below the chip, and its area is slightly larger than the area of the gradient structure.
  • the width of the flow channel detection area is 3 mm
  • the bending radius of the flow channel detection area is 32 mm
  • the radian is 2.09 rad.
  • the detection chip When preparing the detection chip, first remove the adhesive film on the side of the chip intermediate layer 300 and stick it on the lower layer 200. As shown in FIG. 11, add 0.05 ng of the quality control antibody spot area 5 in the exposed sample fluid channel. For goat anti-mouse polyclonal antibody, add 0.02 ng of procalcitonin detection antibody to the first spot 6 of the detection antibody, and add 0.02 ng of procalcitonin capture antibody to the fluorescent microsphere spot area of the coated antibody. Fluorescent microspheres with a solid content of 0.01%. After drying at 37 ° C for 4 hours, the upper cover of the chip was stuck and pressed.
  • the sample reaction ended and flowed into the waste liquid pool 201.
  • the procalcitonin content in the sample can be calculated, and the detection performance is the same as that in the first embodiment.
  • the structure of the microfluidic detection chip in this embodiment is the same as that in the second embodiment, and is used to simultaneously detect the content of procalcitonin and C-reactive protein in whole blood.
  • the preparation steps of the microfluidic detection chip are basically the same as those in the second embodiment, and only the substance added in the spot area is changed. As shown in Figure 11, spotting in the sample fluid channel, adding 0.05ng sheep anti-mouse polyclonal antibody to the quality control antibody spotting area 5 and 0.02ng procalcitonin to the first spotting area 6 of the detection antibody.
  • the sample reaction ended and flowed into the waste liquid tank 201.
  • the sensitivity of this method to detect procalcitonin can reach 0.05ng / mL, and the detection range is 0.05-100ng / mL.
  • the sensitivity of C-reactive protein detection is 0.5mg / L, and the detection range is 0.5-100mg / L.
  • This embodiment provides a fixing device for a microfluidic detection chip, for clamping and fixing the microfluidic detection chip according to the first embodiment.
  • the fixing device 2 includes a rotating tray 401 and a central fixing plate 402.
  • the rotating tray 401 is coaxial with the central fixing plate 402, and its axis is a centrifugal axis 3.
  • the rotating tray 401 is positioned above it.
  • the central fixed plate 402 is divided into three regions that match the shape of the microfluidic detection chip described in the present disclosure.
  • the rotating tray has a light source transmission region 404, and the light source transmission region 404 is in accordance with the present disclosure.
