CN109682962B - Immunofluorescence detection system and detection method based on microfluidic chip - Google Patents
Immunofluorescence detection system and detection method based on microfluidic chip Download PDFInfo
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Classifications
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- G—PHYSICS
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- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
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- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
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Abstract
The invention discloses an immunofluorescence detection system and a detection method based on a microfluidic chip, wherein the immunofluorescence detection system comprises a chip body, wherein one or more test flow channels which are mutually independent and used for testing a sample to be tested are formed in the chip body, and each test flow channel comprises a reaction tank, and a first liquid inlet channel, a second liquid inlet channel and a liquid outlet channel which are communicated with the reaction tank. Wherein the height of the liquid outlet channel is smaller than the diameter of the microsphere. The first liquid inlet channel is used for introducing a sample solution, and the second liquid inlet channel is used for introducing a microsphere solution modified by a capture antibody and a detection antibody. The height of the liquid outlet channel is smaller than the diameter of the microsphere, so that the microsphere can only flow in the second liquid inlet channel and stay in the reaction tank to react with the subsequent sample and the reactant, and the microsphere can not be sucked out of the liquid outlet channel, thereby eliminating special structures such as a micro weir or a micro valve, and the chip structure is simpler, easy to manufacture and lower in cost.
Description
Technical Field
The invention belongs to the technical field of microfluidics, and particularly relates to an immunofluorescence detection system and method based on a microfluidic chip.
Background
A point of care testing (POCT) system is an analytical method that can achieve rapid testing in a laboratory. POCT devices can provide useful diagnostic information for critical patients, and have been applied clinically such as pregnancy tests, urinary glucose and ketone tests, and the like. For example, HCG test cards and HBV test cards can achieve rapid detection. However, conventional laboratory tests require complex equipment, complex procedures and long testing times, which do not provide adequate and timely care to the patient. Therefore, there is a need to develop a low cost and portable device to improve the monitoring efficiency and accuracy of biomarkers in biochemical diagnostics. In the past few years, micro total analysis systems (μ -TAS) have been vigorously developed in the field of life sciences due to their miniaturized design. The system can integrate basic operations (including sample preparation, reaction, separation and detection) into a few square centimeters of chips for automated biochemical and medical analysis, and many microfluidic portable devices have been developed for biochemical detection and medical diagnosis. Among them, immunoassay has become one of the most important analytical methods in clinical detection and biochemical research based on its extremely high specificity. Conventional assay methods represented by ELISA are used as standard techniques for detecting various analytes, and such conventional immunoassays are usually performed in 96-well plates, and have disadvantages of high reagent consumption and long detection time. In addition, the traditional microfluidic immunization technology needs to use special structures such as a micro weir, a micro valve and the like when the nonmagnetic microspheres are used as solid phase carriers. These problems may ultimately prevent immunoassays from becoming a practical technique and immediate diagnostic tool in commercial applications,
disclosure of Invention
The first technical problem to be solved by the invention is to provide an immunofluorescence detection system which has a simple structure and can avoid the use of special structures such as a micro weir or a micro valve.
The second technical problem to be solved by the invention is to provide a detection method using the microfluidic chip system.
In order to solve the first technical problem, the invention adopts the following technical scheme:
an immunofluorescence detection system based on a microfluidic chip comprises a chip body, wherein one or more test flow channels which are mutually independent and used for testing a sample to be tested are formed in the chip body;
the test flow channel comprises a reaction tank, a first liquid inlet channel, a second liquid inlet channel and a liquid outlet channel which are communicated with the reaction tank;
the first liquid inlet channel is used for introducing a sample solution, the second liquid inlet channel is used for introducing a microsphere solution modified by a capture antibody and a detection antibody, and the heights of the liquid outlet channels are smaller than the diameters of the microspheres.
Further, the chip body comprises an upper substrate, a middle partition plate and a lower substrate which are sequentially overlapped and in sealing fit; wherein,
the end face of the upper substrate, which is contacted with the middle partition plate, is concavely formed with the first liquid inlet channel, the first cavity and the liquid outlet channel which are sequentially communicated;
the end face of the lower base plate, which is contacted with the middle partition plate, is concaved inwards to form the second liquid outlet channel and the second cavity which are sequentially communicated;
the first chamber and the second chamber are directly communicated through a through hole penetrating through the middle partition plate so as to form the reaction tank.
Further, the reaction tank is circular in shape.
Further, the height of the first liquid inlet channel is smaller than the diameter of the microsphere.
Further, the microsphere is a polystyrene microsphere.
