CN113769806B - Micro-fluidic chip, preparation method thereof and application of micro-fluidic chip in C-reactive protein detection by combining two-step microsphere competition method - Google Patents
Micro-fluidic chip, preparation method thereof and application of micro-fluidic chip in C-reactive protein detection by combining two-step microsphere competition method Download PDFInfo
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Abstract
The invention discloses a micro-fluidic chip and a preparation method thereof, and application of the micro-fluidic chip in C-reactive protein detection by combining a two-step microsphere competition method. The micro-fluidic chip disclosed by the invention is combined with the application of a two-step microsphere competition method in C-reactive protein detection, and can be used for detecting the high-precision quantification of the biomarker with the concentration range of 10 pg/mL-100 mu g/mL; the detection method provided by the invention is simple and convenient to operate, low in cost, high in detection precision and high in practicability and universality.
Description
Technical Field
The invention belongs to the technical field of blood detection, and particularly relates to a micro-fluidic chip, a preparation method thereof and application of a two-step microsphere competition method in C-reactive protein detection.
Background
C-reactive protein (CRP) is an acute phase-reactive protein synthesized by the liver, has a relative molecular mass of about 120000, and is composed of 5 identical monomers in a non-covalent bond. CRP has been widely used clinically in the diagnosis, treatment and prognosis of many diseases as a sensitive index for early detection of systemic inflammatory response. According to the CRP concentration level in serum, plasma or whole blood, the infection, the risk degree of diseases and the active period of diseases can be effectively judged.
Current traditional detection methods for CRP include: (1) a one-way immunodiffusion method: the one-way immunodiffusion method has good repeatability, does not need a special instrument for detection, is easy to generate false negative, and needs dilution and re-detection; (2) immunoturbidimetry: the sensitivity is 5.0mg/L, large-scale instruments such as an automatic biochemical analyzer, an automatic immunoassay analyzer and the like are needed, the time consumption is long, the operation is complex, and the required sample amount is large; (3) latex agglutination method: the sensitivity is 1.0 mg/L, and the method is a semi-quantitative detection method and is rarely used at present; (4) a chemiluminescence method: the method has strong specificity and wide linear range, but has long detection time and needs specific environment and operators; (5) a radioimmunoassay method comprises: the radioimmunoassay has a high sensitivity of 3. mu.g/L, but has a problem of isotope contamination. In addition, the immunochromatography test strip is based on an immunochromatography method. Although the immunochromatographic test paper is convenient to carry, the sensitivity is not high, and part of the test paper can only realize qualitative detection or semi-quantitative detection (CN206235627U, CN201096787 and the like).
The micro-fluidic chip is rapidly popularized in recent years due to the advantages of small structure size, high flux, small consumption of samples and reagents, high analysis speed, low cost and the like. The research on the microfluidic chip is the leading edge of the development of the current analytical instrument, and the technology for processing the microfluidic chip by using different materials is continuously developed. Conventional microfluidic chips are made of PDMS (Polydimethylsiloxane) or silicon wafers, on which various fine structures are formed by photolithography or templates are fabricated by photolithography and etching (CN1699984A, CN203663854U, etc.). However, the technology requires expensive lithography machines or laser engraving, and also requires the use of chemical reagents such as photoresist and developer, which is complicated in operation process, radiation is generated during the lithography process, and the chemical reagents also have certain harm to the bodies of the experimenters and certain requirements for laboratories.
Visualization has evolved on many platforms as a simple, straightforward, and fast method. For example, colorimetric methods are one of the most widely used methods in enzyme-linked immunosorbent assays (ELISA), which use simple visual color changes to obtain results. However, most of the current visualization platforms are qualitative results, and if quantitative results are realized, other instruments and equipment are needed for detection (CN101017176A and the like), which is difficult to realize in resource-poor areas.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide a microfluidic chip which can quantify the quantity of microspheres by naked eyes and directly read the quantity of the microspheres.
The second purpose of the invention is to provide the preparation method of the microfluidic chip, and the processing method of the microfluidic microsphere accumulation chip has good economy and strong adaptability.
The third purpose of the invention is to provide the application of the microfluidic chip in the detection of C-reactive protein by combining a two-step microsphere competition method.
The primary purpose of the invention is realized by the following technical scheme:
a micro-fluidic chip comprises a PDMS chip positioned at the top, a PDMS film in the middle and a substrate at the bottom, wherein micro-channels or micro-channel arrays in different shapes, a solution outlet and a solution inlet are arranged in the PDMS chip, the solution inlet is positioned at the starting end of the micro-channel or the micro-channel array, and the solution outlet is positioned at the tail end of the micro-channel or the micro-channel array.