  • the flow channel detection area 304 of the flow control detection chip is coaxial and is an arc-shaped through hole.
  • the rotating tray 401 is provided with a foot post hole 403 corresponding to the foot post of the chip lower layer of the microfluidic detection chip.
  • the present disclosure is provided with a positioning chute 405 on the side of the central fixing plate 402, and the positioning chute 405 is evenly arranged along the side of the central fixing plate.
  • the side of the microfluidic detection chip 1 of the present disclosure is provided with a chute 8 to form a clamping structure with the positioning chute 405.
  • each microfluidic detection chip and two positioning chute 405 Cooperate to prevent circumferential movement between the microfluidic detection chip and the central fixed disk.

Abstract

一种微流控检测芯片（1）及其制备方法、固定装置（2）和离心式检测装置，检测芯片（1）包含叠置的三层，芯片上层（100）包含加样区（101）和通气孔（103，104）；芯片下层（200）包括废液池（201），废液池（201）内设有坡度结构（203）或凹槽（201）；芯片中间层（300）为双面胶层，在双面胶层（300）上用有胶区（301）和无胶区（302）分隔出样本流动通道；采用自驱动和短时离心相结合的微流控检测芯片（1）的技术，可以解决传统纸基材的固有问题，进一步提高免疫检测中样本利用率以及检测速度和检测灵敏度，微流控检测芯片（1）制备和组装简单，对检测设备要求低，能方便地应用于临床检验。

Description

一种微流控检测芯片及其制备方法、固定装置和离心式检测装置 技术领域
本公开属于医疗检测领域，尤其涉及一种免疫指标检测微流控检测芯片及其应用其的检测装置。
背景技术
免疫层析技术自诞生以来，已经历了由胶体金定性和半定量到荧光定量的发展，其主要方法是以硝酸纤维素膜、玻璃纤维膜和吸水纸组合为流动基材，将不同标记物包被抗体固定其上进行双抗体夹心或者竞争性反应。因其使用方便、检测快速、可以直接使用滤膜对全血进行分离，且对制造设备和检测设备要求较低等特点目前在POCT(即时诊断)领域应用较广。
然而这种纸质基材制备的试剂条难以克服其固有缺陷，主要来自于纸质基材本身对免疫反应的影响，硝酸纤维素膜对蛋白固定能力有限、手工组装步骤多等因素，普遍存在批内和批间精密度较大、灵敏度较低等缺陷，导致在实际临床检验使用中存在问题。尤其不适用在一些临床对灵敏度要求较高的检测项目，如：降钙素原(PCT)、N末端脑钠肽前体(NT-proBNP)、肌钙蛋白I(cTnI)等。
20世纪90年代发展起来的微流控检测芯片是指利用几十到几百微米的沟道处理和控制10 -9～10 -8升体积的液体的科学与技术系统，其具有体积小、试剂消耗少、分析速度快、分析过程自动化、易于集成化以及高通量等许多优点。微流控检测芯片对于免疫检测的益处有：PMMA和PS等基材本身对蛋白具有较强吸附力，再利用流道毛细管作用力或施加驱动力，引导液体流动，可以方便的实现免疫层析反应，再结合检测设备可以方便的对样本中待测物质进行定量测定。
专利文献1公开一种用于荧光免疫检测的微流控芯片及其制备方法，其包括芯片基板，基板上开设微流控通道，微流控通道包括依次连通的样本滴加区、全血过滤区、抗体包被区、反应区、检测区、质控区和废液收集区，全血过滤区设有红细胞滤血膜。检测时，在离心力驱动下，全血样本经过过滤区去除血细胞随后进行检测。该方案一方面全血过滤膜分离时会带来检测干扰和样本浪费，另一方面蛇形管道状的反应区很容易残留样本，对检测灵敏度产生不利的影响。此外，直接在基板上开设通道的制备方法加工成本较高，且单纯用离心力控制液体可能液体流速过快，不利于层析反应的进行。
专利文献2公开定量检测全血中脑钠肽的磁微粒化学发光微流控芯片，其由顶部胶带、芯片基板和底部胶带构成，其中芯片基板上的过滤区、磁颗粒标记BNP抗体包被区、反应区、清洗区、检测区、流体释放通道依次连接。该方法中采用抗体修饰的磁颗粒，制备方法相对复杂，微流控芯片在检测中需要设备电磁铁牵引芯片中磁颗粒运动，对设备复杂性要求高。此外，过滤区包含滤血膜，也会存在检测干扰和样本浪费等问题。
专利文献3公开了一种全自动微流控芯片荧光免疫检测系统，其包括芯片卡存储盘、样品管盘和离心反应盘，离心反应盘有一个或多个芯片卡槽，芯片卡槽的大小与芯片检测卡相匹配，芯片检测卡依次排列有加样槽、荧光探针槽、反应检测槽和废液槽以及连接上述槽的微通道，通过该系统可以从根本上实现全自动化、多样本、高通量、快速检测的要求。但是该系统组装相对复杂，芯片检测卡装夹并固定到离心反应盘芯片卡槽过程的可靠性和便捷性还有待提高。
专利文献4公开了一种叠置的三层芯片结构，第二层为双面粘覆薄膜，上刻蚀有微结构，操作简单、生产效率高。不足之处在于并未具体描述双面粘覆薄膜上的微结构如何设置，此外第一层和第三层上没有设计其他微结构，该方法制备的芯片需要配合微泵微阀和特殊检测设备等，适用于某些科学研究领域，不适合临床对生物样本的即时检测。
专利文献1：CN106807461A
专利文献2：CN105259162A
专利文献3：CN207036852U
专利文献4：CN203663854U。
发明内容
发明要解决的问题
