CN109499634B - Microfluidic chip and preparation method and detection method thereof - Google Patents

Microfluidic chip and preparation method and detection method thereof Download PDF

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CN109499634B
CN109499634B CN201811523924.4A CN201811523924A CN109499634B CN 109499634 B CN109499634 B CN 109499634B CN 201811523924 A CN201811523924 A CN 201811523924A CN 109499634 B CN109499634 B CN 109499634B
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detection
chip
pipeline
reagent
sample
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CN109499634A (en
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顾悦
蒋理国
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Dialab Zhangjiagang Biotechnology Co ltd
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Dialab Zhangjiagang Biotechnology Co ltd
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Priority to CN202311423864.XA priority patent/CN117443474A/en
Priority to CN202311421940.3A priority patent/CN117258862A/en
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G01N33/5304—Reaction vessels, e.g. agglutination plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

The invention discloses a microfluidic chip and a preparation method and a detection method thereof, wherein the microfluidic chip comprises a chip substrate and a chip cover plate, a sample adding port for adding a detection sample is formed in the chip cover plate, a reaction cavity, a mixing pipeline, a detection pipeline and a waste liquid tank are sequentially formed in the chip substrate, the mixing pipeline is communicated with the reaction cavity, the detection pipeline is communicated with the mixing pipeline, fluorescent microsphere marking reagents for providing fluorescent detection signals are coated in the mixing pipeline, a carrier film is loaded in the detection pipeline, and at least one capture antibody reagent is coated on the carrier film. The microfluidic chip can realize strict and controllable reagent redissolution volume and reaction time, and improves detection repeatability; the packaging is simple, the chip production process is greatly simplified, and the chip yield is improved; more than one capture antibody can be coated in different areas of the carrier film, so that multi-project combined detection is realized.

Description

Microfluidic chip and preparation method and detection method thereof
Technical Field
The invention belongs to the technical field of medical instrument in-vitro diagnosis, and particularly relates to a microfluidic chip for bedside diagnosis (POCT), a preparation method and a detection method of the microfluidic chip.
Background
Bedside diagnosis (Point of Care Testing, POCT) is a rapid diagnosis of patients on site by means of miniaturized or moderate desktop devices or reagents. The POCT detection method has the characteristics of simple and convenient operation, easy maintenance of a detection system, capability of detecting at any time and any place, low detection cost and the like, and POCT detection products are gradually accepted and popularized by the market.
POCT test products for disease diagnosis in the prior art can be broadly classified into two categories according to test methodologies or product materials: chromatography POCT detection technology and microfluidic POCT detection technology.
The development of the chromatography POCT detection technology has been history for more than thirty years, and the dominant product of POCT detection in the market is developed based on the chromatography technology at present. The POCT product developed based on the chromatographic technology has low technical threshold, but is easy to be interfered by external environment due to the fact that the structure is not closed, the sample flow is uncontrolled, and the difference between detected individuals is large, so that the detection sensitivity and the detection repeatability are affected, and the detection performance requirement of clinical diagnosis by clinical laboratory doctors cannot be met.
The microfluidic chip POCT detection technology is a rapid detection technology developed in the last decade. The microfluidic technology has the characteristics of sample flow control, chip sealing, tiny pipeline, strong controllability and the like, so that the detection sensitivity and the detection repeatability of the microfluidic chip POCT detection technology are improved to different degrees compared with those of the chromatographic POCT detection technology, and the microfluidic chip POCT detection technology is more and more favored by the market.
In the prior art, the POCT detection products of the microfluidic chip can be roughly divided into two types according to different sample driving modes: one type is a passive microfluidic detection chip, i.e. a flow of sample in the chip is driven by capillary forces provided by micro-channels in the chip. The passive microfluidic chip POCT has the advantages of simple detection mode and low requirements on a detection system, but the whole sample detection reaction has low controllability and large reagent reaction randomness. The reagent reaction cavity in the passive microfluidic chip is filled with dry reagent in advance, and the dry reagent is redissolved after the sample flows into the reagent cavity. The re-dissolution process is random, the re-dissolution volume of the sample and the re-dissolution process are uncontrollable, so that the detection repeatability is poor, and the detection repeatability is not obviously improved compared with the chromatographic POCT detection technology.
Another type of microfluidic chip POCT detection products are active microfluidic detection chips, i.e. the flow of samples in the chip is driven by external forces such as air pressure, mechanical forces, centrifugal forces, electrodynamic forces, etc. The POCT of the active microfluidic chip is used for accurately controlling the flowing and uniform mixing reaction process of the detection sample and the reagent, so that the detection repeatability and the detection sensitivity of the reagent are greatly improved. However, with the active microfluidic chip POCT integrated with the magnetic particle detection technology, the magnetic particles coated with different antibodies cannot be separated by a magnetic field, so that the detection technology has the main defects that only single-item detection can be performed, and multiple-item detection cannot be performed simultaneously in a single-channel microfluidic chip. The active microfluidic chip POCT with integrated pressure valve has the problem that the whole chip packaging process becomes very complex due to the need of integrating an elastic film in the chip, resulting in low chip manufacturing yield.
Disclosure of Invention
In view of the above, in order to overcome the defects in the prior art, one of the purposes of the present invention is to provide a microfluidic chip capable of realizing rapid, accurate, high-sensitivity and multi-project quantitative detection on site.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the utility model provides a micro-fluidic chip, includes chip base plate and chip apron, set up the application of sample mouth that is used for adding the detection sample on the chip apron, set gradually on the chip base plate be located reaction chamber, with the mixing pipeline that the reaction chamber is linked together, with the detection pipeline and the waste liquid groove that the mixing pipeline is linked together, the coating has the fluorescent microsphere marking reagent that is used for providing fluorescence detection signal in the mixing pipeline, the carrier membrane is loaded in the detection pipeline, the coating has at least one kind to catch antibody reagent on the carrier membrane. In some embodiments, the detection conduit has a depth of 50 μm to 1000 μm; the depth of the waste liquid tank is 0.2mm-2mm.
When the microfluidic chip is particularly used, the sample adding port can be connected to a driving device, the driving mode of the driving device can be pneumatic, hydraulic or electric, and the like, and the driving device provides driving force for the added detection sample to flow back and forth in a pipeline of the chip so as to enable the sample to fully react with the reagent in the pipeline.
