CN110988332A - Multicolor fluorescence microfluidic chip detection method and microfluidic chip for realizing method - Google Patents

Multicolor fluorescence microfluidic chip detection method and microfluidic chip for realizing method Download PDF

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CN110988332A
CN110988332A CN201911152694.XA CN201911152694A CN110988332A CN 110988332 A CN110988332 A CN 110988332A CN 201911152694 A CN201911152694 A CN 201911152694A CN 110988332 A CN110988332 A CN 110988332A
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bin
sample
antibody
channel
micro
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CN110988332B (en
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唐勇
廖政
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Sichuan Park Lan Medical Technology Co ltd
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Sichuan Park Lan Medical Technology Co ltd
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    • 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
    • G01N33/54326—Magnetic particles
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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
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
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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
    • 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
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—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 multiphase flow arrangements
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • 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
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0883—Serpentine channels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00178—Special arrangements of analysers
    • G01N2035/00277—Special precautions to avoid contamination (e.g. enclosures, glove- boxes, sealed sample carriers, disposal of contaminated material)

Abstract

The invention provides a multicolor fluorescence micro-fluidic chip detection method and a micro-fluidic chip for realizing the method, the method is based on the micro-fluidic technology, the mixing, the flowing mixing and capturing reaction, the magnetic separation, the cleaning, the detection and the like of a sample and a reagent are integrated on the chip, and all reagent components required by the reaction are integrated on the chip, so that the operation of a client is simple and convenient, and the detection can be completed at any time and any place. A closed micro-channel for a sample to flow is formed in the micro-fluidic chip, and the micro-channel comprises a mixing bin, a reaction bin, a delay channel, a capturing bin and a waste liquid bin which are sequentially communicated.

Description

Multicolor fluorescence microfluidic chip detection method and microfluidic chip for realizing method
Technical Field
The invention relates to the technical field of microfluidic instantaneous detection, in particular to a multicolor fluorescence microfluidic chip detection method and a microfluidic chip for realizing the method.
Background
The microfluidic technology is the most rapidly developed detection method in recent years, and the method automatically completes the analysis process by integrating basic operation units of sample preparation, reaction, separation, detection and the like in the biological, chemical and medical analysis processes on a micron-scale chip. All operation steps are integrated through the matching combination of the size and the curvature of the flow channel, the micro valve and the cavity design, and finally the whole detection integration is miniaturized and automated. The POCT (Point-of-care Testing) diagnostic reagent developed by the microfluidic chip technology can effectively and quickly finish accurate diagnosis and treatment of diseases.
However, most of the current microfluidic chips can only complete detection of a single index through a single chip, for example, patent CN201510696773 realizes the purpose of accurate detection of diseases, but has low detection efficiency, and it is difficult to simultaneously detect multiple biological targets of the same disease in the same chip.
Patent CN200910159718.4 by the company of micropoint mentions that antibodies of different targets are coated at different positions in the same microfluidic chip channel, the same fluorescent substance is used to label the labeled antibodies of different targets, and then the detection of different targets can be completed in one channel at the same time. However, the use of the same fluorescence to label different antibodies may lead to false positive or non-specific results due to possible cross-reactivity between the different antibodies.
The research and development of a multicolor fluorescence micro-fluidic immunodetection chip product for simultaneously detecting multiple indexes are very important by integrating the prior art and the development prospect.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a multicolor fluorescence microfluidic chip detection method. Compared with the prior art, the method has the advantages of short detection time, high sensitivity and good repeatability, and can complete simultaneous detection of multiple indexes.
A second object of the invention is to provide a microfluidic chip for implementing the method.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention relates to a microfluidic chip detection method based on a magnetic bead technology and a reagent freeze-drying technology, which comprises the following steps:
(1) sample adding: injecting a sample into a sample filling port of a micro-fluidic chip, wherein the micro-fluidic chip is internally provided with a micro-channel, the micro-channel comprises a mixing bin, a reaction bin, a time delay channel, a capturing bin and a waste liquid bin which are sequentially communicated, the sample enters the mixing bin to dissolve a reagent ball to obtain a mixed sample,
the reagent balls are pre-loaded into the mixing bin and comprise capture reagent balls and marking reagent balls, the capture reagent balls are magnetic beads coated with antibodies, antigens or secondary antibodies, the marking reagent balls are fluorescent microspheres coated with the antibodies, the antigens or the secondary antibodies, and the fluorescent microspheres contain multiple rare earth elements; the reagent ball is used for immunoreaction with the test sample;
(2) reaction: the mixed sample enters a reaction bin from the mixing bin and further enters a delay channel, so that the immunoreaction is fully carried out;
(3) capturing: after the immunoreaction is finished, the mixed sample enters a capturing bin from the delay channel, a magnet positioned on the bottom wall of the capturing bin adsorbs a conjugate of magnetic beads coated with an antibody, an antigen or a secondary antibody, which is generated by the immunoreaction, to the capturing bin, and the rest of the sample continuously flows into a waste liquid bin;
(4) cleaning: injecting cleaning fluid into the micro-channel, enabling the residual mixed sample to enter a waste fluid bin, and capturing the magnetic bead conjugate only contained in the bin;
(5) and (3) detection: and pushing the microfluidic chip into a detection module for detection.
Preferably, the capture reagent ball is prepared by the following method:
[1] coating an antibody, an antigen or a secondary antibody on the surface of the magnetic bead;
[2] and freezing the coated magnetic beads in liquid nitrogen, and then vacuumizing and drying.
Preferably, the magnetic beads are superparamagnetic particles, and the material is ferric oxide and/or ferroferric oxide.
Preferably, the particle size of the magnetic beads is 0.1-10 μm, preferably 1 μm.
Preferably, the magnetic induction intensity of the magnetic beads is 1000-30000 gauss, and preferably 2000-10000 gauss.
Preferably, the labeled reagent ball is prepared by the following method:
[1] coating an antibody, an antigen or a secondary antibody on the surface of the fluorescent microsphere;
[2] freezing the coated fluorescent microspheres in liquid nitrogen, and then vacuumizing and drying.
Preferably, the fluorescent microspheres include polystyrene fluorescent microspheres containing Eu, polystyrene fluorescent microspheres containing Tb, and polystyrene fluorescent microspheres containing Sm. The ratio between different fluorescent microspheres can be adjusted according to the concentration of different antigens in blood and the titer of antibodies.