为了解决传统纸基材的固有问题以及上述现有技术中所存在的问题，利用微流控技术进一步提高免疫检测中样本利用率以及检测速度和检测灵敏度，本公开设计了一种自驱动和短时离心相结合的免疫指标检测用微流控检测芯片及其制备方法，芯片制备和组装简单，对检测设备要求低，能方便地应用于临床对生物样本的即时检测。
本公开还提供基于该微流控检测芯片的固定装置以实现多个芯片装夹和固定的稳定性和便捷性，此外，本公开也提供了基于该微流控检测芯片的离心式检测装置。
用于解决问题的方案
本发明人为了解决上述问题经过努力研究，设计出一种微流控检测芯片，具体而言， 本公开包括如下技术方案：
[1]一种微流控检测芯片，所述检测芯片包含叠置的三层，芯片上层包含加样区和通气孔；芯片下层设有坡度结构或凹槽；芯片中间层为双面胶层，在所述双面胶层上用有胶区和无胶区分隔出样本流动通道，所述样本流动通道包括样本槽区、流道检测区、废液槽区，其中所述样本槽区与所述芯片上层的加样区相对应，所述废液槽区至少覆盖所述芯片下层的坡度结构或凹槽，所述流道检测区为弧形。
[2]根据[1]所述的微流控检测芯片，所述加样区也设有坡度结构，所述坡度结构的夹角为15°-45，所述坡度结构上分布有一个或多个圆柱形凸起状引流点。
[3]根据[1]或[2]所述的微流控检测芯片，所述芯片下层还包括与所述加样区对应的引流槽。
[4]根据[1]-[3]任一技术方案所述的微流控检测芯片，所述芯片上层和芯片下层的材料选自聚苯乙烯(PS)、聚二甲基硅氧烷(PDMS)、聚甲基丙烯酸甲酯(PMMA)、聚对苯二甲酸乙二醇酯(PET)、玻璃或聚碳酸酯(PC)中的一种；优选的，所述芯片上层和芯片下层的材料选自聚甲基丙烯酸甲酯(PMMA)、聚苯乙烯(PS)或聚碳酸酯(PC)；所述芯片中间层为聚对苯二甲酸乙二醇酯(PET)胶或聚甲基丙烯酸甲酯(PMMA)胶。
[5]根据[1]或[4]任一技术方案所述的微流控检测芯片，所述凹槽对应于所述废液槽区的下半部分，深度为1-2mm。
[6]根据[1]-[5]任一技术方案所述的微流控检测芯片，所述芯片上层和所述芯片下层的厚度均为1.5-2.5mm，所述芯片中间层的厚度为0.05-0.5mm。
[7]根据[1]-[6]任一技术方案所述的微流控检测芯片的制备方法，该方法至少包括1)通过激光在所述双面胶层刻蚀样本流体通道，2)将所述双面胶层一侧的剥离层撕下，并将其粘贴在芯片下层表面的上方，3)在样本流体流道内进行点样，干燥后粘上芯片上层，压紧。
[8]一种微流控检测芯片的固定装置，其特征在于，所述固定装置用以同时固定多个如[1]-[6]任一技术方案所述的微流控检测芯片，所述固定装置包括一个中央固定盘和一个旋转托盘，所述旋转托盘被其上方的所述中央固定盘划分为多个如[1]-[6]任一技术方案所述的微流控检测芯片形状匹配的区域，所述固定装置主要利用所述中央固定盘的各个侧面分别将多个如[1]-[6]任一技术方案所述的微流控检测芯片固定在所述旋转托盘上，所述旋转托盘内具有光源透过区，所述光源透过区与[1]-[6]任一技术方案所述的微流控检测 芯片的流道检测区同轴心。
[9]一种如[8]所述的微流控检测芯片的固定装置，所述中央固定盘的侧面设置有定位斜槽，所述定位斜槽沿所述中央固定盘的侧面均匀设置。
[10]一种离心式检测装置，所述离心式检测装置至少包括以下模块：
微流控模块，包括[1]-[6]任一技术方案所述的微流控检测芯片；
固定模块，包括[8]或[9]所述的固定装置；
转动模块，连接于所述固定装置，用于带动所述固定装置转动；及
检测模块，用于通过所述固定装置的光源透过区对所述微流控检测芯片的流道检测区进行检测，并输出检测结果。
发明的效果
本公开提供一种自驱动和短时离心相结合的微流控检测芯片，其优点包括：1)没有全血过滤步骤，避免全血过滤膜分离中产生的干扰和样本浪费，全血样本直接参与反应，对样本利用效率更高；2)自驱动和短时离心结合可以很好地控制流体流速，利于层析反应的进行，通过毛细管作用和离心作用驱动样本在流体通道流动，利用离心甩干流道检测区内残余液体，并且在芯片下层设置凹槽便于收集离心时的废液，该设计从一定程度上降低了非特异性结合和检测本底，提高了检测灵敏度；3)通道表面可以通过设计固定更多的荧光微球和抗体，提高了检测灵敏度；4)芯片制备和组装较简单，在双面胶层上设置微流体通道的同时在第一层和第三层上也设计有微结构，这样就无需配合微泵微阀等，且对检测设备要求较小，可以非常方便的应用于免疫学单项目或多项目测定。
本公开的微流控检测芯片的固定装置通过中央固定盘的限位，保证了芯片在高速离心的状态下不会发生离心偏离的现象，侧面的弧形设计充分考虑了人手取拿芯片的可操作性和舒适度。
本公开基于微流控检测芯片的离心式检测装置，组装方便，操作简易，检测灵敏度高，适用于大中小型医疗机构、急诊科、社区医院、家庭检测和科学研究等。
附图说明
图1：本公开提供的微流控检测芯片的第一实施例的外形图。
图2：本公开提供的微流控检测芯片的第一实施例的上层结构图。
图3：本公开提供的微流控检测芯片的第一实施例的下层结构图。
图4：本公开提供的微流控检测芯片的第一实施例的中间层结构图。
图5：本公开提供的微流控检测芯片的第一实施例的试剂固定区域示意图。
图6：本公开提供的微流控检测芯片的第一实施例固定在本公开固定装置并检测的示意图。
图7：本公开提供的微流控检测芯片的第二实施例的外形图。
图8：本公开提供的微流控检测芯片的第二实施例的上层结构图。
图9：本公开提供的微流控检测芯片的第二实施例的下层结构图。
图10：本公开提供的微流控检测芯片的第二实施例的中间层结构图。
图11：本公开提供的微流控检测芯片的第二实施例或第三实施例的试剂固定区域示意图。
图12：本公开提供的微流控检测芯片的第二实施例或第三实施例固定在本公开固定装置并检测的示意图。
图13：本公开提供的微流控检测芯片的固定装置的第四实施例装配示意图。
图14：本公开提供的微流控检测芯片的固定装置的第四实施例定位斜槽配合示意图。
附图标记说明
微流控检测芯片 1
芯片上层 100
芯片下层 200
芯片中层 300
加样区 101
加样孔 102
加样区通气孔 103
废液池通气孔 104
卡柱 105
引流点 106
芯片下层的凹槽 201
芯片下层的卡柱孔 202
芯片下层的坡度结构 203
引流槽 204
有胶区 301
无胶区 302
样本槽区 303
流道检测区 304
废液槽区 305
中间层卡柱孔 306
固定装置 2
离心轴心 3
包被抗体的荧光微球点样区 4
质控抗体点样区 5
检测抗体第一点样区 6