According to some preferred aspects of the invention, a super-hydrophobic pipeline is further arranged between the mixing pipeline and the detection pipeline. The super-hydrophobic pipeline comprises a groove microstructure and a first micron column of the microstructure, wherein the surface of the groove microstructure is provided with a super-hydrophobic reagent. The superhydrophobic pipeline can play a role in detecting a transition function that a sample reacts with a reagent to form a reaction complex and the reaction complex flows to the detection pipeline. The depth of each groove in the groove microstructure is 20-500 mu m, and the width is 50-4000 mu m; the diameter of the first micrometer column is 20-500 micrometers, and the height is 20-500 micrometers; the superhydrophobic reagent is fluorosilane dissolved in an electronic fluorination solution.
The super-hydrophobic pipeline comprises a groove microstructure, a first micron column and a super-hydrophobic reagent covered on the surface of the groove microstructure, the super-hydrophobic reagent and the super-hydrophobic surface formed by the groove microstructure can realize a passive flow blocking valve, when a detection sample flows to the super-hydrophobic surface by a driving force, the liquid can be effectively blocked from continuing to flow, the liquid can be left in the groove and cannot be spread, the driving force must be improved to drive the liquid to continue to flow forwards, and the controllability of the liquid flow is further enhanced.
According to some preferred aspects of the invention, a second micrometer column with a micrometer structure is arranged in the mixing pipeline, and the fluorescent microsphere marking reagent is coated on the second micrometer column. The second micrometer column has a diameter of 10-500 μm and a height of 10-500 μm.
The mixing pipeline is communicated with the reaction cavity, and can provide a back-and-forth flowing space for a detection sample, and the detection sample is uniformly mixed with the fluorescent microsphere labeled reagent coated by the redissolution of the fluorescent microsphere labeled reagent, so that liquid homogeneous phase immunoreaction occurs to form a reaction compound. After the detection sample enters the mixing pipeline, the fluorescent microsphere marking reagent is redissolved, then the detection sample and the fluorescent microsphere marking reagent are uniformly mixed, and finally the detection sample and the fluorescent microsphere marking reagent are subjected to incubation reaction, wherein the redissolution and the uniform mixing basically occur in the mixing pipeline, and the incubation mainly occurs in the reaction cavity. I.e. the reaction chamber may store the test sample and provide a reaction site for the test sample and the reagents.
More preferably, the fluorescent microsphere labeling reagent is a detection antibody labeled with a fluorescent microsphere.
More preferably, the capture antibody reagent is a capture antibody labeled with a nano-polystyrene microsphere.
According to some preferred aspects of the invention, a transition pipeline is arranged between the mixing pipeline and the reaction cavity, and a third micrometer column with a micrometer structure and a boss are arranged in the transition pipeline. The third micrometer column and the boss in the transition pipeline can further play a role in uniformly mixing liquid flowing between the reaction cavity and the uniformly mixing channel, so that the reaction between the detection sample and the reagent is more sufficient. The diameter of the third micrometer column is 20-500 micrometers, and the height is 20-500 micrometers; the height of the boss is 20-1000 μm, and the width is 100-4000 μm.
The carrier film is a nitrocellulose film. If only one capture antibody reagent is coated in the carrier film, the capture antibody reagent can be dispersed randomly in the carrier film; if two or more capture antibody reagents are coated in the carrier film, the carrier film needs to be divided into several regions, and different capture antibody reagents are coated in different regions. In this way, in the final detection, the fluorescent signal generated by the complex corresponding to the different regions can be resolved.
The invention also provides a preparation method of the microfluidic chip, which comprises the following steps:
step 1) preparation of fluorescent microsphere marking reagent: marking the purified detection antibody raw material by adopting a time-resolved fluorescence microsphere analysis method, and collecting fluorescence microsphere markers, namely fluorescence microsphere marking reagents;
Step 2) preparation of capture antibody reagent: diluting the purified capture antibody raw material by adopting a diluent, and marking the diluted capture antibody raw material on the nanometer polystyrene microsphere to prepare a capture antibody reagent;
step 3) super-hydrophilic modification of the chip surface material: performing super-hydrophilic modification on the chip surface material by adopting a vacuum plasma bombardment or atmospheric plasma bombardment method;
and 4) spraying and drying the sealing liquid: after finishing the super-hydrophilic modification of the chip surface in the step 3), spraying a layer of sealing liquid on the surface of the microfluidic chip to seal the surface, and then drying the chip;
step 5) preparation of a super-hydrophobic pipeline: after the chip is dried in the step 4), performing superhydrophobic modification on the surface of a specific pipeline in the chip, namely the surface of the groove microstructure by using a precise sample application instrument and a superhydrophobic reagent, and then drying the chip to obtain a superhydrophobic pipeline;
step 6) drying the fluorescent microsphere labeling reagent: after the chip in the step 5) is dried, adding the fluorescent microsphere markers collected in the step 1) into a uniformly mixing pipeline in a chip substrate, and then drying to enable the fluorescent microsphere markers to be dried in the chip and coated on a second micrometer column;
Step 7) immobilization of capture antibodies: after the chip in the step 6) is dried, coating the capture antibody reagent prepared in the step 2) on a carrier film by adopting a precise sample application instrument, then drying to fix the capture antibody marked by the polystyrene microsphere on the carrier film, and placing the carrier film coated with the capture antibody reagent on a detection pipeline of a chip substrate;
if only one capture antibody reagent is coated in the carrier film, the capture antibody reagent can be dispersed randomly in the carrier film; if two or more capture antibody reagents are coated in the carrier film, the carrier film needs to be divided into a plurality of areas, and different capture antibody reagents are coated in different areas, so that fluorescent signals generated by the complexes corresponding to different areas can be distinguished in the final detection;
step 8) packaging the microfluidic chip: and (3) placing water absorbing paper in the waste liquid tank, and assembling and bonding the chip cover plate and the chip substrate to obtain the microfluidic chip. The chip substrate and the cover plate are bonded together through pressure glue, ultrasonic waves, laser and the like to form the microfluidic chip for diagnosis beside the closed land.