Preferably, in step (1), before injecting the sample, the microfluidic chip is subjected to a hydrophilic treatment, wherein the hydrophilic treatment is at least one selected from plasma surface cleaning treatment, chemical surface modification, polymer hydrophilic substance treatment, Chemical Vapor Deposition (CVD) and surface self-assembly.
Preferably, in the step (4), the cleaning solution contains a buffer system, a surfactant, a sealant and a preservative, wherein the buffer system is at least one selected from phosphate, carbonate and tris-HCL, and the pH value of the cleaning solution is 5-11;
the sealant is at least one of bovine serum albumin, casein, fish skin gelatin and synthetic sealant;
the surfactant is at least one selected from Tween 20 and Triton x-100;
the preservative is at least one selected from Proclin-300 and sodium azide.
In the examples of the present invention, the washing solution used contained tween 20, casein, and a phosphate buffer solution of Proclin-300 having a pH of 7.4.
Preferably, in step (5), the fluorescence intensity in the capture chamber is detected by using a time-resolved fluorescence detector, and then the concentration of the antigen or antibody in the analyte is determined by using a standard curve.
The invention also relates to a micro-fluidic chip for realizing the detection method of the multicolor fluorescence micro-fluidic chip, which has the following structure:
the microfluidic chip comprises a substrate and an upper cover, wherein the substrate is provided with a concave cavity, the upper cover seals the concave cavity and forms a closed micro-channel for a sample to flow,
the micro flow channel comprises a mixing bin, a reaction bin, a capturing bin and a waste liquid bin which are sequentially communicated, the micro flow channel is provided with a vent hole communicated with the outside atmosphere and a sample filling port communicated with the mixing bin, a first communicating channel is arranged between the mixing bin and the reaction bin, a second communicating channel is arranged between the capturing bin and the waste liquid bin,
a delay passage communicated with the reaction chamber and the capture chamber is arranged between the reaction chamber and the capture chamber, the delay passage is of a zigzag structure and is used for the sample to zigzag flow in the delay passage, a solid reagent ball used for immunoreaction with the sample is arranged in the mixing chamber,
and a magnet for adsorbing the magnetic beads is arranged on the bottom wall of the capturing bin.
The invention has the beneficial effects that:
the invention provides a multicolor fluorescence micro-fluidic chip detection method, which integrates the mixing process of a sample and a reagent ball, the flow reaction and capture process, the magnetic separation process, the cleaning and detection process on a chip and integrates all reagent components required by the reaction on the chip on the basis of the micro-fluidic technology, so that the client is simple and convenient to operate and can finish the detection anytime and anywhere. And the method can package a plurality of fluorescent microspheres containing a plurality of rare earth elements in the same chip, thereby completing the simultaneous detection of a plurality of indexes.
Furthermore, the freeze-dried reagent ball and the microfluidic chip are combined, so that the freeze-dried reagent can be quickly released and mixed, and the reagent ball and a sample do not need to be sucked and mixed back and forth when the freeze-dried reagent ball is used. In addition, the whole chip can be stored for a long time at normal temperature due to the use of the freeze-dried reagent balls, the requirements that reagents in the traditional liquid-phase chip must be stored at 4 ℃, and need to be transported by a cold chain are met, and the transportation and storage cost of the reagents is greatly reduced.
Drawings
FIG. 1 is a schematic overall view of a single-channel fluoroimmunoassay microfluidic chip according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of the composition of the upper cover and the substrate in an embodiment of the present invention;
FIG. 3 is a schematic view showing the structure of a microchannel according to an embodiment of the present invention;
FIG. 4 is a schematic vertical cross-sectional view of a single-channel fluoroimmunoassay microfluidic chip according to an embodiment of the present invention;
FIG. 5 is a schematic top view of the top cover in an embodiment of the present invention;
fig. 6 is a schematic view of the lower surface of the upper cover in the embodiment of the present invention.
In fig. 1-6:
the device comprises an upper cover-1, a connecting column-101, a substrate-2, a jack-201, a micro flow channel-3, a mixing bin-301, a reaction bin-302, a delay channel-303, a capturing bin-304, a waste liquid bin-305, a sample filling port-306, a vent hole-307, a first communicating channel-308, a second communicating channel-309, a cleaning liquid placing bin-4, an opening-401, an injection channel-5, a puncture column-501, a cleaning liquid bubble-6 and a reagent ball-7.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The invention relates to a multicolor fluorescence micro-fluidic chip detection method, which is carried out in a micro-fluidic chip with the following structure.
[ microfluidic chip ]
As shown in fig. 1 to 6, the microfluidic chip of the present invention includes a substrate 2 and an upper cover 1, wherein the substrate 2 has a cavity, and the cavity is sealed by the upper cover 1 to form a closed microchannel 3 for a sample to flow.
The microchannel 3 includes a mixing chamber 301, a reaction chamber 302, a capturing chamber 304, and a waste liquid chamber 305 which are connected in this order, and the microchannel 3 is provided with a vent hole 307 communicating with the outside atmosphere and a sample filling port 306 communicating with the mixing chamber 301. After the sample is injected, the sample flows in the micro flow channel 3, and the immune reaction, the sample separation and other preparation processes occur in the flow process. A first communication channel 308 is arranged between the mixing bin 301 and the reaction bin 302, and a second communication channel 309 is arranged between the capturing bin 304 and the waste liquid bin 305.
A delay channel 303 for communicating the reaction chamber 302 and the capture chamber 304 is arranged between the reaction chamber 302 and the capture chamber, the delay channel 303 is of a zigzag structure for the sample to flow in the zigzag manner in the delay channel 303, and a solid reagent ball 7 for immunoreaction with the sample is arranged in the mixing chamber 301.
The reagent ball 7 comprises a capture reagent ball and a marking reagent ball, the capture reagent ball is a magnetic bead coated with an antibody, an antigen or a secondary antibody, the marking reagent ball is a fluorescent microsphere coated with the antibody, the antigen or the secondary antibody, and the fluorescent microsphere contains a plurality of rare earth elements.
A magnet for adsorbing magnetic beads is arranged on the bottom wall of the capturing chamber 304.