检测抗体第二点样区 7
斜槽 8
旋转托盘 401
中央固定盘 402
旋转托盘的脚柱孔 403
光源透过区 404
定位斜槽 405
具体实施方式
为了更加清楚地阐述本公开的上述目的、特征和优点，在该部分结合附图详细说明本公开的具体实施方式。除了在本部分描述的各个实施方式以外，本公开还能够通过其他不同的方式来实施，在不违背本公开精神的情况下，本领域技术人员可以做相应的改进、变形和替换，因此本公开不受该部分公开的具体实施例的限制。本公开的保护范围应以权利要求为准。
本公开的说明书和权利要求书及上述附图中的术语“包括”以及它们任何变形，意图在于覆盖不排他的包含。例如包含一系列步骤或单元的过程、方法或系统、产品或设备没有限定于已列出的步骤或单元，而是可选地还包括没有列出的步骤或单元，或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤或单元。
另外，为了更好的说明本公开，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本公开同样可以实施。在一些实例中， 对于本领域技术人员熟知的方法、手段、元件和电路未作详细描述，以便于凸显本公开的主旨。
<微流控检测芯片>
微流控检测芯片包含叠置的三层：芯片上层、芯片下层、芯片中间层。芯片的形状可以是类椭圆形、方形、长方形、多边形或者圆形，优选地，其形状为类椭圆形以实现更好地握持。芯片上下层的厚度均为1.5～2.5mm，如果厚度太薄，则芯片加载样本量过小并且容易变形，如果厚度太厚，透光性会受到影响，影响检测结果，同时也不符合芯片小型化的需求。芯片中间层的厚度为0.05～0.5mm。芯片上层和下层之间靠中间层粘贴或者中间层粘贴和卡扣共同进行密合固定。
芯片上层和芯片下层的材料选自聚苯乙烯、聚二甲基硅氧烷、聚甲基丙烯酸甲酯、聚对苯二甲酸乙二醇酯、玻璃或聚碳酸酯中的一种；优选的，所述芯片上层和芯片下层的材料选自聚甲基丙烯酸甲酯、聚苯乙烯或聚碳酸酯；所述芯片中间层为聚对苯二甲酸乙二醇酯胶或聚甲基丙烯酸甲酯胶。
芯片上层主要用于将样本引入检测区域。芯片上层包含加样区，加样区上设有加样孔，用于添加样本，加样孔可设计成可与生物实验中常规实验的标准规格的移液枪枪头严密匹配的圆形，直径可在2mm～3mm之间，通过加样孔添加的样本可以沿着样本流动通道流动。由于本公开采用的重力和毛细管作用自驱动和短时离心相结合，本公开的加样区中无需固定有全血过滤装置如全血滤膜等，可以减少样本浪费，提高样本利用效率。加样区的形状与芯片中间层的样本槽区形状相对应。在一种可能的实施方式中，加样区为不规则扇形。在另一种可能的实施方式中，加样区内有夹角为15°-45°的坡度结构，加样区内分布数个圆柱形凸起状引流点，引流点直径为0.2-0.6mm，高为0.2-0.6mm，引流点间距为1-5mm，引流点数目为3-10个，起储存样本和引流作用。
芯片上层还包括通气孔，即加样区通气孔和废液池通气孔。所述通气孔为与大气相通的通孔，优选是圆形通孔以改善样本溶液的流动性，直径可在0.5～2.0mm范围内。在一种可能的实施方式中，芯片上层设置1个加样区通气孔和1个废液池通气孔。在另一种可能的实施方式中，加样区通气孔可以设置多个，取决于加样区的个数。
为了有利于芯片的组装和固定，芯片上层设置卡柱，卡柱的个数一般根据实际需要，可以设置2-4个，为了组装的密合性更好，进一步优选设置4个卡柱。
芯片下层设置坡度结构或凹槽可以用来引导和存储废液。在可能的实施方式中，所述坡度结构的夹角为15°-45°，所述坡度结构内分布数个圆柱形凸起状引流点用于引流 废液，引流点直径为0.2-0.6mm，高为0.2-0.6mm，引流点间距为1-5mm，引流点数目为3-10个。在另一种可能的实施方式中，芯片下层设置凹槽以更便于收集离心时流道检测区甩出的残余废液，凹槽对应于芯片中间层的废液槽区的下半部分，为异形，深度在1-2mm。在其他可能的实施方式中，芯片下层还包括对应芯片上层的加样区位置设有引流槽，引流槽为宽度0.2-0.6mm，深度0.1-0.5mm的槽道，以便于引导样本流入流道检测区。另外，芯片下层还可以包括一定数量的卡柱孔，与芯片上层的卡柱相对应以加固芯片三层的密合性。
芯片中间层为双面胶层，在所述双面胶层上用有胶区和无胶区分隔出样本流动通道，所述样本流动通道包括样本槽区、流道检测区、废液槽区，其中所述样本槽区与所述芯片上层的加样区相对应，所述样本槽区的形状可以与所述芯片上层的加样区相同。当将芯片上下层和中间层密合后，所述废液槽区的主要作用在于存储废液，即相当于废液池，所述废液槽区的形状至少完全覆盖所述芯片下层的坡度结构或凹槽，所述废液槽区可以与所述芯片下层的坡度结构或凹槽的大小相同或者大于坡度结构或凹槽的大小。所述流道检测区为弧形，所述流道检测区的弯曲半径25～35mm(以固定装置的旋转托盘离心轴心为圆心的半径)，弧度在1.8～2.2rad范围内，宽度为2-4mm。在所述样本流体流道的表面通过固定荧光微球-抗体标记物和抗体以进行免疫指标检测，增加荧光微球-抗体标记物和抗体的数量可以进一步提高检测灵敏度。芯片中间层也还可以包括一定数量的卡柱孔，与芯片上层的卡柱相对应以加固芯片三层的密合性。
<微流控检测芯片的制备方法>
本公开所述的微流控检测芯片的制备方法至少包括：
1)通过激光在双面胶层刻蚀样本流体通道；
具体而言，可以采用激光雕刻机对双面胶层进行刻蚀。一般而言传统的微流控检测芯片都是在芯片层如PDMS或PMMA上利用光刻机来刻蚀通道结构，光刻机昂贵且光刻过程存在光源污染，本公开采用在双面胶层上刻蚀通道结构，可以提高生产效率、降低生产成本且制备时候减少对环境的污染；
2)将双面胶层一侧的剥离层撕下，将所述双面胶层粘贴在芯片下层表面的上方；
3)在样本流体流道内进行点样，干燥后粘上芯片上层，压紧；
具体而言，在流道检测区和样本槽区的交接位置处设置包被抗体的荧光微球点样区，顺着样本流动的方向，依次设置检测抗体第二点样区、检测抗体第一点样区和质控抗体点样区，通过设立质控抗体点样区以减少产品批间和/或批内变异，提高产品质量。在上 述点样区内加入相应的抗体和荧光微球-抗体标记物，在35-40℃下干燥3-6小时，优选4小时，粘上芯片上层，压紧，如芯片上层设有卡柱，则将芯片上层的卡柱插入芯片中间层和芯片下层的卡柱孔，通过粘合作用和卡扣作用共同进行密合固定。
<微流控检测芯片的用途>