Preferably, the blocking solution comprises 0.05% -0.5% BSA, 0.01% -0.5% Tween 20 and 10mM-200mM PBS; the dilution was 10mM PBS. Wherein BSA is bovine serum albumin, and the total volume ratio of the mass of BSA to the blocking solution is preferably 0.1%; tween 20 is a surfactant, preferably the ratio of the volume to the total volume of the blocking solution is 0.01%; PBS is a buffer, preferably at a concentration of 10mM.
The invention also provides a detection method of the microfluidic chip, which comprises the following steps:
step A, adding samples and mixing evenly: adding a sample by a pipette gun, absorbing a detection sample, adding the detection sample into a sample adding hole of the micro-fluidic chip subjected to bonding, then placing the micro-fluidic chip subjected to sample adding into a clamping groove of a detection instrument, combining the sample adding hole of the micro-fluidic chip with a gas circuit driving device of the instrument, controlling the gas circuit driving device to generate alternate positive pressure and negative pressure in the sample adding hole, driving the detection sample to flow back and forth in a reaction cavity and a mixing pipeline in the chip, uniformly mixing the detection sample with a fluorescent microsphere marking reagent in the mixing pipeline while re-dissolving the fluorescent microsphere marking reagent, stopping generating positive or negative pressure after the mixing is finished, and incubating the mixed solution to enable immune complex reaction to occur, so as to form a fluorescent microsphere marked antibody/antigen complex;
capturing of antibody/antigen complex: after the step A is completed, the gas circuit driving device generates a high positive gas pressure at the sample adding hole, drives a uniformly mixed sample containing the antibody/antigen compound to flow through the super-hydrophobic pipeline, reduces the high positive gas pressure to be low positive gas pressure, drives the uniformly mixed sample to flow into a carrier film in the chip detection pipeline, and simultaneously carries out immune reaction on a capture antibody reagent coated in the carrier film and the antibody/antigen compound in the uniformly mixed sample to form a capture antibody-antigen-labeled antibody compound, and other matters which are not captured flow into a waste liquid tank along with liquid and are absorbed by absorbent paper;
Step C, data reading: and B, after the step B is finished, the optical device of the detection instrument reads the fluorescence intensity on the carrier film in the microfluidic chip, calculates the concentration of the capture antibody-antigen-labeled antibody and generates a detection result.
The positive pressure and the negative pressure generated by the driving device in the detection step and the magnitudes and time of the high positive pressure and the low positive pressure are set according to actual requirements.
Due to the implementation of the technical scheme, the microfluidic chip has the following advantages compared with the prior art:
1. the microfluidic chip is provided with the reaction cavity with fixed reaction volume and the mixing pipeline, and the active driving mode is combined to drive the detection sample and the reagent to flow in the pipeline, so that the re-dissolution and mixing process of the sample and the reagent is accurately controlled, the reagent re-dissolution volume and the reaction time are strictly controllable, and the detection repeatability is greatly improved;
2. the super-hydrophobic pipeline designed by the microfluidic chip forms a passive flow blocking valve, is simple to package, greatly simplifies the chip production process and improves the chip yield;
3. the microfluidic chip disclosed by the invention is coated with more than one capture antibody in different areas of the carrier film, so that multi-project combined detection is realized.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a perspective view of a microfluidic chip in a preferred embodiment 1 of the present invention;
fig. 2 is a laminated view of a microfluidic chip according to a preferred embodiment 1 of the present invention;
FIG. 3 is an enlarged view of section I of FIG. 2;
FIG. 4 is an enlarged view of section II of FIG. 2;
in the accompanying drawings: the chip comprises a chip cover plate-1, a chip substrate-2, a sample adding port-3, a reaction cavity-4, a transition pipeline-5, a third micrometer column-51, a boss-52, a uniform mixing pipeline-6, a second micrometer column-61, a super-hydrophobic pipeline-7, a first micrometer column-71, a groove microstructure-72, a detection pipeline-8, a carrier film-9, a waste liquid tank-10 and absorbent paper-11.
Detailed Description
In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that, the terms "first" and "second" are used herein for convenience in distinguishing a plurality of objects, and are not limited. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, apparatus, article, or device that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed or inherent to such process, method, article, or device.
Example 1 microfluidic chip
As shown in fig. 1-4, a microfluidic chip of the present embodiment includes a chip substrate 2 and a chip cover plate 1, a sample adding port 3 for adding a detection sample is provided on the chip cover plate 1, a reaction chamber 4 located below the sample adding port 3, a mixing pipeline 6 communicated with the reaction chamber 4, a detection pipeline 8 communicated with the mixing pipeline 6, and a waste liquid tank 10 are sequentially provided on the chip substrate 2, and a water absorbing paper 11 matched with the waste liquid tank 10 is transferred in the waste liquid tank 10. The depth of the detection pipe 8 in this embodiment is 200 μm, and the depth of the waste liquid tank 10 is 1mm; in other embodiments, the depth of the detection conduit 8 may be in the range of 50 μm to 1000 μm and the depth of the waste liquid tank 10 may be in the range of 0.2mm to 2mm.
The mixing pipeline 6 is coated with fluorescent microsphere marking reagent for providing fluorescent detection signals, the detection pipeline 8 is provided with a carrier film 9, and the carrier film 9 is coated with at least one capture antibody reagent. Wherein the fluorescent microsphere labeling reagent is a detection antibody marked by fluorescent microsphere, and the capture antibody reagent is a capture antibody marked by nanometer polystyrene microsphere.
The mixing pipeline 6 is communicated with the reaction cavity 4, and can provide a space for the detection sample to flow back and forth, and the detection sample is uniformly mixed with the fluorescent microsphere marked reagent coated by the redissolution of the fluorescent microsphere marked reagent, so that liquid homogeneous phase immunoreaction occurs to form a reaction compound. After the detection sample enters the mixing pipeline 6, the fluorescent microsphere marking reagent is redissolved, then the detection sample and the fluorescent microsphere marking reagent are uniformly mixed, and finally the detection sample and the fluorescent microsphere marking reagent are subjected to incubation reaction, wherein the redissolution and the mixing basically occur in the mixing pipeline 6, and the incubation mainly occurs in the reaction cavity 4. The reaction chamber 4 may store a detection sample and provide a reaction site for the detection sample and a reagent.