[ reagent ball ]
The detection using the capture reagent ball and the labeled reagent ball is based on a double antibody sandwich method and a double antigen sandwich method in a non-competitive binding assay, and a competitive method for measuring an antibody or an antigen. Taking the traditional double antibody sandwich method as an example, the method comprises the following steps: 1) linking the specific antibody with a solid phase carrier to form a solid phase antibody, and washing to remove the unbound antibody and impurities; 2) adding a sample to be detected, and carrying out heat preservation reaction. Combining the antigen in the sample with the solid phase antibody to form a solid phase antigen-antibody compound, and washing to remove other unbound substances; 3) adding enzyme-labeled antibody, and keeping the temperature for reaction. The antigen on the solid phase antigen-antibody complex is combined with the enzyme-labeled antibody, and the unbound enzyme-labeled antibody is removed by washing. The amount of enzyme present on the solid support is then related to the amount of antigen detected in the sample. 4) Adding substrate for color development. The enzyme on the solid phase catalyzes the substrate to a colored product and the amount of antigen in the sample is determined colorimetrically.
The labeling method adopted by the invention comprises physical adsorption, chemical crosslinking or specific action between biological molecules, and the ligand of the analyte is connected to the surface of the magnetic bead or the fluorescent microsphere. Obtaining the fluorescent microsphere marked by the ligand or the magnetic bead marked by the ligand. Wherein the ligand is capable of specifically binding to or competing with the analyte. The magnetic beads have a large specific surface area as a carrier for capturing the antibody, are used together with fluorescent microspheres, and are subjected to liquid phase flow capture together with a sample to be detected, so that the magnetic particles are enriched and then specific fluorescent signals are detected, and the method has the advantages of low background, high sensitivity and wide linear range. Meanwhile, thousands of magnetic particles are enriched and then fluorescence is measured, so that the fluorescence signal is stronger, the method is simpler than the method of measuring the fluorescence signal of a single magnetic particle in a traditional liquid phase chip and then accumulating 100 magnetic particles for calculation, the requirement on an instrument is lower, and the cost of the instrument is greatly reduced.
The physical adsorption mainly utilizes the hydrophobic surface of magnetic beads or fluorescent microspheres to perform non-specific adsorption with hydrophobic groups of protein, so that magnetic particles or fluorescent particles are marked on the surface of the protein.
The chemical crosslinking is mainly characterized in that functional groups on the surfaces of magnetic beads or fluorescent microspheres are activated by a crosslinking agent and then are combined with corresponding groups on the surfaces of proteins such as antibodies or antigens, and the magnetic beads or the fluorescent microspheres and the antibodies or the antigens are specifically crosslinked together.
In the embodiment of the invention, the magnetic beads are labeled on the surface of the protein by a chemical crosslinking method: carboxyl groups of functional groups on the surface of magnetic beads are activated by carbodiimide (EDC) and N-hydroxysuccinimide (NHS) crosslinking agents, and then the activated carboxyl groups are connected with amino groups on the surface of protein molecules such as antibodies and antigens.
In the embodiment of the invention, the fluorescent microspheres are marked on the surface of the protein by adopting a chemical crosslinking method: activating functional group carboxyl on the surface of the fluorescent marker by using an EDC + NHS cross-linking agent, and then connecting the functional group carboxyl with surface amino of protein molecules such as antibodies or antigens.
The specific biological effect includes, but is not limited to, biotin-avidin system, etc. coupling avidin with magnetic particle or fluorescent marker via chemical cross-linking, labeling antibody or antigen with biotin, incubating avidin-magnetic particle or avidin-fluorescent marker and biotin-antibody or antigen in the system, and linking the antibody or antigen with avidin-magnetic particle or avidin-fluorescent marker via the specific affinity of biotin and avidin.
According to the invention, the capture reagent ball can be prepared by the following method:
[1] and coating the surface of the magnetic bead with an antibody, an antigen or a secondary antibody. In the examples, carboxyl groups are bonded to the surface of the magnetic beads in advance. Taking the coated antibody as an example, after the capture antibody is added into the buffer solution for reaction, the blocking solution is added to obtain the capture antibody marked by the magnetic beads.
[2] And freezing the coated magnetic beads in liquid nitrogen, and then vacuumizing and drying to obtain the freeze-dried capture reagent balls.
Furthermore, the magnetic beads are superparamagnetic particles, and the material is ferric oxide and/or ferroferric oxide. The magnetic beads used in the embodiments of the present invention are particles with polystyrene as a shell and ferroferric oxide as a core.
Further, the particle size of the magnetic beads is 0.1 to 10 μm, preferably 1 μm.
Furthermore, the magnetic induction intensity of the magnetic beads is 1000-30000 gauss, preferably 2000-10000 gauss. The magnetic induction intensity has obvious influence on the detection result, if the magnetic induction intensity is smaller, the capture efficiency of the magnet on the magnetic beads is low, and the capture amount is not uniform; if the magnetic induction intensity is too high, the magnetic capture speed is too high, which may cause the shape of the capture line to be non-uniform, and the number of magnetic beads in different regions to be different, which also causes the deviation of the detection result.
According to the invention, the labeled reagent ball can be prepared by the following method:
[1] and coating the surface of the fluorescent microsphere with an antibody, an antigen or a secondary antibody. Similar to the coating of magnetic beads, the surface of the fluorescent microsphere is pre-bonded with carboxyl in the example. Taking the coated antibody as an example, after the capture antibody is added into the buffer solution for reaction, the blocking solution is added to obtain the capture antibody marked by the fluorescent microspheres.
[2] Freezing the coated fluorescent microspheres in liquid nitrogen, and then vacuumizing and drying.
The labeled reagent ball of the present invention includes, but is not limited to, time-resolved fluorescent latex particles, quantum dots, fluorescent microspheres, colloidal gold, colored latex, and the like. Preferably, time-resolved fluorescent latex particles, i.e., fluorescent microspheres, are used.
The rare earth element is used as a novel fluorescent substance, has the advantages of narrow light-emitting spectral band, high color purity, high quantum yield, wide emission wavelength distribution area and the like, and can be coated by the nano material and modified with active groups on the surface by embedding different rare earth complexes in the nano material such as polystyrene microspheres. Currently, the most commonly used is polystyrene rare earth fluorescent microsphere. The fluorescent probe is often applied to the field of immunochromatography and used as a fluorescent marker to mark an antibody so as to realize target detection. The rare earth fluorescent microspheres should have the same excitation wavelength and different emission wavelengths, and the emission peaks of the fluorescent materials should not overlap.