在本公开的微流控检测芯片使用时，可以直接将新鲜血液100-300μl直接滴加于加样孔中，样本在重力作用和毛细管作用下在样本流动通道内流动，先与包被了捕获抗体的荧光微球混合，之后与检测抗体和多抗反应，产生荧光信号。一般而言，从样本加入加样孔起算10-15分钟，样本可以反应结束流入废液池。将微流控检测芯片置于检测设备上并进行固定，在本公开中可以将多个芯片固定在同一固定装置上，施加离心力1-3分钟，转速在2000-6000rpm范围内，目的在于甩干流道检测区的残余废液。之后读取检测区内检测质控抗体点样区和检测抗体点样区的荧光强度，通过检测标准品拟合定标曲线，即可以计算出样本中免疫指标的含量。
<微流控检测芯片的固定装置>
由于本公开的微流控检测芯片需要在高速离心情况下工作，为了解决微流控检测芯片在离心力作用下的装夹和固定牢固性问题，本公开还设计了配合本公开微流控检测芯片使用的固定装置。
所述固定装置用以同时固定多个本公开所述的微流控检测芯片，所述固定装置包括一个中央固定盘和一个旋转托盘，所述旋转托盘被其上方的所述中央固定盘划分为多个与本公开所述的微流控检测芯片形状匹配的区域，所述固定装置主要利用所述中央固定盘的各个侧面分别将多个本公开所述的微流控检测芯片固定在所述旋转托盘上，所述旋转托盘内具有光源透过区，所述光源透过区与本公开所述的微流控检测芯片的流道检测区同轴心。在可能的实施方式中，光源透过区为弧形。中央固定盘和旋转托盘之间利用螺丝固定即可。
为了增强固定的稳定性，本公开在所述中央固定盘的侧面设置有定位斜槽，所述定位斜槽沿所述中央固定盘的侧面均匀设置，本公开的微流控检测芯片侧面设有斜槽，与定位斜槽形成卡紧结构，一般而言，每个微流控检测芯片与两个定位斜槽配合以防止微流控检测芯片与中央固定盘之间发生周向移动。作为可替换的另一种实施方式，若微流控检测芯片的侧面设计有凹陷结构，则可以在中央固定盘的侧面设有弹性球头柱塞与微流控检测芯片相对应的凹陷结构形成弹性限制装配，对应每个微流控检测芯片，弹性球头柱塞一般可以设置2个。
在本公开的一个可能的实施方式中，所述固定装置可以一次同时固定3个本公开所述的微流控检测芯片，所述中央固定盘具有3段弧形侧面，每段弧形侧面设置有2个定位斜槽，所述中央固定盘与所述旋转托盘通过外围卡扣固定，所述中央固定盘位于所述旋转托盘的上方，所述旋转托盘因此被划分出三块与所述微流控检测芯片形状相近的区域，所述微流控检测芯片通过与所述定位斜槽形成卡紧结构从而固定在该区域。
在可能的实施方式中，由于微流控检测芯片属于小型的薄塑料片，旋转托盘外侧面的两边可以设置成弧形以与微流控检测芯片两侧的弧形结构相互匹配，充分考虑了人手取拿芯片的可操作性和舒适性。
在另一种可能的实施方式中，如果微流控检测芯片的芯片底座设置了脚柱，则对应地可在旋转托盘设置脚柱孔，有利于芯片在托盘上的牢固性。
<基于微流控检测芯片的离心式检测装置>
本公开还提供一种基于本公开的微流控检测芯片的离心式检测装置，其至少包括以下模块：
微流控模块，包括本公开所述的微流控检测芯片；
固定模块，包括本公开所述的固定装置以用于固定若干个微流控检测芯片；
转动模块，连接于所述固定装置，用于带动所述固定装置转动进而实现微流控检测芯片的高速离心，在一种可能的实现方式中，所述转动模块包括步进电机例如可以是高速微型步进电机；及检测模块，用于通过所述固定装置的光源透过孔对所述微流控检测芯片的流道检测区进行检测，并输出检测结果。
在一种可能的实现方式中，所述检测模块包括：
发光单元，所述发光单元用于发出第一光线；
光传播单元，所述光传播单元用于将所述第一光线传播到所述光源透过区；当所述转动模块带动所述微流控检测芯片的流道反应区转动到所述光源透过区上方，所述第一光线通过所述光源透过区照射所述流道反应区的样本时，所述光传播单元传播所述样本受所述第一光线激发而发出的第二光线；及
检测单元，所述检测单元用于检测所述第二光线，并根据所述第二光线生成第一信号，所述第一信号为电信号，所述第一信号用于分析所述样本，并将所述第一信号作为所述检测结果。
在可能的实施方式中，所述离心式检测装置还包括温控模块和分析模块。温控模块用于控制流道检测区的温度。分析模块用于接收所述检测结果，并根据所述检测结果对 所述样本进行分析，并输出分析结果。
通过各个模块的配合，本公开的离心式检测装置可以实现对免疫学单项目或多项目测定，精密度和检测灵敏度较传统纸基材免疫层析方法高。如对降钙素原指标的检测，纸基材免疫层析产品通常分析灵敏度为0.2ng/mL，本方法可达0.05ng/mL。本方法制备的免疫检测试剂，批内和批间精密度<10％,而传统方法为15％-20％。
由下面的实施例进一步说明本公开，但不构成限制。
实施例
实施例一
本实施例提供一种微流控检测芯片，用于检测全血中降钙素原含量。
图1示出了本实施例微流控检测芯片的外形图。由图1-图4可知，微流控检测芯片1形状为类椭圆形，长宽高的最大尺寸为55mm×35mm×4mm。芯片1包含叠置的三层：芯片上层100、芯片下层200、芯片中间层300。
芯片上层100的材质是PMMA，形状为类椭圆形形，上层100的厚度为2.0mm。图2显示的芯片上层100的结构，由此可见芯片上层100包含加样区101，加样区101上设有加样孔102，用于添加样本，加样孔102直径为4mm。加样区101的形状为不规则扇形。芯片上层100还包括2个通气孔，即图1所示的加样区通气孔103和废液池通气孔104。所述通气孔为与大气相通的圆形通孔以改善样本溶液的流动性，直径为1.0mm。
为了有利于芯片的组装和固定，芯片上层100设置4个卡柱105。
芯片下层200的材质是PMMA，形状与芯片上层相匹配，芯片下层200的厚度为2.0mm。图3显示的芯片下层200的结构，其包括一收集离心时残余废液的凹槽201，为异形，长宽深的最大尺寸为32mm×3.3mm×1.5mm。芯片下层还包括芯片下层的卡柱孔202。
芯片中间层为双面胶层，材质为PET胶，厚度为0.05mm。图4显示的芯片中间层300的结构，在所述双面胶层上用有胶区301(深色区域)和无胶区302(白色区域)分隔出样本流动通道(即无胶区302)，所述样本流动通道又可以细分为样本槽区303、流道检测区304、废液槽区305三个区域，其中所述样本槽区303与所述芯片上层的加样区101形状相同，所述废液槽区305完全覆盖所述芯片下层的凹槽201，其面积大于凹槽的面积。所述流道检测区的宽度为3mm，长度为30mm，所述流道检测区的弯曲半径32mm，弧度2.09rad。芯片中间层还包括芯片中间层的卡柱孔306。