Further, the device also comprises a power module; wherein,
the power module is used for providing power for the microsphere solution, the detection antibody, the sample solution introduction and the waste liquid discharge of the chip body.
Further, the top of the chip body is provided with a sample inlet communicated with the first liquid inlet channel, a microsphere sample inlet communicated with the second liquid inlet channel and a waste liquid outlet communicated with the liquid outlet channel.
Further, the reaction tanks are distributed in the chip body in a rectangular array, the second liquid inlet channels of the reaction tanks on each horizontal row share one microsphere sample inlet, the first liquid inlet channels of the reaction tanks on each vertical row share one sample inlet, and the liquid outlet channels of all the reaction tanks share one waste liquid outlet.
Further, the power module comprises a vacuum pump and a pressure regulating valve, wherein the vacuum pump is connected with the waste liquid outlet through a pipeline, and the pressure regulating valve is arranged on the pipeline.
Further, the device also comprises a fluorescence microscope, and the microfluidic chip is arranged on an objective table of the fluorescence microscope.
In order to solve the second technical problem, the invention adopts the following technical scheme:
according to the immunofluorescence detection method using the immunofluorescence detection system, a sample solution and a microsphere solution modified by a capture antibody are respectively led into a reaction tank through a first liquid inlet channel and a second liquid inlet channel by a power module, the microsphere solution modified by the capture antibody and an antigen are incubated in a reaction tank in a crossing way, a detection antibody is led in through the second liquid inlet channel after incubation is completed, whether the sample solution is provided with the antigen is judged by observing whether the microsphere is provided with fluorescence or not under a fluorescence microscope, and further qualitative and quantitative analysis of the sample solution is completed.
Compared with the prior art, the invention has the following advantages:
(1) The height of the liquid outlet channel is smaller than the diameter of the microsphere, so that the microsphere can only flow in the second liquid inlet channel and stays in the reaction tank to react with the subsequent sample and the reactant, thus completing the whole immunoassay, eliminating the special structures such as a micro weir or a micro valve and the like when the nonmagnetic microsphere is used as a solid phase carrier, simplifying the chip structure and reducing the operation cost.
(2) The device has a plurality of independent test flow channels, can realize directional delivery of microspheres carrying different antibodies and parallel sample injection of various biological samples, achieves high-speed simultaneous detection of various samples, and greatly saves analysis time and cost.
(3) The microfluidic chip and the vacuum pump are adopted as driving forces, so that the device has the advantages of portability, easiness in use and the like; the biological sample and reagent adding method is simple, the equipment is sealed well, and the method is safe and reliable.
Drawings
FIG. 1 is a front view of a microfluidic chip of the present invention;
FIG. 2 is an exploded view of a microfluidic chip of the present invention;
FIG. 3 is a perspective view of a microfluidic chip of the present invention;
FIG. 4 is a schematic diagram of a microfluidic chip system according to the present invention;
FIG. 5 is a fluorescent image obtained after 4 samples are reacted with 4 microspheres modified with different capture antibodies, respectively;
FIG. 6 relationship between human IgG sample and fluorescence intensity in microfluidic chip.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1 and 3, an immunofluorescence detection system based on a microfluidic chip includes a chip body 1 having one or more test channels formed therein for testing a sample to be tested independently of each other; the test flow channel comprises a reaction tank 2, a first liquid inlet channel 3, a second liquid inlet channel 4 and a liquid outlet channel 5 which are communicated with the reaction tank 2; the first liquid inlet channel 3 is used for introducing a sample solution, the second liquid inlet channel 4 is used for introducing a microsphere solution modified by a capture antibody and a detection antibody, and the height of the liquid outlet channel 5 is smaller than the diameter of the microsphere. In order to save analysis time and cost, the test flow channel is preferably designed to be multiple, so that different samples can be subjected to immunodetection at the same time. The top of the chip body 1 is provided with a sample inlet 6 communicated with the first liquid inlet channel 3, a microsphere sample inlet 7 communicated with the second liquid inlet channel 4 and a waste liquid outlet 8 communicated with the liquid outlet channel 5.
Specifically, the microspheres are polystyrene microspheres with a diameter of 40 μm, capture antibodies of 500 μg/ml are immobilized on the microspheres, and the surfaces of the microspheres are silanized and blocked with BSA before use to improve the immobilization efficiency of the antibodies.