In one embodiment of the present invention, the fluorescent microspheres include polystyrene fluorescent microspheres containing Eu, polystyrene fluorescent microspheres containing Tb, and polystyrene fluorescent microspheres containing Sm. The rare earth elements have stronger absorption capacity under the excitation light with the wavelength of 365nm, and different rare earth elements correspond to different emission wavelength intervals, but the emission wavelengths are within the range of 500-650 nm. Wherein the excitation wavelength of the carboxyl polystyrene microsphere containing the Eu complex is 350-360nm, and the emission wavelength is 615 nm; the excitation wavelength of the carboxyl polystyrene microsphere containing the Tb complex is 350-360nm, and the emission wavelength is 547 nm; the excitation wavelength of the carboxyl polystyrene microsphere containing the Sm complex is 350-360nm, and the emission wavelength is 645 nm. Further, the ratio between different fluorescent microspheres is adjusted according to the concentration of different antigens in blood and the titer of antibodies. Preferably, the number ratio of rare earth elements of the Eu-containing polystyrene fluorescent microsphere, Tb-containing polystyrene fluorescent microsphere and Sm-containing polystyrene fluorescent microsphere is 1 (1-5) to (1-5).
The reagent ball 7 can be inserted into the groove of the mixing bin 301 during production, and then the upper cover 1 and the substrate 2 are buckled and connected. The reagent ball 7 is integrally packaged in the chip body, and integrated transportation is realized. In order to prevent the reagent balls 7 from being changed in quality during transportation, the reagent balls 7 can be freeze-dried and then plugged into the mixing bin 301.
[ detection method ]
The method for detecting by using the microfluidic chip comprises the following steps:
(1) sample adding: the sample is injected into the sample filling port 306, and the sample enters the mixing chamber 301 to melt the reagent ball 7, thereby obtaining a mixed sample. Compared with the prior art, the mixing bin 301 and the built-in reagent ball 7 are arranged in the microfluidic chip, so that the reagent ball 7 can be completely dissolved in a sample and fully mixed with the sample, and the full implementation of immune reaction is ensured. And the reagent ball 7 can flow together with the sample, and immune reaction can be carried out in the whole flowing process.
In one embodiment of the present invention, the microfluidic chip needs to be subjected to a hydrophilic treatment before the sample is injected. Hydrophilic treatment methods include, but are not limited to, plasma surface cleaning, chemical surface modification, polymer hydrophilic treatment, Chemical Vapor Deposition (CVD), surface self-assembly, and the like. In the embodiment of the invention, the hydrophilic treatment is realized by spraying the PEG modified polymer aqueous solution.
In one embodiment of the present invention, the volume of the injected sample is 10 to 300. mu.l, preferably 20 to 100. mu.l. In the specific embodiment of the invention, for the same microfluidic chip, the sample addition amount is 40. mu.l, and the diluent addition amount is also 40. mu.l, totaling 80. mu.l. The injection sample of the prior art microfluidic chip is usually 250. mu.l. Therefore, the sample consumption of the microfluidic chip is greatly reduced.
(2) Reaction: the mixed sample enters the reaction chamber 302 from the mixing chamber 301 through the first communication channel 308, and is further fully mixed and subjected to immune reaction. The mixed sample then enters the delay channel 303 to allow the immune reaction to proceed sufficiently. The delay path 303 may be a helical meander, a V-meander, or other shaped meander.
The first communicating passage 308 may be a communicating hole or a communicating opening, and the cross-sectional area of the whole is narrow. E.g. a slit structure. The converging and circulating action of the fluid liquid by the first communicating channel 308 can achieve the effect of uniform mixing.
As shown in fig. 2 or fig. 3, the delay channel 303 is provided with a plurality of capillary holes, the plurality of capillary holes are arranged along the direction from the reaction bin 302 to the capture bin 304, and the plurality of capillary holes are sequentially communicated end to form the delay channel 303 in an S-shaped structure. This arrangement results in a longer time for the mixed sample to flow through. The time for fully carrying out the immunoreaction is ensured, and the flowing process can promote the reagent ball 7 and the sample to be kept in a fully and uniformly mixed state, thereby being beneficial to fully carrying out the immunoreaction.
(3) Capturing: after the immunoreaction is completed, the mixed sample enters the capturing chamber 304 from the delay channel 303, the magnet located at the bottom wall of the capturing chamber 304 adsorbs the binding substance of the magnetic beads coated with the antibody, the antigen or the secondary antibody generated by the immunoreaction to the capturing chamber 304, and the rest of the sample continues to flow into the waste liquid chamber 305.
Compared with the prior art, the flow-type capture structure also enables the amount of the combination to flow into the waste liquid bin 305 to be obviously reduced, ensures the detection dosage, reduces the required sample dosage and saves the sample cost. Compared with the microfluidic chip which is solidified with the liquid reagent into a whole in the prior art, the single-channel fluorescence immunoassay microfluidic chip provided by the invention has the advantages that the structure is simple, the production difficulty is reduced, and the production qualification rate is improved; and the reagent and the sample can be fully mixed, the duration of the immunoreaction is ensured, the immunoreaction is fully carried out, the conjugate is easy to capture, and the detection correctness is ensured.
The immunoreaction time in the present invention is 1 to 30 minutes, preferably 5 minutes. The immunoreaction time is the time of the sample flowing through the mixing bin, the reaction bin and the delay channel. The existing microfluidic chip is generally 15-20min, so the immunoreaction time of the microfluidic chip is also obviously shortened.
(4) Cleaning: after the mixed sample flows through delay channel 303, the immunoreaction is complete. When the mixed sample flows through the capture chamber 304, the magnet disposed on the bottom wall of the capture chamber 304 adsorbs the binding substance of the magnetic beads coated with the antibody, antigen or secondary antibody produced by the immunoreaction to the capture chamber 304, and the rest of the sample continues to flow into the waste chamber 305. At this time, it is necessary to inject a cleaning liquid into the microchannel 3 so that the remaining sample can completely enter the waste liquid chamber 305 and the capturing chamber 304 and only the adsorbed conjugate remains. The washing time is generally 2 minutes.