制备本检测芯片时，先将芯片中间层300一侧胶覆膜撕下，粘贴在芯片下层200，如图5所示在暴露出的样本流体通道中的质控抗体点样区5加入0.05ng羊抗鼠多克隆抗体，在检测抗体第一点样区6加入0.02ng的降钙素原检测抗体，包被抗体的荧光微球点样区4加入包被了0.02ng降钙素原捕获抗体的0.01％固含量的荧光微球，37℃干燥4小时后粘上芯片上层盖板，压紧。
检测时，直接滴加100ul全血在加样孔102，等待10分钟后，样本反应结束全部流入废液池201。将微流控检测芯片1置于固定装置上，如图6所示，可以3个微流控检测芯片固定在固定装置2上，在以离心轴心3施加5000rpm逆时针离心力1分钟，然后读取检测区内检测质控抗体点样区和检测抗体点样区荧光强度。通过检测标准品拟合定标曲线，即可计算出样本内降钙素原含量，用本方法检测全血中降钙素原，从加样到读出结果只需要10-15分钟，分析灵敏度可达0.05ng/mL，检测范围为0.05ng/mL-100ng/mL,接近化学发光法检测性能。
实施例二
本实施例提供另一种结构的微流控检测芯片，用于检测全血中降钙素原含量。
图7示出了本实施例微流控检测芯片的外形图。由图7-图10可知，微流控检测芯片形状为类椭圆形，长宽高的最大尺寸为55mm×35mm×4mm，芯片1包含叠置的三层：芯片上层100、芯片下层200、芯片中间层300。
芯片上层100的材质是PMMA，形状为类椭圆形，上层100的厚度为2.0mm。图8显示的芯片上层100的结构，由此可见芯片上层100包含加样区101，加样区101上设有加样孔102，用于添加样本，加样孔102直径为3mm。加样区101内有夹角为25°斜坡，其上分布数个直径为0.4mm，高度为0.4mm的圆柱形凸起状引流点106，引流点间距为4mm，对样本起引流作用。加样区101的形状为不规则扇形。芯片上层100还包括2个通气孔，即图8所示的加样区通气孔103和废液池通气孔104。所述通气孔为与大气相通的圆形通孔以改善样本溶液的流动性，直径为1.0mm。
芯片下层200的材质是PMMA，形状与芯片上层相匹配，芯片下层200的厚度为2.0mm。图9显示的芯片下层200的结构，其包括一个夹脚为20°坡度结构203，为异形，最长41mm，最宽8mm，坡度结构203上分布有数个直径为0.4mm，高度为0.4mm的圆柱形凸起状引流点106，引流点间距为4mm，对样本起到引流作用。芯片下层200在对应芯片上层100的加样区101的位置设有引流槽204，引流槽宽度0.4mm，深度0.1mm，作用是引导样本流入样本流体通道内。
芯片中间层为粘合芯片上层和芯片下层并形成流体通道的双面胶层，材质为PET胶，厚度为0.05mm。图10显示的芯片中间层300的结构，芯片中间层为双面胶层，在所述双面胶层上用有胶区301和无胶区302分隔出样本流动通道，所述样本流动通道包括样本槽区303、流道检测区304、废液槽区305，其中所述样本槽区303与所述芯片上层的加样区101相对应，所述废液槽区305与所述芯片下层的坡度结构203相对应，废液槽区305能完全覆盖所述芯片下层的坡度结构203，其面积略大于坡度结构的面积。所述流道检测区的宽度为3mm，所述流道检测区的弯曲半径32mm，弧度2.09rad。
制备本检测芯片时，先将芯片中间层300一侧胶覆膜撕下，粘贴在芯片下层200，如图11所示在暴露出的样本流体通道中的质控抗体点样区5加入0.05ng羊抗鼠多克隆抗体，在检测抗体第一点样区6加入0.02ng的降钙素原检测抗体，包被抗体的荧光微球点样区4加入包被了0.02ng降钙素原捕获抗体的0.01％固含量的荧光微球，37℃干燥4小时后粘上芯片上层盖板，压紧。
检测时，直接滴加100ul全血在加样孔102，等待10分钟后，样本反应结束全部流入废液池201。将微流控检测芯片1置于固定装置上，如图12所示，可以3个微流控检测芯片固定在固定装置2上，在以离心轴心3施加5000rpm逆时针离心力1分钟，然后读取检测区内检测质控抗体点样区和检测抗体点样区荧光强度。通过检测标准品拟合定标曲线，即可计算出样本内降钙素原含量，检测性能同实施例一。
实施例三
本实施例中微流控检测芯片的结构同实施例二，用于同时检测全血中降钙素原和C反应蛋白含量。
微流控检测芯片的制备步骤与实施例二基本相同，仅改变点样区加入的物质。如图11所示，在样本流体通道中点样，分别在质控抗体点样区5加入0.05ng羊抗鼠多克隆抗体、在检测抗体第一点样区6加入0.02ng的降钙素原检测抗体，在检测抗体第二点样区7加入0.05ng的C反应蛋白检测抗体，在包被抗体的荧光微球点样区4加入荧光抗体混合物，具体是包被了0.02ng降钙素原捕获抗体的0.05％固含量的荧光微球和包被了0.05ng C反应蛋白捕获抗体的0.05％固含量的荧光微球混合物，37℃下干燥4小时后，粘上上层盖板，压紧。
检测时，直接滴加120ul全血在加样孔102，等待15分钟后，样本反应结束全部流入废液池201。将微流控检测芯片1置于固定装置上，如图12所示，可以3个微流控检测芯片固定在固定装置2上，在以离心轴心3施加2000rpm逆时针离心力2分钟，然后读取检测区 内检测质控抗体点样区和检测抗体点样区荧光强度。通过检测标准品拟合定标曲线，即可计算出样本内降钙素原和C反应蛋白含量。用该方法检测降钙素原分析灵敏度可达0.05ng/mL，检测范围为0.05-100ng/mL；对C反应蛋白检测分析灵敏度为0.5mg/L，检测范围为0.5-100mg/L。
实施例四
本实施例提供一种微流控检测芯片的固定装置，用于装夹和固定实施例一所述的微流控检测芯片。
由图13可见，所述固定装置2包括旋转托盘401和中央固定盘402，所述旋转托盘401与中央固定盘402同轴，轴心均为离心轴心3，所述旋转托盘401被其上方的中央固定盘402划分为3个与本公开所述的微流控检测芯片形状匹配的区域，所述旋转托盘内具有光源透过区404，所述光源透过区404与本公开所述微流控检测芯片的流道检测区304同轴心，为弧形通孔。所述旋转托盘401上有与微流控检测芯片的芯片下层脚柱相对应的脚柱孔403。
为了增强固定的稳定性，本公开在所述中央固定盘402的侧面设置有定位斜槽405，所述定位斜槽405沿所述中央固定盘的侧面均匀设置。如图14所示，本公开的微流控检测芯片1侧面设有斜槽8，与定位斜槽405形成卡紧结构，一般而言，每个微流控检测芯片与两个定位斜槽405配合以防止微流控检测芯片与中央固定盘之间发生周向移动。

Claims (7)