In this embodiment, the antibody modified microsphere solution and the sample solution are respectively introduced into the reaction tank 2 through the sample inlet 6 and the microsphere inlet 7, and are subjected to intersection incubation in the reaction tank 2, after incubation is completed, the detection antibody is introduced from the microsphere inlet 7, and whether the sample solution has an antigen or not is determined by observing whether the microsphere has fluorescence under a fluorescence microscope, so that qualitative and quantitative analysis of the sample solution can be completed. Because the height of the liquid outlet channel 5 is smaller than the diameter of the microsphere, the microsphere can only flow in the second liquid inlet channel 4 and stay in the reaction tank 2 to react with the subsequent sample and the reactant, thus completing the whole immune analysis without sucking out from the liquid outlet channel, and omitting the special structures such as a micro weir or a micro valve used when the nonmagnetic microsphere is used as a solid phase carrier. Therefore, the manufacturing process of the chip is simplified, and the operation cost is reduced.
In order to prevent the microspheres from entering the first liquid inlet channel, the height of the first liquid inlet channel is designed to be smaller than the diameter of the microspheres. Furthermore, in order to facilitate the processing and manufacturing of the chip, the cross section shape and the size of the first liquid inlet channel 3 are the same as those of the liquid outlet channel.
Specifically, as shown in fig. 2, the chip body 1 of the microfluidic chip in this embodiment includes an upper substrate 101, a middle spacer 102, and a lower substrate 103 that are stacked and sealed in order. Wherein, the end surface of the upper base plate 101 contacted with the middle baffle plate 102 is concavely formed with a first liquid inlet channel 3, a first chamber 9 and a liquid outlet channel 5 which are communicated in sequence; the end surface of the lower base plate 103, which is contacted with the middle partition plate 102, is concavely formed with a second liquid outlet channel 4 and a second chamber 10 which are communicated in sequence; the first chamber 9 and the second chamber 10 are directly communicated through a through hole penetrating through the middle partition plate to form the reaction tank 2, and the first chamber 9, the second chamber 10 and the through hole are coaxially arranged. In this embodiment, the second liquid inlet channel 4 is used for introducing microspheres capable of adsorbing capture antibodies. The first liquid inlet channel 3 is used for leading in a sample solution, the middle partition plate 102 is used for blocking cross mixing of the solution, pollution is avoided, and the chip body 1 adopts a layered structure, so that the processing of a micro-channel in the chip body 1 can be facilitated.
In practical design, it is conceivable that the upper substrate 101, the middle spacer 102, and the lower substrate 103 may be made of a polymer material such as Polydimethylsiloxane (PDMS), polycarbonate (PC), cyclic Olefin Copolymer (COC), silica gel, polytetrafluoroethylene, polymethyl methacrylate (PMMA), or the like.
The specific manufacturing process of the chip body is as follows: in this embodiment, the upper substrate 101, the middle partition 102 and the lower substrate 103 are made of Polydimethylsiloxane (PDMS) and are manufactured by photolithography, while the male mold for casting the microfluidic chip is manufactured by wet mask photolithography, and the technology has the advantages of high flexibility, low manufacturing cost, short period, etc., and the micro-channel structure is designed by using Adobe Illustrator CS software, and then printed on a high-resolution transparent film to obtain a mask with a required size, and manufactured by using UV exposure and wet chemical etching technologies.
The positive film is prepared as follows: the ITO film is used as a substrate of the micro-channel shaping mold, the DuPont dry film and the ITO film with proper sizes are cut respectively, the protective films on the outer layers of the DuPont dry film and the ITO film are peeled off respectively, and the dry film is slowly and uniformly stuck on the substrate under a darkroom red light, so that the surface of the stuck dry film is smooth and bubble-free. And (3) protecting the substrate with the dry film by white paper, and slowly passing through a plastic packaging machine for 3 times to enable the substrate and the substrate to be tightly attached. And then, tightly attaching the printed mask with the micro-channel pattern on the dry film, and putting the dry film into an ultraviolet exposure box for exposure, wherein the exposure time is set according to the number of attached layers. The light transmitted by the mask pattern under the irradiation of ultraviolet light denatures the dry film thereunder, resulting in a resist pattern. Removing the microchannel mask and the DuPont dry film, and removing the protective film with 0.85% Na 2 CO 3 Developing, dissolving and removing the part shielded by the micro-channel mask in the exposure process to leave only the dry film pattern, and repeatedly cleaning the residual Na with ultrapure water 2 CO 3 Drying the aqueous solution, and heating in a 60 ℃ oven to remove residual moisture to obtain the microchip shaping mold.