In one embodiment of the present invention, a cleaning solution placing chamber 4 for containing a cleaning solution is disposed on the microfluidic chip, and the cleaning solution placing chamber 4 is communicated with the reaction chamber 302 through an injection channel 5. And the chip body is also provided with a blocking piece which can close the injection channel 5 so as to close the injection channel 5 when the immune reaction of the sample occurs and then conduct the injection channel 5 when the cleaning solution needs to be injected. As shown in fig. 1, the cleaning liquid placing chamber 4 is located in the upper lid 1, and is formed by a groove recessed in the upper lid 1, and the cleaning liquid placing chamber 4 is provided with an opening 401 through which the cleaning liquid flows out. As shown in fig. 3, the injection channel 5 is located on the substrate 2. As shown in fig. 4, after the upper cover 1 is fastened to the substrate 2, one end of the injection channel 5 is communicated with the opening 401, and the other end is communicated with the reaction chamber 302. The blocking piece can be a baffle or a plug with the cross section matched with that of the injection passage 5, and can be inserted into and pulled out of the injection passage 5; alternatively, the barrier may be a film bag, such as a film bag made of aluminum foil or plastic film, which encloses the cleaning fluid. The film bag is wrapped with a cleaning solution to form a cleaning solution bubble 6. The injection passage 5 is provided with a piercing pin 501 for piercing the film bag and extends from the opening 401 into the cleaning solution storage compartment 4. With such an arrangement, the cleaning liquid can be packaged integrally with the chip body, and when the cleaning liquid needs to be injected into the micro flow channel 3, the cleaning liquid bubble 6 is squeezed to make the piercing column 501 pierce the film bag. The cleaning liquid automatically flows out, flows into the injection channel 5 through the opening 401 and then is injected into the reaction chamber 302, so that the cleaning liquid is prevented from being manually injected or automatically injected through a complex structure by using a matched full-automatic detector.
In the present invention, the cleaning liquid functions to clean the micro flow channel 3 and the captured magnetic beads and remove substances that affect the detection result due to nonspecific adsorption. The cleaning solution in the embodiment contains a buffer system, a surfactant, a sealant and a preservative, wherein the buffer system comprises but is not limited to phosphate, carbonate, tris-HCL and the like, and the pH value of the cleaning solution is 5-11; blocking agents include, but are not limited to, bovine serum albumin, casein, fish skin gelatin, synthetic blocking agents, and the like; surfactants include, but are not limited to, Tween 20, Triton x-100, and the like; preservatives include, but are not limited to, Proclin-300, sodium azide, and the like. In the examples of the present invention, the washing solution used contained tween 20, casein, and a phosphate buffer solution of Proclin-300 having a pH of 7.4.
(5) And (3) detection: and pushing the microfluidic chip into a detection module for detection.
In one embodiment of the present invention, the detection process is to detect the fluorescence intensity in the capture chamber 304 by using a time-resolved fluorescence detector, and then determine the concentration of the antigen or antibody in the analyte by using a standard curve.
Because the magnetic beads have larger specific surface area as carriers for capturing the antibodies, the magnetic beads are combined with the fluorescent microspheres to carry out immunoreaction with a sample to be detected, and then the magnetic beads are enriched and specific fluorescent signals are detected. The detection process has the advantages of low background noise, high sensitivity and wide linear range. Meanwhile, thousands of magnetic beads are enriched and then detected, so that the fluorescence signal is stronger, the method is simpler than the method of calculating by accumulating 100 magnetic particles in the fluorescence signal for detecting a single magnetic particle in the traditional luminex liquid-phase chip, the requirement on the instrument is lower, and the cost of the instrument is greatly reduced.
Example 1: the same microfluidic chip is adopted to simultaneously detect cTnI, MYO and CK-MB
First, antibody labeling
1. cTnI, MYO and CK-MB labeled antibodies are obtained by adopting multicolor rare earth fluorescent microspheres
10mg of Eu ligand carboxyl polystyrene microspheres with the diameter of 300nm can be obtained by self-making or commercial purchase. 10ml of MES coating buffer pH 6.0 was added to the microspheres, and after resuspension with sonication, 10ml of coating buffer containing 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was added. Mixing the two solutions, and shaking and mixing on shaking table for 30 min. Taking out the mixed solution, centrifuging to remove the supernatant, and resuspending with 10ml of coating buffer solution to obtain 10ml of activated Eu ligand carboxyl polystyrene microspheres. To this, 10ml of coating buffer containing 2mg of a cardiac troponin (cTnI) -labeled antibody was added, and the mixture was coated on a shaker for 4 hours with shaking. The cTnI antibody marked with the Eu ligand polystyrene microsphere is obtained through the cleaning and sealing processes.
10mg of Tb ligand carboxyl polystyrene microspheres with the diameter of 300nm were added into 10ml of MES coating buffer with pH 6.0, and 10ml of coating buffer containing 10mg of EDC was added after ultrasonic resuspension. Mixing the two solutions, and shaking and mixing on shaking table for 30 min. Taking out the mixed solution, and centrifuging to remove the supernatant to obtain 10mg of the Tb ligand polystyrene microspheres subjected to heavy suspension and activation. To this was added 10ml of coating buffer containing 2mg of Myoglobin (MYO) marker antibody, and the coating was shaken on a shaker for 4 hours. And obtaining the MYO antibody marked with the Tb ligand polystyrene microspheres through the cleaning and sealing processes.
10mg of Sm ligand carboxyl polystyrene microspheres with the diameter of 300nm are added into 10ml of MES coating buffer solution with the pH value of 6.0, and 10ml of coating buffer solution containing 10mg of EDC is added after ultrasonic resuspension. Mixing the two solutions, and shaking and mixing on shaking table for 30 min. Taking out the mixed solution, centrifuging and removing supernatant fluid to obtain 10mg of the Sm ligand polystyrene microsphere which is well resuspended and activated. To this, 10ml of a coating buffer containing 2mg of creatine phosphokinase-isozyme (CK-MB) -labeled antibody was added, and the mixture was coated on a shaker for 4 hours with shaking. The CK-MB antibody marked with Sm ligand polystyrene microspheres is obtained through the cleaning and sealing processes.
And mixing the cTnI antibody marked with the Eu ligand polystyrene microsphere, the MYO antibody marked with the Tb ligand polystyrene microsphere and the CK-MB antibody marked with the Sm ligand polystyrene microsphere according to the quantity ratio of 1:2:2 to obtain the multi-color fluorescence-marked microsphere for detection.