As shown in fig. 2-3, in this embodiment, a superhydrophobic pipeline 7 is further disposed between the mixing pipeline 6 and the detecting pipeline 8. The superhydrophobic conduit 7 includes a groove microstructure 72 and a first micron post 71 of the microstructure, and the surface of the groove microstructure 72 has a superhydrophobic agent. The superhydrophobic conduit 7 may serve the transitional function of the detection sample reacting with the reagent to form a reaction complex, the reaction complex flowing to the detection conduit 8. The depth of each groove in the groove microstructure 72 in this embodiment is 50 μm and the width is 4mm; the first micrometer-column 71 has a diameter of 100 μm and a height of 100 μm; the superhydrophobic reagent is fluorosilane dissolved in an electronic fluorination solution. In other embodiments, the depth of each groove in groove microstructure 72 ranges from 20-500 μm and the width ranges from 50-4000 μm; the first micrometer-column 71 has a diameter in the range of 20-500 μm and a height in the range of 20-500 μm.
The superhydrophobic surface formed by the superhydrophobic reagent and the groove microstructure 72 can realize a passive flow blocking valve, when a detection sample is driven to flow to the superhydrophobic surface, the liquid can be effectively blocked from continuing to flow, the liquid can be left in the groove and can not be spread out, the driving force must be increased to drive the liquid to continue to flow forwards, and the controllability of the liquid flow is further enhanced.
As shown in fig. 2-3, a second micrometer column 61 with a micrometer structure is arranged in the mixing pipeline 6, and the fluorescent microsphere marking reagent is coated on the second micrometer column 61. The second micrometer-column 61 in this embodiment has a diameter of 200 μm and a height of 200 μm. In other embodiments, the second micron posts 61 have a diameter in the range of 10-500 μm and a height in the range of 10-500 μm.
As shown in fig. 2 and 4, in this embodiment, a transition pipe 5 is disposed between the mixing pipe 6 and the reaction chamber 4, and a third micrometer post 51 and a boss 52 with micrometer structures are disposed in the transition pipe 5. The third micrometer column 51 and the boss 52 in the transition pipeline 5 can further play a role in uniformly mixing the liquid flowing between the reaction cavity 4 and the uniform mixing channel 6, so that the reaction between the detection sample and the reagent is more sufficient. The third micrometer-column 51 has a diameter of 200 μm and a height of 200 μm; the height of the boss 52 is 200 μm and the width is 200 μm. In other embodiments, the third micron posts 51 have a diameter in the range of 20-500 μm and a height in the range of 20-500 μm; the height of the boss 52 is in the range of 20-1000 μm and the width is in the range of 100-4000 μm.
The carrier film 9 in this embodiment is a nitrocellulose film. Only one capture antibody reagent is coated in the carrier film 9, and the capture antibody reagent can be randomly dispersed in the carrier film 9; if two or more capture antibody reagents are coated in the carrier film 9, it is necessary to divide the carrier film 9 into several regions, and different capture antibody reagents are coated in different regions. In this way, in the final detection, the fluorescent signal generated by the complex corresponding to the different regions can be accurately distinguished.
When the microfluidic chip in this embodiment is specifically used, the sample loading port may be connected to a driving device, and the driving mode of the driving device may be a pneumatic, hydraulic or electric mode, and the driving device provides a driving force for the applied detection sample to flow back and forth in the pipeline of the chip, so that the detection sample fully reacts with the reagent in the pipeline.
The microfluidic chip in the embodiment is provided with a reaction cavity with a fixed reaction volume and a mixing pipeline, and the active driving mode is combined to drive the detection sample and the reagent to flow in the pipeline, so that the re-dissolution and mixing process of the sample and the reagent is accurately controlled, the reagent re-dissolution volume and the reaction time are strictly controllable, and the detection repeatability is greatly improved; the super-hydrophobic pipeline is designed to form a passive flow blocking valve, so that the packaging is simple, the chip production process is greatly simplified, and the chip yield is improved; more than one capture antibody can be coated in different areas of the carrier film, so that multi-project combined detection is realized.
Example 2 preparation method of microfluidic chip
The embodiment provides a preparation method of the microfluidic chip based on embodiment 1, which specifically comprises the following steps:
step 1) preparation of fluorescent microsphere labeling reagent
And (3) marking the purified detection antibody raw material by adopting a time-resolved fluorescence microsphere analysis method, and collecting fluorescence microsphere markers, namely the fluorescence microsphere marking reagent.
Step 2) preparation of Capture antibody reagent
Diluting the purified capture antibody raw material by adopting a diluent, and marking the diluted capture antibody raw material on the nanometer polystyrene microsphere to prepare the capture antibody reagent.
The dilution in this example was 10mM PBS.
Step 3) super-hydrophilic modification of chip surface material
The super-hydrophilic modification is carried out on the chip surface material by adopting a vacuum plasma bombardment or atmospheric plasma bombardment method.
Step 4) spraying and drying the sealing liquid
After the chip surface super-hydrophilicity modification in the step 3) is finished, spraying a layer of sealing liquid on the surface of the microfluidic chip to seal the surface, and then drying the chip.
The blocking solution in this example included 0.1% BSA, 0.01% Tween 20 and 10mM PBS. Wherein BSA is bovine serum albumin, tween 20 is a surfactant, and PBS is a buffer solution.
Step 5) preparation of superhydrophobic pipeline
And (3) after the chip in the step (4) is dried, performing superhydrophobic modification on the surface of a specific pipeline in the chip, namely the surface of the groove microstructure by using a precise sample application instrument and a superhydrophobic reagent, and then drying the chip to obtain the superhydrophobic pipeline.
Step 6) drying the fluorescent microsphere labeling reagent
And 5) after the chip is dried in the step 5), adding the fluorescent microsphere markers collected in the step 1) into a uniformly mixing pipeline in a chip substrate, and then drying to enable the fluorescent microsphere markers to be dried in the chip and coated on a second micrometer column.