The upper substrate, the middle partition plate and the lower substrate are compounded as follows: uniformly coating 5% of PVA on a bottom plate made of PDMS, putting the bottom plate into a baking oven at 60 ℃ for evaporating water to form a PVA thin layer, fully mixing a silicone rubber prepolymer Sylgard 184 and a curing agent in a ratio of 10:1, casting the mixture on a micro-channel die, vacuumizing and degassing the mixture for 30 minutes by a circulating water vacuum pump, covering the die with the PDMS bottom plate coated with PVA after bubbles in the mixture completely disappear, and placing a heavy object on the PDMS bottom plate so as to obtain an intermediate layer with uniform thickness meeting the requirement. The mixture was placed in an oven at 60℃for 2h to accelerate curing of the PDMS preform. After cooling, the cured PDMS thin layer was peeled off the template, at which point the PDMS interlayer was bonded to PVA. And (3) punching holes at the tail end of the micro-channel by using a puncher to serve as an inlet and an outlet of the solution, performing oxygen plasma activation on one surface of the PDMS substrate with the pipeline and the middle layer by using an arc gun, and completing the combination of the two chips under the auxiliary positioning of a microscope. And then, putting the combined chip into distilled water to dissolve the PVA layer and drying, carrying out oxygen plasma activation on the chip and another chip containing a micro-channel structure again, wherein the micro-channel is an upper-layer PDMS chip (upper substrate) with the size of 25 mu m, the middle-layer PDMS chip (middle partition board) with the size of 200 mu m and a lower-layer PDMS chip (lower substrate) with the size of 100 mu m are used for precisely positioning the bonding points of the three-layer micro-structure PDMS under the microscopic auxiliary positioning, and then, irreversibly and tightly adhering the three-layer micro-structure PDMS to complete the manufacture of the three-layer micro-structure chip.
For convenient observation under a microscope, the shape of the reaction cell 3 is preferably designed to be circular.
Referring to fig. 4, an immunofluorescence detection system based on a microfluidic chip further comprises a power module; the power module is used for providing power for microspheres, detection antibodies, sample solutions and derived waste liquid led in by the microfluidic chip.
Referring to fig. 2 and 3, it is conceivable that in practical application, the reaction cells 2 are distributed in a rectangular array in the chip body 1, the second liquid inlet channels of the reaction cells 2 on each horizontal row share a microsphere sample inlet, the first liquid inlet channels of the reaction cells 2 on each vertical row share a sample inlet, and the liquid outlet channels of all the reaction cells share a waste liquid outlet. In this embodiment, the number of reaction tanks is 16, and the reaction tanks are distributed in four rows and four columns, and correspondingly, the number of sample inlets and microsphere sample inlets is four.
Referring to fig. 4, the power module comprises a vacuum pump 11, a pressure regulating valve 12 and a waste liquid storage 13, wherein the vacuum pump 11 is communicated with the waste liquid storage 13 through a pipeline 14, the waste liquid storage 13 is connected with a waste liquid outlet 8 through a latex tube, the pressure regulating valve 12 is arranged on the waste liquid storage 13, negative pressure is provided through the vacuum pump 11, microsphere solution and sample solution can be adsorbed in a reaction tank, meanwhile, waste liquid after reaction is completed is sucked into the waste liquid storage 13 through the latex tube through the negative pressure provided by the vacuum pump 11, and meanwhile, the adsorption pressure can be regulated through regulating the pressure regulating valve 12.
Specifically, the microfluidic chip system of the present embodiment further includes a fluorescence microscope 15, the microfluidic chip body 1 is disposed on a stage of the fluorescence microscope 15, and the specific structure of the fluorescence microscope 15 is not an improvement point of the present invention and is not described herein.
Application examples
And respectively introducing microsphere solutions modified by different capture antibodies (goat anti-chicken IgG, goat anti-rabbit IgG, goat anti-human IgG and goat anti-mouse IgG) into four microsphere sample inlets 7, introducing different sample solutions (chicken IgG, rabbit IgG, human IgG and mouse IgG) into four sample inlets 6, incubating the microsphere solutions modified by the capture antibodies with the antigens in a reaction chamber, controlling the pressure of a vacuum pump through a pressure regulating valve, introducing detection antibodies from the microsphere sample inlets 7 after incubation is completed, and judging whether the sample solution is provided with the antigens by observing whether the microspheres are provided with fluorescence under a fluorescence microscope, so as to complete qualitative and quantitative analysis of the sample solution, as shown in fig. 5 and 6. In FIG. 5, (a), (b), (c) and (d) represent fluorescent images of antigen chicken IgG, rabbit IgG, human IgG, mouse IgG, respectively, at a concentration of 0.1. Mu.g/mL, and 1,2,3 and 4 represent microspheres modified with the capture antibodies goat anti-chicken IgG, goat anti-rabbit IgG, goat anti-human IgG and goat anti-mouse IgG, respectively.