2. Magnetic beads are adopted to obtain cTnI, MYO and CK-MB capture antibodies
10mg of carboxyl magnetic beads having a diameter of 1 μm were added to 10ml of MES coating buffer solution having a pH of 6.0, and after ultrasonic resuspension, 10ml of coating buffer solution containing 10mg of EDC was added. Mixing the two solutions, and shaking and mixing on shaking table for 30 min. The mixture was removed and centrifuged to remove the supernatant, yielding 10mg of activated carboxyl magnetic beads that were resuspended. To this was added 10ml of coating buffer containing 1mgcTnI capture antibody, and the mixture was coated for 4 hours with shaking on a shaker. And obtaining the capture magnetic beads coated with the cTnI antibody through the cleaning and sealing processes.
10mg of carboxyl magnetic beads having a diameter of 1 μm were added to 10ml of MES coating buffer solution having a pH of 6.0, and after ultrasonic resuspension, 10ml of coating buffer solution containing 10mg of EDC was added. Mixing the two solutions, and shaking and mixing on shaking table for 30 min. The mixture was removed and centrifuged to remove the supernatant, yielding 10mg of activated carboxyl magnetic beads that were resuspended. To this was added 10ml of coating buffer containing 1mg MYO capture antibody, and the coating was shaken on a shaker for 4 hours. And obtaining the capture magnetic beads coated with the MYO antibody through the cleaning and sealing processes.
10mg of carboxyl magnetic beads having a diameter of 1 μm were added to 10ml of MES coating buffer solution having a pH of 6.0, and after ultrasonic resuspension, 10ml of coating buffer solution containing 10mg of EDC was added. Mixing the two solutions, and shaking and mixing on shaking table for 30 min. The mixture was removed and centrifuged to remove the supernatant, yielding 10mg of activated carboxyl magnetic beads that were resuspended. To this, 10ml of a coating buffer containing 1mg of CK-MB capture antibody was added, and the mixture was coated on a shaker for 4 hours with shaking. And obtaining the capture magnetic beads coated with the CK-MB antibody through the cleaning and sealing processes.
And mixing the cTnI antibody-coated capture magnetic beads, the MYO antibody-coated capture magnetic beads and the CK-MB antibody-coated capture magnetic beads according to the quantity ratio of 1:2:2 to obtain the capture magnetic beads containing various capture antibodies.
Preparation of freeze-dried microspheres
And (3) respectively spraying the prepared multicolor fluorescent microspheres and the capture magnetic beads into liquid nitrogen by using an ink-jet spotting machine according to the speed of 5 mu l/drop to form frozen beads. And putting the frozen beads into a freeze dryer for vacuum drying to obtain two freeze-dried reagent beads, namely the freeze-dried beads of the multicolor fluorescent microspheres and the freeze-dried beads of the captured magnetic beads.
Preparation of three-color and multi-color fluorescent micro-fluidic chip
And spraying an aqueous solution containing 0.01 percent of PEG modified polymer on the surfaces of the substrate and the upper cover of the microfluidic chip, and drying in an oven for 1 hour. And obtaining the micro-fluidic chip with the modified surface hydrophilicity. After the two lyophilized reagent spheres are loaded into the mixing bin 301, the substrate 2 and the upper cover 1 of the microfluidic chip are built into a closed chip by an ultrasonic welding machine. The cleaning liquid bubble 6 is attached to the opening 401 through which the cleaning liquid flows out. A magnet is mounted to the bottom wall of the capture chamber 304. After the assembly is completed, the microfluidic chip and the drying agent are placed in an aluminum foil bag for vacuum packaging.
Fourth, sample detection
Mu.l of a blood plasma sample from a myocardial infarction patient was added to 90. mu.l of a dilution of PBST containing 0.1% BSA. After mixing well, 90. mu.l of sample was added to the sample fill port 306 of the chip. The chip is placed in an incubation detector. After the freeze-dried reagent ball 7 is rapidly dissolved by the sample, the mixed sample enters the reaction bin 302 from the mixing bin 301 through the first communicating channel 308, and forms uniform mixed liquid in the reaction bin 302, and further enters the time delay channel 303, so that the immune reaction is fully performed. After mixing the samples with the test samples, the concentration of cTnI was in the pg-ng scale, and the concentrations of MYO and CK-MB were in the ng scale. After the immunoreaction is completed, the mixed sample enters the capture chamber 304, the magnet located at the bottom wall of the capture chamber 304 adsorbs the magnetic bead conjugate coated with the antibody generated by the immunoreaction to the capture chamber 304, and the rest of the sample continues to flow into the waste liquid chamber 305. After the mixed sample finishes the flow capture, the chip is put into an incubation detector and reacts in the instrument for 5 min. Then, the pressurizing device in the incubation detector pushes the cleaning solution 6, so that the cleaning solution bubble 6 is punctured by the puncturing column 501 at the lower part, the cleaning solution enters the micro flow channel 3 under the action of pressure, the cleaning solution starts to clean the micro flow channel 3 and the magnetic beads captured by the capturing bin 304, and the residual unreacted fluorescent microsphere labeled antibody is flushed into the waste liquid bin 305, thereby completing the cleaning. The cleaning time is 3min, and then the chip is detected by the irradiation of an ultraviolet LED lamp.
The magnetic beads in the capture bin 304 respectively emit fluorescent light with different wavelengths, and the fluorescent light is focused by the light path and then identified by the detection module. The fluorescence values read at different wavelengths represent the contents of cTnI, MYO and CK-MB contained in the sample, respectively. After calibration by a standard curve, the concentrations of the three markers in blood were obtained. The stronger the fluorescence signal, the higher the amount of marker in the sample.
Compared with a single item detection chip, the correlations of the cTnI, the MYO and the CK-MB are respectively 98.1%, 99.5% and 98.8%. The detection result shows that the multi-fluorescence microfluidic chip can realize the simultaneous detection of three items, and meanwhile, the three items do not have obvious mutual interference.
Example 2: PCT, CRP and SAA are simultaneously detected by adopting the same micro-fluidic chip
First, antibody labeling
1. PCT, CRP and SAA labeled antibodies are obtained by adopting multicolor rare earth fluorescent microspheres
10mg of Eu ligand carboxyl polystyrene microspheres with the diameter of 300nm can be obtained by self-making or commercial purchase. 10ml of MES coating buffer pH 6.0 was added to the microspheres, and after resuspension with sonication, 10ml of coating buffer containing 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was added. Mixing the two solutions, and shaking and mixing on shaking table for 30 min. Taking out the mixed solution, centrifuging to remove the supernatant, and resuspending with 10ml of coating buffer solution to obtain 10ml of activated Eu ligand carboxyl polystyrene microspheres. To this, 10ml of coating buffer containing 2mg of Procalcitonin (PCT) -labeled antibody was added, and the mixture was coated on a shaker for 4 hours with shaking. The PCT antibody marked with the Eu ligand polystyrene microsphere is obtained through the cleaning and sealing processes.