Step 7) immobilization of Capture antibody
And 6) after the chip is dried in the step 6), coating the capture antibody reagent prepared in the step 2) on a carrier film by adopting a precise sample application instrument, then drying to fix the capture antibody marked by the polystyrene microsphere on the carrier film, and placing the carrier film coated with the capture antibody reagent on a detection pipeline of a chip substrate.
If only one capture antibody reagent is coated in the carrier film, the capture antibody reagent can be dispersed randomly in the carrier film; if two or more capture antibody reagents are coated in the carrier film, the carrier film needs to be divided into a plurality of areas, and different capture antibody reagents are coated in different areas, so that fluorescent signals generated by complexes corresponding to different areas can be distinguished in the final detection.
Step 8) microfluidic chip packaging
And (3) placing water absorbing paper in the waste liquid tank, and assembling and bonding the chip cover plate and the chip substrate to obtain the microfluidic chip.
The chip substrate and the cover plate are bonded together through pressure glue, ultrasonic wave, laser and the like to form the microfluidic chip for diagnosis beside the closed land.
Example 3 detection method of microfluidic chip
The embodiment provides a detection method based on the microfluidic chip described in embodiment 1, which specifically includes the following steps:
step A, adding samples and mixing uniformly
And (3) adding samples by a pipette gun, sucking the detection samples, adding the detection samples into sample adding holes of the microfluidic chip subjected to bonding, then placing the microfluidic chip subjected to sample adding into a clamping groove of a detection instrument, combining the sample adding holes of the microfluidic chip with an air path driving device of the instrument, controlling the air path driving device to generate alternate positive pressure and negative pressure in the sample adding holes, driving the detection samples to flow back and forth in a reaction cavity and a mixing pipeline in the chip, uniformly mixing the detection samples with fluorescent microsphere labeled reagents in the mixing pipeline while re-dissolving the fluorescent microsphere labeled reagents, stopping generating positive air pressure or negative air pressure after the mixing is finished, and incubating the mixed solution to enable immune complex reaction to occur, so as to form the fluorescent microsphere labeled antibody/antigen complex.
Step B. Capture of antibody/antigen complexes
After the step A is completed, the gas path driving device generates a high positive gas pressure at the sample adding hole, drives the uniformly mixed sample containing the antibody/antigen compound to flow through the super-hydrophobic pipeline, reduces the high positive gas pressure to be low positive gas pressure, drives the uniformly mixed sample to flow into a carrier film in the chip detection pipeline, and simultaneously carries out immune reaction on the capture antibody reagent coated in the carrier film and the antibody/antigen compound in the uniformly mixed sample to form the capture antibody-antigen-labeled antibody compound, and other matters which are not captured flow into a waste liquid tank along with liquid and are absorbed by absorbent paper.
Step C, data reading
And B, after the step B is finished, the optical device of the detection instrument reads the fluorescence intensity on the detection pipeline of the microfluidic chip, calculates the concentration of the capture antibody-antigen-labeled antibody and generates a detection result.
The positive air pressure and the negative air pressure generated by the driving device in the detection step and the high positive air pressure and the low positive air pressure are set according to actual requirements in time.
EXAMPLE 4 detection of Procalcitonin (PCT) by microfluidic chip
Antibody labelling
A. Fluorescent microsphere marked detection antibody
1. A20 mg/ml EDC, 10mM PBS solution was prepared.
2. 90ul of 10mM PBS buffer solution is taken, 10ul of 300nm fluorescent microspheres are added, and shaking and mixing are carried out.
3. Adding 5ul EDC activating solution into the buffer solution containing fluorescent microspheres prepared in the step 2, shaking and mixing uniformly, and placing in a shaking table for 15min (room temperature or 20 ℃ C., 250 rpm).
4. Preparing 100ul of labeled antibody: PCT detection monoclonal antibody and rabbit IgG antibody solutions were formulated with 10mM PBS buffer at a final concentration of 0.5mg/ml.
5. And (3) placing the activated fluorescent microsphere solution prepared in the step (3) in a centrifugal machine, centrifuging at 15000rpm for 15min, and discarding the supernatant.
6. And (3) adding the PCT antibody and rabbit IgG antibody solution with the concentration of 0.5mg/ml obtained in the step (4) into the centrifuged activated fluorescent microsphere solution obtained in the step (5), and shaking and uniformly mixing. Ultrasonic treatment for 2-3min, shaking, mixing, and incubating in a shaker for 2 hr under the following conditions: at room temperature, 250rpm.
7. Adding 20ul of blocking solution (1% bovine serum albumin) into the solution obtained in the step 6, and placing the solution in a shaking table for blocking for 2 hours, wherein the blocking conditions are as follows: at room temperature, 250rpm.
8. The solution obtained in step 7 was placed in a centrifuge, centrifuged at 15000rpm for 15min, and the supernatant was discarded.
9. Adding 500ul of 10mM PBS buffer solution into the substance obtained in the step 8, redissolving the fluorescent microspheres, shaking, mixing uniformly, placing in a centrifuge at 15000rpm, centrifuging for 15min, and discarding the supernatant.
10. Adding 200ul microsphere preservation solution (0.1% BSA, 5% sucrose, 10mM PBS) into the material obtained in step 9, shaking, mixing, and ultrasound for 3min, and preserving at 2-8deg.C.
B. Polystyrene microsphere marked capture antibody
1. A20 mg/ml EDC, 10mM PBS solution was prepared.
2. 90ul of 10mM PBS buffer solution is taken, 10ul of 150nm polystyrene microsphere is added, and shaking and mixing are carried out.
3. Adding 5ul EDC activated solution into the buffer solution with polystyrene microsphere prepared in step 2, shaking, mixing, and shaking for 15min (room temperature or 20deg.C, 250 rpm).
4. Preparing 100ul capture antibody: PCT coated monoclonal antibody and goat anti-rabbit IgG antibody solutions were formulated with 10mM PBS buffer at a final concentration of 0.5mg/ml.
5. Placing the activated polystyrene microsphere solution prepared in the step 3 into a centrifugal machine, centrifuging at 15000rpm for 15min, and discarding the supernatant.
6. And (3) adding the PCT antibody and goat anti-rabbit IgG antibody solution prepared in the step (4) into the substance obtained in the step (5), and shaking and mixing uniformly. Ultrasonic treatment for 2-3min, shaking, mixing, and incubating in a shaker for 2 hr under the following conditions: at room temperature, 250rpm.