  1. 一种微流控检测芯片，其特征在于，所述微流控检测芯片包含叠置的三层，芯片上层包含加样区和通气孔；芯片下层设有坡度结构或凹槽；芯片中间层为双面胶层，在所述双面胶层上用有胶区和无胶区分隔出样本流动通道，所述样本流动通道包括样本槽区、流道检测区、废液槽区，其中所述样本槽区与所述芯片上层的加样区相对应，所述废液槽区至少覆盖所述芯片下层的坡度结构或凹槽，所述流道检测区为弧形；所述芯片下层的坡度结构的夹角为15°-45°，所述芯片下层的坡度结构内分布数个圆柱形凸起状引流点，所述凹槽对应于所述废液槽区的下半部分，深度为1-2mm；所述芯片上层和所述芯片下层的厚度均为1.5-2.5mm，所述芯片中间层的厚度为0.05-0.5mm。
  2. 根据权利要求1所述的微流控检测芯片，其特征在于，所述加样区内设有坡度结构，所述加样区内的坡度结构的夹角为15°-45°，所述加样区内的坡度结构上分布有一个或多个圆柱形凸起状引流点。
  3. 根据权利要求1或2所述的微流控检测芯片，其特征在于，所述芯片下层还包括与所述加样区对应的引流槽。
  4. 根据权利要求1或2所述的微流控检测芯片，其特征在于，所述芯片上层和芯片下层的材料选自聚苯乙烯、聚二甲基硅氧烷、聚甲基丙烯酸甲酯、聚对苯二甲酸乙二醇酯、玻璃或聚碳酸酯中的一种；所述芯片中间层为聚对苯二甲酸乙二醇酯胶或聚甲基丙烯酸甲酯胶。
  5. 权利要求1-4任一项所述的微流控检测芯片的制备方法，其特征在于，该方法至少包括1)通过激光在所述双面胶层刻蚀样本流体通道，2)将所述双面胶层一侧的剥离层撕下，并将其粘贴在芯片下层表面的上方，3)在样本流体流道内进行点样，干燥后粘上芯片上层，压紧。
  6. 一种微流控检测芯片的固定装置，其特征在于，所述固定装置用以同时固定多个如权利要求1-4任一项所述的微流控检测芯片，所述固定装置包括一个中央固定盘和一个旋转托盘，所述旋转托盘被其上方的所述中央固定盘划分为多个与权利要求1-4任一项所述的微流控检测芯片形状匹配的区域，所述固定装置利用所述中央固定盘的各个侧面分别将多个如权利要求1-4任一项所述的微流控检测芯片固定在所述旋转托盘上，微流控检测芯片所述旋转托盘内具有光源透过区，所述光源透过区与权利要求1-4任一项所述的微流控检测芯片的流道检测区同轴心；所述中央固定盘的侧面设置有定位斜槽，所述定位斜槽沿所述中央固定盘的侧面均匀设置。
  7. 一种离心式检测装置，其特征在于，所述离心式检测装置至少包括以下模块：
    微流控模块，包括权利要求1-4任一项所述的微流控检测芯片；
    固定模块，包括权利要求6所述的固定装置；
    转动模块，连接于所述固定装置，用于带动所述固定装置转动；及
    检测模块，用于通过所述固定装置的光源透过区对所述微流控检测芯片的流道检测区进行检测，并输出检测结果。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111604095A (zh) * 2020-05-09 2020-09-01 东莞市东阳光诊断产品有限公司 微流控芯片、芯片控制装置及其操作方法
CN112578111A (zh) * 2020-12-25 2021-03-30 扬州初心科技服务有限公司 一种食品抗生素蠕动多联免疫检测器

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108414773B (zh) * 2018-07-12 2018-11-02 上海速创诊断产品有限公司 一种微流控检测芯片及其制备方法、固定装置和离心式检测装置
CN109709316A (zh) * 2018-12-27 2019-05-03 天津昌和生物医药技术有限公司 一种多靶项微流测试卡及制备方法
CN109499636B (zh) * 2019-01-14 2024-02-27 大连大学 一种病原体免疫检测数字微流控芯片及其制作方法
CN110180611B (zh) * 2019-07-05 2024-03-08 宋禹 微流控芯片组件
CN110568203B (zh) * 2019-09-12 2022-05-24 重庆科技学院 一种多通道荧光免疫层析微流控芯片的使用方法
CN110773246B (zh) * 2019-11-01 2021-12-14 上海速创诊断产品有限公司 一种微流控芯片及用于高敏肌钙蛋白检测的试剂盒
CN110773245A (zh) * 2019-11-01 2020-02-11 上海速创诊断产品有限公司 一种微流控芯片及其处理方法
CN111426847A (zh) * 2020-04-10 2020-07-17 上海速创诊断产品有限公司 一种微流控芯片、检测试剂盒、微流控检测系统及其用途
US11813608B2 (en) 2020-09-22 2023-11-14 Oregon State University Fiber substrate-based fluidic analytical devices and methods of making and using the same
CN114011480B (zh) * 2021-11-04 2023-04-07 上海速创诊断产品有限公司 一种电化学发光微流控检测芯片及用于蛋白检测的试剂盒
CN114088951A (zh) * 2021-11-19 2022-02-25 太原理工大学 心肌梗塞多指标联合检测的微流控芯片及装置和检测方法
CN114768896B (zh) * 2022-03-11 2023-12-29 南方科技大学 集成全血分离和分子诊断的离心式微流控芯片及制备方法

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20080090667A (ko) * 2007-04-05 2008-10-09 삼성전자주식회사 원심력 기반의 미세유동 시스템 및 상기 미세유동 시스템용바이오 카트리지
CN103055981A (zh) * 2012-12-31 2013-04-24 苏州汶颢芯片科技有限公司 一种聚二甲基硅氧烷微流控芯片及其制备方法
CN203663854U (zh) 2013-07-01 2014-06-25 香港大学深圳医院 新型的微流控芯片
CN204679514U (zh) * 2015-05-12 2015-09-30 天津微纳芯科技有限公司 一种用于微流控芯片检测装置的锁紧装置