10mg of Tb ligand carboxyl polystyrene microspheres with the diameter of 300nm were added into 10ml of MES coating buffer with pH 6.0, and 10ml of coating buffer containing 10mg of EDC was added after ultrasonic resuspension. Mixing the two solutions, and shaking and mixing on shaking table for 30 min. Taking out the mixed solution, and centrifuging to remove the supernatant to obtain 10mg of the Tb ligand polystyrene microspheres subjected to heavy suspension and activation. To this, 10ml of a coating buffer containing 2mg of a C-reactive protein (CRP) -labeled antibody was added, and the mixture was coated for 4 hours with shaking on a shaker. The CRP antibody marked with Tb ligand polystyrene microspheres is obtained through the cleaning and sealing processes.
10mg of Sm ligand carboxyl polystyrene microspheres with the diameter of 300nm are added into 10ml of MES coating buffer solution with the pH value of 6.0, and 10ml of coating buffer solution containing 10mg of EDC is added after ultrasonic resuspension. Mixing the two solutions, and shaking and mixing on shaking table for 30 min. Taking out the mixed solution, centrifuging and removing supernatant fluid to obtain 10mg of the Sm ligand polystyrene microsphere which is well resuspended and activated. To this, 10ml of coating buffer containing 2mg of Serum Amyloid A (SAA) -labeled antibody was added, and the mixture was coated for 4 hours on a shaker. The SAA antibody marked with Sm ligand polystyrene microspheres is obtained through the cleaning and sealing processes.
Mixing the PCT antibody marked with the Eu ligand polystyrene microspheres, the CRP antibody marked with the Tb ligand polystyrene microspheres and the SAA antibody marked with the Sm ligand polystyrene microspheres according to the quantity ratio of 1:3:2 to obtain the multicolor fluorescence-marked microspheres for detection.
2. Obtaining PCT, CRP and SAA capture antibodies by using magnetic beads
10mg of carboxyl magnetic beads having a diameter of 1 μm were added to 10ml of MES coating buffer solution having a pH of 6.0, and after ultrasonic resuspension, 10ml of coating buffer solution containing 10mg of EDC was added. Mixing the two solutions, and shaking and mixing on shaking table for 30 min. The mixture was removed and centrifuged to remove the supernatant, yielding 10mg of activated carboxyl magnetic beads that were resuspended. To this was added 10ml of coating buffer containing 1mg of PCT capture antibody, and the mixture was coated for 4 hours on a shaker. And obtaining the capture magnetic beads coated with the PCT antibody through the processes of washing and sealing.
10mg of carboxyl magnetic beads having a diameter of 1 μm were added to 10ml of MES coating buffer solution having a pH of 6.0, and after ultrasonic resuspension, 10ml of coating buffer solution containing 10mg of EDC was added. Mixing the two solutions, and shaking and mixing on shaking table for 30 min. The mixture was removed and centrifuged to remove the supernatant, yielding 10mg of activated carboxyl magnetic beads that were resuspended. To this, 10ml of coating buffer containing 1mg of CRP capture antibody was added, and the mixture was coated for 4 hours with shaking on a shaker. And obtaining the capture magnetic beads coated with the CRP antibody through the washing and sealing processes.
10mg of carboxyl magnetic beads having a diameter of 1 μm were added to 10ml of MES coating buffer solution having a pH of 6.0, and after ultrasonic resuspension, 10ml of coating buffer solution containing 10mg of EDC was added. Mixing the two solutions, and shaking and mixing on shaking table for 30 min. The mixture was removed and centrifuged to remove the supernatant, yielding 10mg of activated carboxyl magnetic beads that were resuspended. To this was added 10ml of coating buffer containing 1mg of SAA capture antibody, and the mixture was coated for 4 hours with shaking on a shaker. And obtaining the capture magnetic beads coated with the SAA antibody through the cleaning and sealing processes.
And mixing the PCT antibody-coated capture magnetic beads, the CRP antibody-coated capture magnetic beads and the SAA antibody-coated capture magnetic beads according to the quantity ratio of 1:3:3 to obtain the capture magnetic beads containing various capture antibodies.
Preparation of freeze-dried microspheres
And (3) respectively spraying the prepared multicolor fluorescent microspheres and the capture magnetic beads into liquid nitrogen by using an ink-jet spotting machine according to the speed of 5 mu l/drop to form frozen beads. And putting the frozen beads into a freeze dryer for vacuum drying to obtain two freeze-dried reagent beads, namely the freeze-dried beads of the multicolor fluorescent microspheres and the freeze-dried beads of the captured magnetic beads.
Preparation of three-color and multi-color fluorescent micro-fluidic chip
And spraying an aqueous solution containing 0.01 percent of PEG modified polymer on the surfaces of the substrate and the upper cover of the microfluidic chip, and drying in an oven for 1 hour. And obtaining the micro-fluidic chip with the modified surface hydrophilicity. After the two lyophilized reagent spheres are loaded into the mixing bin 301, the substrate 2 and the upper cover 1 of the microfluidic chip are built into a closed chip by an ultrasonic welding machine. The cleaning liquid bubble 6 is attached to the opening 401 through which the cleaning liquid flows out. A magnet is mounted to the bottom wall of the capture chamber 304. After the assembly is completed, the microfluidic chip and the drying agent are placed in an aluminum foil bag for vacuum packaging.
Fourth, sample detection
10 μ l of plasma samples from infected patients were added to 200 μ l of dilution in PBST containing 0.1% BSA. After mixing well, 90. mu.l of sample was added to the sample fill port 306 of the chip. The chip is placed in an incubation detector. After the freeze-dried reagent ball 7 is rapidly dissolved by the sample, the mixed sample enters the reaction bin 302 from the mixing bin 301 through the first communicating channel 308, and forms uniform mixed liquid in the reaction bin 302, and further enters the time delay channel 303, so that the immune reaction is fully performed. After the sample and the sample are mixed, the concentration of PCT is in the grade of 10pg-100ng/ML, the concentration of CRP is in the grade of 0.1ug/ML-200ug/ML, and the concentration of SAA is in the grade of 1-100 ng/ML. After the immunoreaction is completed, the mixed sample enters the capture chamber 304, the magnet located at the bottom wall of the capture chamber 304 adsorbs the magnetic bead conjugate coated with the antibody generated by the immunoreaction to the capture chamber 304, and the rest of the sample continues to flow into the waste liquid chamber 305. After the mixed sample finishes the flow capture, the chip is put into an incubation detector and reacts in the instrument for 5 min. Then, the pressurizing device in the incubation detector pushes the cleaning solution 6, so that the cleaning solution bubble 6 is punctured by the puncturing column 501 at the lower part, the cleaning solution enters the micro flow channel 3 under the action of pressure, the cleaning solution starts to clean the micro flow channel 3 and the magnetic beads captured by the capturing bin 304, and the residual unreacted fluorescent microsphere labeled antibody is flushed into the waste liquid bin 305, thereby completing the cleaning. The cleaning time is 3min, and then the chip is detected by the irradiation of an ultraviolet LED lamp.