7. Adding 20ul of blocking solution (1% bovine serum albumin) into the substance prepared in the step 6, and placing the mixture in a shaking table for blocking for 2 hours, wherein the blocking conditions are as follows: at room temperature, 250rpm.
8. Placing the substance prepared in the step 7 into a centrifugal machine, centrifuging at 15000rpm for 15min, and discarding the supernatant.
9. 500ul of 10mM PBS buffer solution is added into the substance prepared in the step 8, the polystyrene microspheres are redissolved, the mixture is placed in a centrifuge after shaking and mixing, the centrifuge is centrifuged at 15000rpm for 15min, and the supernatant is discarded.
10. Adding 200ul of 10mM PBS to the substance prepared in the step 9, shaking, mixing uniformly, performing ultrasonic treatment for 3min, and storing at 2-8 ℃.
(II) microfluidic chip Assembly
1. The super-hydrophilic modification is carried out on the chip substrate surface material by adopting a vacuum plasma bombardment or atmospheric plasma bombardment method.
2. After the chip surface super-hydrophilicity modification is finished, spraying a layer of sealing liquid on the surface of the microfluidic chip to seal the surface, and then drying the chip. The blocking solution contained 0.1% BSA, 0.01% Tween 20 and 10mM PBS.
3. And (2) after the chip is dried, performing superhydrophobic modification on the surface of a specific pipeline in the chip, namely the surface of the groove microstructure by using a precise sample application instrument and a superhydrophobic reagent, and then drying the chip to obtain the superhydrophobic pipeline.
4. And (3) after the middle chip in the step (3) is dried, adding the PCT monoclonal antibody and the rabbit IgG antibody marked by the fluorescent microspheres collected in the step (one) into a mixing pipeline in a chip substrate, and then drying to dry the fluorescent microsphere markers in the mixing pipeline of the chip.
5. And (3) after the middle chip in the step (4) is dried, coating the PCT capture antibody reagent and the goat anti-rabbit IgG antibody reagent which are marked by the polystyrene microspheres and prepared in the step (one) on a carrier film by adopting a precise sample application instrument, then drying, fixing the capture antibody marked by the polystyrene microspheres on the carrier film, and placing the carrier film coated with the capture antibody reagent on a detection pipeline of a chip substrate.
6. And (3) placing water absorbing paper in the waste liquid tank, assembling the chip cover plate and the chip substrate, and bonding through pressure glue to obtain the microfluidic chip.
(III) sample detection
1. And (3) a liquid-transfering gun samples serum samples containing PCT antigen, absorbing 150 mu L of detection samples, adding the detection samples into sample-adding holes of the micro-fluidic chip subjected to bonding, then placing the micro-fluidic chip subjected to sample-adding into a clamping groove of a detection instrument, combining the sample-adding holes of the micro-fluidic chip with an air channel driving device of the instrument, controlling the air channel driving device to generate alternate positive pressure and negative pressure in the sample-adding holes, driving the detection samples to flow back and forth in a reaction cavity and a mixing pipeline in the chip, uniformly mixing the detection samples and the fluorescent microsphere-labeled PCT monoclonal antibody and rabbit IgG antibody reagent in the mixing pipeline, stopping generating positive air pressure or negative air pressure after the mixing is completed, and incubating the mixed solution to perform immune complex reaction to form the fluorescent microsphere-labeled PCT monoclonal antibody/PCT antigen complex.
2. After the step 1 is completed, the gas path driving device generates high positive gas pressure in the sample adding hole, drives the uniformly mixed sample containing the PCT antibody/PCT antigen complex to flow through the super-hydrophobic pipeline, reduces the high positive gas pressure to low positive gas pressure, drives the uniformly mixed sample to flow into a carrier film in the chip detection pipeline, and simultaneously carries out immunoreaction with the PCT-labeled antibody/PCT antigen complex and the rabbit IgG antibody in the uniformly mixed sample to form the PCT-labeled antibody-PCT antigen-PCT-labeled antibody complex and the rabbit IgG-goat anti-rabbit IgG antibody complex, wherein other matters which are not captured flow into a waste liquid tank along with liquid and are absorbed by absorbent paper.
3. After the step 2 is completed, an optical device of the detection instrument reads the fluorescence intensity (T1 value) of the PCT capture antibody-PCT antigen-PCT labeled antibody complex and the fluorescence intensity (C value) of the rabbit IgG-sheep anti-rabbit IgG antibody complex on a microfluidic chip detection pipeline to obtain a fluorescence intensity ratio (T/C value), and then the concentration of the PCT capture antibody-PCT antigen-PCT labeled antibody is calculated through a calibration curve to generate a detection result.
The positive air pressure and the negative air pressure generated by the driving device in the detection step, the high positive air pressure and the low positive air pressure and the driving time are set according to actual requirements.
Comparative example 1
This comparative example provides a conventional passive bedside diagnostic microfluidic chip. The microfluidic chip provided in this comparative example was substantially the same as in example 1, except that the superhydrophobic tubing and the carrier film were not present in this comparative example. The microfluidic chip of the comparative example comprises a chip substrate and a chip cover plate, wherein a sample adding port for adding a detection sample is formed in the chip cover plate, and a reaction cavity communicated with the sample adding port, a detection pipeline communicated with the reaction cavity and a waste liquid tank communicated with the detection pipeline are formed in the chip substrate. In the comparative example, a fluorescent microsphere marking reagent for providing a fluorescent detection signal is coated in a reaction cavity, and a polystyrene marked capture antibody reagent is coated in a detection pipeline.
Antibody labelling
A. Fluorescent microsphere marked detection antibody
1. A20 mg/ml EDC, 10mM PBS solution was prepared.
2. 90ul of 10mM PBS buffer solution is taken, 10ul of 300nm fluorescent microspheres are added, and shaking and mixing are carried out.
3. Adding 5ul EDC activating solution into the buffer solution containing fluorescent microspheres prepared in the step 2, shaking and mixing uniformly, and placing in a shaking table for 15min (room temperature or 20 ℃ C., 250 rpm).