CN105259162A (zh) 2015-10-26 2016-01-20 深圳华迈兴微医疗科技有限公司 定量检测全血中脑钠肽的磁微粒化学发光微流控芯片
CN106434302A (zh) * 2016-09-18 2017-02-22 华中科技大学 一种便携式无动力源的微流控细胞分离芯片
CN106807461A (zh) 2017-01-10 2017-06-09 北京华科泰生物技术有限公司 一种用于荧光免疫检测的微流控芯片及其制备方法
CN107126753A (zh) * 2017-05-25 2017-09-05 苏州岚轩生物科技有限公司 主动式微流控芯片及其全血过滤装置
CN206701297U (zh) * 2017-03-10 2017-12-05 山东华芯电子有限公司 一种多指标检测微流控芯片
CN207036852U (zh) 2017-07-03 2018-02-23 沈阳微流控生物科技有限公司 一种全自动微流控芯片荧光免疫检测系统
CN108414773A (zh) * 2018-07-12 2018-08-17 上海速创诊断产品有限公司 一种微流控检测芯片及其制备方法、固定装置和离心式检测装置
CN208526658U (zh) * 2018-07-12 2019-02-22 上海速创诊断产品有限公司 一种微流控检测芯片、固定装置和离心式检测装置

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7318912B2 (en) * 2001-06-07 2008-01-15 Nanostream, Inc. Microfluidic systems and methods for combining discrete fluid volumes
CN100547403C (zh) * 2005-08-12 2009-10-07 宗小林 一种微流控芯片分析仪及配套芯片
TWI411779B (zh) * 2011-05-18 2013-10-11 Univ Nat Sun Yat Sen 微流體生物晶片及其自動化反應偵測系統
US10166541B2 (en) * 2014-12-10 2019-01-01 The Regents Of The University Of California Centrifugal microfluidic platform for automated media exchange
CN104841500B (zh) 2015-05-12 2016-08-17 天津微纳芯科技有限公司 一种用于样品检测的芯片及其封装方法
CN106238113B (zh) * 2016-08-31 2019-04-09 香港大学深圳医院 一种微流控芯片及其制作工艺
CN106442443B (zh) * 2016-09-12 2018-12-07 北京天健惠康生物科技有限公司 一种微液滴荧光检测系统
CN106914287B (zh) * 2017-03-14 2018-12-28 同昕生物技术（北京）有限公司 一种微流控芯片及其制备方法与应用
CN107102131B (zh) * 2017-07-03 2019-02-05 沈阳微流控生物科技有限公司 一种全自动微流控芯片荧光免疫检测系统及其检测方法

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20080090667A (ko) * 2007-04-05 2008-10-09 삼성전자주식회사 원심력 기반의 미세유동 시스템 및 상기 미세유동 시스템용바이오 카트리지
CN103055981A (zh) * 2012-12-31 2013-04-24 苏州汶颢芯片科技有限公司 一种聚二甲基硅氧烷微流控芯片及其制备方法
CN203663854U (zh) 2013-07-01 2014-06-25 香港大学深圳医院 新型的微流控芯片
CN204679514U (zh) * 2015-05-12 2015-09-30 天津微纳芯科技有限公司 一种用于微流控芯片检测装置的锁紧装置
CN105259162A (zh) 2015-10-26 2016-01-20 深圳华迈兴微医疗科技有限公司 定量检测全血中脑钠肽的磁微粒化学发光微流控芯片
CN106434302A (zh) * 2016-09-18 2017-02-22 华中科技大学 一种便携式无动力源的微流控细胞分离芯片
CN106807461A (zh) 2017-01-10 2017-06-09 北京华科泰生物技术有限公司 一种用于荧光免疫检测的微流控芯片及其制备方法
CN206701297U (zh) * 2017-03-10 2017-12-05 山东华芯电子有限公司 一种多指标检测微流控芯片
CN107126753A (zh) * 2017-05-25 2017-09-05 苏州岚轩生物科技有限公司 主动式微流控芯片及其全血过滤装置
CN207036852U (zh) 2017-07-03 2018-02-23 沈阳微流控生物科技有限公司 一种全自动微流控芯片荧光免疫检测系统
CN108414773A (zh) * 2018-07-12 2018-08-17 上海速创诊断产品有限公司 一种微流控检测芯片及其制备方法、固定装置和离心式检测装置
CN208526658U (zh) * 2018-07-12 2019-02-22 上海速创诊断产品有限公司 一种微流控检测芯片、固定装置和离心式检测装置

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3674713A4

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111604095A (zh) * 2020-05-09 2020-09-01 东莞市东阳光诊断产品有限公司 微流控芯片、芯片控制装置及其操作方法
CN111604095B (zh) * 2020-05-09 2022-02-15 东莞市东阳光诊断产品有限公司 微流控芯片、芯片控制装置及其操作方法
CN112578111A (zh) * 2020-12-25 2021-03-30 扬州初心科技服务有限公司 一种食品抗生素蠕动多联免疫检测器

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