The invention adopts the vacuum degree of 0.02MPa to ensure that microspheres are successfully introduced into a chip reaction chamber, 4 microspheres modified with different capture antibodies and 4 antigen solutions are respectively injected into 4 microsphere sample inlets and 4 sample inlets at the top of an upper substrate, after antigen-antibody reaction, the unreacted complete antigen is washed for 2min by using a buffer solution, after incubation is finished, corresponding detection antibodies are injected, each capture antibody only reacts with corresponding specific antigen, and the three antibodies meet to realize real-time detection of target antigens. The ELISA test chip system can perform rapid and efficient qualitative and quantitative analysis on various biological samples at the same time, immunological reactions of the multiple biological samples can be determined by one-time operation, the high-speed parallel analysis capability of the ELISA test chip system can provide efficient and convenient instant detection, and the development technology of the micro-fluidic chip system can be finally used for developing a portable micro-fluidic chip disease diagnosis system, so that diagnosis and treatment equipment is further miniaturized, portable and easy to use.
The above examples are only illustrative of the invention and are not intended to be limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. Nor is it necessary or impossible to exhaust all embodiments herein. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (7)
1. The utility model provides an immunofluorescence detecting system based on micro-fluidic chip, includes the chip body, and its inside is formed with one or more test flow channels that are used for testing the sample that awaits measuring each other, its characterized in that:
the test flow channel comprises a reaction tank, a first liquid inlet channel, a second liquid inlet channel and a liquid outlet channel which are communicated with the reaction tank;
the first liquid inlet channel is used for introducing a sample solution, the second liquid inlet channel is used for introducing a microsphere solution modified by a capture antibody and a detection antibody, and the height of the liquid outlet channel is smaller than the diameter of the microsphere;
the chip body comprises an upper substrate, a middle partition plate and a lower substrate which are sequentially overlapped and in sealing fit; wherein,
the end face of the upper substrate, which is contacted with the middle partition plate, is concavely formed with the first liquid inlet channel, the first cavity and the liquid outlet channel which are sequentially communicated;
the end face of the lower base plate, which is contacted with the middle partition plate, is concaved inwards to form the second liquid inlet channel and the second chamber which are sequentially communicated;
the first chamber and the second chamber are directly communicated through a through hole penetrating through the middle partition plate so as to form the reaction tank;
the height of the first liquid inlet channel is smaller than the diameter of the microsphere;
the reaction tanks are distributed in the chip body in a rectangular array, the second liquid inlet channels of the reaction tanks on each horizontal row share one microsphere sample inlet, the first liquid inlet channels of the reaction tanks on each longitudinal row share one sample inlet, and the liquid outlet channels of all the reaction tanks share one waste liquid outlet.
2. The immunofluorescence detection system of claim 1, wherein: the reaction tank is round in shape.
3. The immunofluorescence detection system of any one of claims 1 or 2, wherein: the device also comprises a power module; wherein,
the power module is used for providing power for the microsphere solution, the detection antibody, the sample solution introduction and the waste liquid discharge of the chip body.
4. The immunofluorescence detection system of claim 3, wherein: the top of the chip body is provided with a sample inlet communicated with the first liquid inlet channel, a microsphere sample inlet communicated with the second liquid inlet channel and a waste liquid outlet communicated with the liquid outlet channel.
5. The immunofluorescence detection system of claim 3 or 4, wherein: the power module comprises a vacuum pump and a pressure regulating valve, the vacuum pump is connected with the waste liquid outlet through a pipeline, and the pressure regulating valve is arranged on the pipeline.
6. The immunofluorescence detection system of claim 3, wherein: the chip body is arranged on an objective table of the fluorescence microscope.
7. A detection method using the immunofluorescence detection system of claim 6, characterized in that: and introducing the sample solution and the microsphere solution modified by the capture antibody into a reaction tank through a first liquid inlet channel and a second liquid inlet channel respectively by a power module, crossing and incubating the microsphere solution modified by the capture antibody and the antigen in the reaction tank, introducing a detection antibody through the second liquid inlet channel after incubation is completed, and judging whether the sample solution has the antigen or not by observing whether the microsphere has fluorescence under a fluorescence microscope, thereby completing qualitative and quantitative analysis of the sample solution.
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