The magnetic beads in the capture bin 304 respectively emit fluorescent light with different wavelengths, and the fluorescent light is focused by the light path and then identified by the detection module. The fluorescence values read at different wavelengths represent the amount of PCT, CRP, SAA contained in the sample, respectively. After calibration by a standard curve, the concentrations of the three markers in blood were obtained. The stronger the fluorescence signal, the higher the amount of marker in the sample.
Compared with the single item detection chip, the correlations of the three items of PCT, CRP and SAA are 99.2%, 97.5% and 98.6%, respectively. The detection result shows that the multi-fluorescence microfluidic chip can realize the simultaneous detection of three items, and meanwhile, the three items do not have obvious mutual interference.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims (10)

1. A multicolor fluorescence microfluidic chip detection method is characterized by comprising the following steps:
(1) sample adding: injecting a sample into a sample filling port of a micro-fluidic chip, wherein the micro-fluidic chip is internally provided with a micro-channel, the micro-channel comprises a mixing bin, a reaction bin, a time delay channel, a capturing bin and a waste liquid bin which are sequentially communicated, the sample enters the mixing bin to dissolve a reagent ball to obtain a mixed sample,
the reagent balls are pre-loaded into the mixing bin and comprise capture reagent balls and marking reagent balls, the capture reagent balls are magnetic beads coated with antibodies, antigens or secondary antibodies, the marking reagent balls are fluorescent microspheres coated with the antibodies, the antigens or the secondary antibodies, and the fluorescent microspheres contain multiple rare earth elements; the reagent ball is used for immunoreaction with the test sample;
(2) reaction: the mixed sample enters a reaction bin from the mixing bin and further enters a delay channel, so that the immunoreaction is fully carried out;
(3) capturing: after the immunoreaction is finished, the mixed sample enters a capturing bin from the delay channel, a magnet positioned on the bottom wall of the capturing bin adsorbs a conjugate of magnetic beads coated with an antibody, an antigen or a secondary antibody, which is generated by the immunoreaction, to the capturing bin, and the rest of the sample continuously flows into a waste liquid bin;
(4) cleaning: injecting cleaning fluid into the micro-channel, enabling the residual mixed sample to enter a waste fluid bin, and capturing the magnetic bead conjugate only contained in the bin;
(5) and (3) detection: and pushing the microfluidic chip into a detection module for detection.
2. The method of claim 1, wherein the capture reagent beads are prepared by:
[1] coating an antibody, an antigen or a secondary antibody on the surface of the magnetic bead;
[2] and freezing the coated magnetic beads in liquid nitrogen, and then vacuumizing and drying.
3. The method according to claim 2, wherein the magnetic beads are superparamagnetic particles, and the material is ferric oxide and/or ferroferric oxide.
4. A method according to claim 3, characterized in that the magnetic beads have a particle size of 0.1-10 μm, preferably 1 μm;
and/or the magnetic induction intensity of the magnetic beads is 1000-30000 gauss, preferably 2000-10000 gauss.
5. The method of claim 1, wherein the labeled reagent beads are prepared by:
[1] coating an antibody, an antigen or a secondary antibody on the surface of the fluorescent microsphere;
[2] freezing the coated fluorescent microspheres in liquid nitrogen, and then vacuumizing and drying.
6. The method of claim 5, wherein the fluorescent microspheres comprise polystyrene fluorescent microspheres comprising Eu, polystyrene fluorescent microspheres comprising Tb, and polystyrene fluorescent microspheres comprising Sm.
7. The method according to claim 1, wherein in step (1), before injecting the sample, the microfluidic chip is subjected to a hydrophilic treatment, wherein the hydrophilic treatment is at least one selected from the group consisting of plasma surface cleaning, chemical surface modification, polymer hydrophilic substance treatment, chemical vapor deposition, and surface self-assembly.
8. The method according to claim 1, wherein in the step (4), the cleaning solution contains a buffer system, a surfactant, a sealant and a preservative, wherein the buffer system is at least one selected from phosphate, carbonate and tris-HCL, and the pH value of the cleaning solution is 5-11;
the sealant is at least one of bovine serum albumin, casein, fish skin gelatin and synthetic sealant;
the surfactant is at least one selected from Tween 20 and Triton x-100;
the preservative is at least one selected from Proclin-300 and sodium azide.
9. The method of claim 1, wherein in step (5), the fluorescence intensity in the capture chamber is detected by a time-resolved fluorescence detector, and then the concentration of the antigen or antibody in the analyte is determined by a standard curve.
10. A microfluidic chip for implementing the multicolor fluorescence microfluidic chip detection method according to any one of claims 1 to 9,
the microfluidic chip comprises a substrate and an upper cover, wherein the substrate is provided with a concave cavity, the upper cover seals the concave cavity and forms a closed micro-channel for a sample to flow,
the micro flow channel comprises a mixing bin, a reaction bin, a capturing bin and a waste liquid bin which are sequentially communicated, the micro flow channel is provided with a vent hole communicated with the outside atmosphere and a sample filling port communicated with the mixing bin, a first communicating channel is arranged between the mixing bin and the reaction bin, a second communicating channel is arranged between the capturing bin and the waste liquid bin,
a delay passage communicated with the reaction chamber and the capture chamber is arranged between the reaction chamber and the capture chamber, the delay passage is of a zigzag structure and is used for the sample to zigzag flow in the delay passage, a solid reagent ball used for immunoreaction with the sample is arranged in the mixing chamber,
and a magnet for adsorbing the magnetic beads is arranged on the bottom wall of the capturing bin.
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