4. Preparing 100ul of labeled antibody: PCT detection monoclonal antibody and rabbit IgG antibody solutions were formulated with 10mM PBS buffer at a final concentration of 0.5mg/ml.
5. And (3) placing the activated fluorescent microsphere solution prepared in the step (3) in a centrifugal machine, centrifuging at 15000rpm for 15min, and discarding the supernatant.
6. And (3) adding the PCT antibody and rabbit IgG antibody solution with the concentration of 0.5mg/ml obtained in the step (4) into the centrifuged activated fluorescent microsphere solution obtained in the step (5), and shaking and uniformly mixing. Ultrasonic treatment for 2-3min, shaking, mixing, and incubating in a shaker for 2 hr under the following conditions: at room temperature, 250rpm.
7. Adding 20ul of blocking solution (1% bovine serum albumin) into the solution obtained in the step 6, and placing the solution in a shaking table for blocking for 2 hours, wherein the blocking conditions are as follows: at room temperature, 250rpm.
8. The solution obtained in step 7 was placed in a centrifuge, centrifuged at 15000rpm for 15min, and the supernatant was discarded.
9. Adding 500ul of 10mM PBS buffer solution into the substance obtained in the step 8, redissolving the fluorescent microspheres, shaking, mixing uniformly, placing in a centrifuge at 15000rpm, centrifuging for 15min, and discarding the supernatant.
10. Adding 200ul microsphere preservation solution (0.1% BSA, 5% sucrose, 10mM PBS) into the material obtained in step 9, shaking, mixing, and ultrasound for 3min, and preserving at 2-8deg.C.
B. Polystyrene microsphere marked capture antibody
1. A20 mg/ml EDC, 10mM PBS solution was prepared.
2. 90ul of 10mM PBS buffer solution is taken, 10ul of 150nm polystyrene microsphere is added, and shaking and mixing are carried out.
3. Adding 5ul EDC activated solution into the buffer solution with polystyrene microsphere prepared in step 2, shaking, mixing, and shaking for 15min (room temperature or 20deg.C, 250 rpm).
4. Preparing 100ul capture antibody: PCT coated monoclonal antibody and goat anti-rabbit IgG antibody solutions were formulated with 10mM PBS buffer at a final concentration of 0.5mg/ml.
5. Placing the activated polystyrene microsphere solution prepared in the step 3 into a centrifugal machine, centrifuging at 15000rpm for 15min, and discarding the supernatant.
6. And (3) adding the PCT antibody and goat anti-rabbit IgG antibody solution prepared in the step (4) into the substance obtained in the step (5), and shaking and mixing uniformly. Ultrasonic treatment for 2-3min, shaking, mixing, and incubating in a shaker for 2 hr under the following conditions: at room temperature, 250rpm.
7. Adding 20ul of blocking solution (1% bovine serum albumin) into the substance prepared in the step 6, and placing the mixture in a shaking table for blocking for 2 hours, wherein the blocking conditions are as follows: at room temperature, 250rpm.
8. Placing the substance prepared in the step 7 into a centrifugal machine, centrifuging at 15000rpm for 15min, and discarding the supernatant.
9. 500ul of 10mM PBS buffer solution is added into the substance prepared in the step 8, the polystyrene microspheres are redissolved, the mixture is placed in a centrifuge after shaking and mixing, the centrifuge is centrifuged at 15000rpm for 15min, and the supernatant is discarded.
10. Adding 200ul of 10mM PBS to the substance prepared in the step 9, shaking, mixing uniformly, performing ultrasonic treatment for 3min, and storing at 2-8 ℃.
(II) microfluidic chip Assembly
1. The super-hydrophilic modification is carried out on the chip substrate surface material by adopting a vacuum plasma bombardment or atmospheric plasma bombardment method.
2. After the chip surface super-hydrophilicity modification is finished, spraying a layer of sealing liquid on the surface of the microfluidic chip to seal the surface, and then drying the chip. The blocking solution contained 0.1% BSA, 0.01% Tween 20 and 10mM PBS.
3. And (2) after the chip in the step (2) is dried, adding the PCT monoclonal antibody and the rabbit IgG antibody marked by the fluorescent microspheres collected in the step (one) into a reaction cavity in a chip substrate, and then drying to dry the fluorescent microsphere markers in the reaction cavity in the chip.
4. And (3) after the middle chip in the step (3) is dried, coating the polystyrene-marked PCT capture antibody and the goat anti-rabbit IgG antibody reagent prepared in the step (one) on a micrometer column detection channel in a chip substrate by adopting a precise sample application instrument, and then drying to fix the polystyrene microsphere-marked capture antibody on the micrometer column detection channel.
5. And assembling the chip cover plate and the chip substrate and bonding the chip cover plate and the chip substrate through pressure glue to obtain the microfluidic chip.
(III) sample detection
1. And (3) a serum sample containing PCT antigen is added by a pipette, 150 mu L of detection sample is sucked and added into a sample adding hole of the microfluidic chip after bonding, then the microfluidic chip after sample adding is placed into a clamping groove of a detection instrument, the detection sample flows into a reaction cavity through capillary suction, and fluorescent microspheres in the reaction cavity are used for marking PCT monoclonal antibody and rabbit IgG antibody reagent and simultaneously carrying out immune complex reaction with the PCT monoclonal antibody and the PCT antigen reagent to form a PCT monoclonal antibody/PCT antigen complex marked by the fluorescent microspheres.
2. The evenly mixed sample containing the PCT antibody/PCT antigen complex flows into a detection pipeline of the chip by self-driving by capillary force, and simultaneously, the PCT capture antibody and the goat anti-rabbit IgG antibody marked by the polystyrene microsphere coated in the pipeline respectively react with the PCT antibody/PCT antigen complex and the rabbit IgG antibody in the sample in an immune way to form the PCT capture antibody-PCT antigen-PCT marked antibody complex and the rabbit IgG-goat anti-rabbit IgG antibody complex, and other matters which are not captured flow into a waste liquid tank along with liquid.
3. After the step 2 is completed, an optical device of the detection instrument reads the fluorescence intensity (T1 value) of the PCT capture antibody-PCT antigen-PCT labeled antibody complex and the fluorescence intensity (C value) of the rabbit IgG-sheep anti-rabbit IgG antibody complex on a microfluidic chip detection pipeline to obtain a fluorescence intensity ratio (T/C value), and then the concentration of the PCT capture antibody-PCT antigen-PCT labeled antibody is calculated through a calibration curve to generate a detection result.
Example 5 results and analysis
10 tests were performed on the same sample of PCT concentration of 0.05ng/mL using the microfluidic chip prepared in example 4 and comparative example 1, and the performance test and analysis comparison are shown in the following table:
table 1 comparison table of test results
The experimental results in table 1 show that when the same sample is tested, the active microfluidic chip in example 1 and the corresponding detection method in example 4 are adopted to be compared with the passive microfluidic chip in comparative example 1 and the detection method thereof, the detection result of the active microfluidic chip in example 1 can be seen to be significantly improved in terms of detection sensitivity from the T/C value, and the detection repeatability can be seen to be greatly improved from the detection variation coefficient.
The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the present invention and to implement the same, but are not intended to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.

Claims (7)

1. The microfluidic chip comprises a chip substrate and a chip cover plate, and is characterized in that a sample adding port for adding a detection sample is formed in the chip cover plate, a reaction cavity, a mixing pipeline, a detection pipeline and a waste liquid tank are sequentially formed in the chip substrate, the mixing pipeline is communicated with the reaction cavity, the detection pipeline is communicated with the mixing pipeline, fluorescent microsphere marking reagents for providing fluorescent detection signals are coated in the mixing pipeline, a carrier film is loaded in the detection pipeline, and at least one capture antibody reagent is coated on the carrier film;
A super-hydrophobic pipeline is also arranged between the mixing pipeline and the detection pipeline; the super-hydrophobic pipeline comprises a groove microstructure and a first micron column of a micron structure, wherein the surface of the groove microstructure is provided with a super-hydrophobic reagent; the super-hydrophobic surface formed by the super-hydrophobic reagent and the groove microstructure is used for realizing a passive flow blocking valve;
a second micron column with a micron structure is arranged in the mixing pipeline, and the fluorescent microsphere marking reagent is coated on the second micron column; a transition pipeline is arranged between the mixing pipeline and the reaction cavity, and a third micrometer column and a boss with micrometer structures are arranged in the transition pipeline;
the mixing pipeline is communicated with the reaction cavity and is used for providing a space for detecting the sample to flow back and forth.
2. The microfluidic chip according to claim 1, wherein the fluorescent microsphere-labeled reagent is a detection antibody labeled with a fluorescent microsphere.
3. The microfluidic chip according to claim 1, wherein the capture antibody reagent is a capture antibody labeled with polystyrene microspheres.
4. The microfluidic chip according to claim 1, wherein the carrier film is a nitrocellulose film.
5. A method of manufacturing a microfluidic chip according to claim 1, comprising the steps of:
step 1) preparation of fluorescent microsphere marking reagent: marking the detection antibody raw material by adopting fluorescent microspheres, and collecting fluorescent microsphere markers, namely fluorescent microsphere marking reagents;
step 2) preparation of capture antibody reagent: labeling a capture antibody raw material on a nanometer polystyrene microsphere to prepare a capture antibody reagent;
step 3) super-hydrophilic modification of the chip surface material: performing super-hydrophilic modification on the chip surface material by adopting a vacuum plasma bombardment or atmospheric plasma bombardment method;
and 4) spraying and drying the sealing liquid: spraying a layer of sealing liquid on the surface of the microfluidic chip to seal the surface, and then drying the chip;
step 5) preparation of a super-hydrophobic pipeline: covering the groove microstructure with a superhydrophobic reagent, performing superhydrophobic modification on the surface of the groove microstructure in the chip, and then performing drying treatment on the chip to obtain a superhydrophobic pipeline;
step 6) drying the fluorescent microsphere labeling reagent: adding the fluorescent microsphere markers collected in the step 1) into a uniformly mixing pipeline in a chip substrate, and then drying to enable the fluorescent microsphere markers to be dried in the chip and coated on a second micrometer column;
Step 7) immobilization of capture antibodies: coating the capture antibody reagent prepared in the step 2) on a carrier film, then drying to fix the capture antibody marked by the polystyrene microsphere on the carrier film, and placing the carrier film coated with the capture antibody reagent on a detection pipeline of a chip substrate;
step 8) packaging the microfluidic chip: and (3) placing water absorbing paper in the waste liquid tank, and assembling and bonding the chip cover plate and the chip substrate to obtain the microfluidic chip.
6. The method of claim 5, wherein the blocking solution comprises 0.05% -0.5% BSA, 0.01% -0.5% Tween 20 and 10mM-200mM PBS.
7. A method of detecting a microfluidic chip according to claim 1, comprising the steps of:
step A, adding samples and mixing evenly: absorbing a detection sample, adding the detection sample into a sample adding hole of a microfluidic chip, then placing the microfluidic chip with the sample adding completion into a clamping groove of a detection instrument, combining the sample adding hole of the microfluidic chip with a driving device of the detection instrument, controlling the driving device to generate alternate positive pressure and negative pressure in the sample adding hole, driving the detection sample to flow back and forth in a reaction cavity and a mixing pipeline in the chip, uniformly mixing the detection sample with a fluorescent microsphere marked reagent in the mixing pipeline while re-dissolving the fluorescent microsphere marked reagent, stopping generating positive pressure or negative pressure after the mixing is completed, and incubating the mixed solution to enable immune complex reaction to occur to form a fluorescent microsphere marked antibody/antigen complex;
Capturing of antibody/antigen complex: the driving device generates a high positive pressure in the sample adding hole, drives the mixed sample containing the antibody/antigen complex to flow through the super-hydrophobic pipeline, reduces the high positive pressure to a low positive pressure, drives the mixed sample to flow into a carrier film in the chip detection pipeline, and simultaneously performs immune reaction between a capture antibody reagent coated in the carrier film and the antibody/antigen complex in the mixed sample to form a capture antibody-antigen-labeled antibody complex, and the non-captured substance flows into the waste liquid tank;
step C, data reading: and an optical device of the detecting instrument reads the fluorescence intensity on the carrier film in the microfluidic chip, calculates the concentration of the capture antibody-antigen-labeled antibody and generates a detection result.
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