Preferably, the thickness of the PDMS film is smaller than the diameter of the microspheres, and a round hole is formed in the PDMS film; the micro-channel or the micro-channel array and the PDMS film form a closed channel, and the tail end of the channel covers the round hole to form an opening; the solution outlet is located in the circular hole part of the PDMS film.
In the working process of the micro-fluidic chip, after the solution is injected from the solution inlet, the solution can only flow from the opening to the outlet of the round hole through the micro-channel or the micro-channel array, and in the process, the diameter of the microsphere is larger than the thickness of the PDMS film and cannot be discharged, so that the microsphere is accumulated from the tail end of the channel to form a visual accumulation line; the substrate is made of glass or silicon wafers and the like.
The second purpose of the invention is realized by the following technical scheme:
a preparation method of a microfluidic chip comprises the following steps:
(1) preparation of female PMMA (Polymethyl methacrylate) mold: preparing a PMMA female die of a microchannel or a microchannel array (the height dimension of the PMMA female die is determined according to the dimension of the piled microspheres, such as 12 mu m microspheres, the width of the PMMA female die can be more than 20 mu m, and the width dimension of the PMMA female die is at least ensured to be more than 100 mu m so as to meet the requirement of human eye visualization) by a traditional machining mode (such as a numerical control machining center);
(2) preparing a PDMS male mold: pouring PDMS onto the PMMA female die obtained in the step (1), and vacuumizing to enable the PDMS to fill the pores without bubbles; drying, curing and molding the PDMS poured into the PMMA female mold, and removing the PDMS from the PMMA female mold to obtain a PDMS male mold with a convex microchannel or microchannel array;
(3) treating the PDMS male mold obtained in the step (2) by using a plasma cleaning machine, and silanizing the PDMS male mold by using 1H,1H,2H, 2H-perfluoro octyl trichlorosilane for chemical vapor deposition so as to facilitate the separation of the subsequent PDMS;
(4) preparing a microsphere stacking chip: pouring PDMS onto the silanized PDMS male mold in the step (3), and vacuumizing to fill the PDMS with the pores without leaving air bubbles; drying, curing and molding PDMS poured on the silanized PDMS male mold, and removing the PDMS to obtain a PDMS chip with a microchannel or a microchannel array;
(5) preparation of PDMS film: placing a smooth disc in the center of a high-speed spin coater, dripping a small amount of PDMS in the center of the disc, setting centrifugal parameters according to the size of the accumulated microspheres (for example, for 12 μm microspheres, the rotating speed of 5800 plus 6000RPM can be adopted, and spin coating is carried out for 30 minutes), uniformly coating PDMS on the surface of the disc after spin coating, and drying to obtain a PDMS film;
(6) perforating the PDMS chip with the micro-channel or the micro-channel array obtained in the step (4), and setting a solution inlet and a solution outlet;
(7) punching the PDMS film obtained in the step (5) to obtain a PDMS film with a round hole;
(8) bonding the PDMS chip provided with the inlet and the outlet and the micro-channel or the micro-channel array in the step (6) with the PDMS film with the circular hole obtained in the step (7) after treatment by a plasma cleaner;
(9) and (4) bonding the PDMS chip bonded with the PDMS film in the step (8) and the substrate glass slide after treatment by using a plasma cleaning machine, and obtaining the microfluidic microsphere stacking chip capable of quantifying the number of microspheres by naked eyes.
Wherein, the micro-channel or micro-channel array in the step (1) can be in any shape and in any number.
Wherein, the drying temperature of the step (2), the step (4) and the step (5) is 70 ℃; the drying time in the step (2) is 8 hours, the drying time in the step (4) is 8 hours, and the drying time in the step (5) is 10 hours.
Wherein, the treatment time of the plasma cleaning machine in the step (3), the step (8) and the step (9) is 5-10 min, 1min and 1min respectively.
And (4) setting the centrifugal rotating speed in the step (5) according to actual requirements.
The third purpose of the invention is realized by the following technical scheme:
an application of a microfluidic chip in combination with a two-step microsphere competition method in C-reactive protein detection.
Preferably, the microfluidic chip is combined with the application of a two-step microsphere competition method in C-reactive protein detection, and the two-step immune microsphere competition strategy specifically comprises the following steps:
1) firstly, preparing microspheres modified with antibodies and magnetic particles modified with antigens;
2) then sucking a sample to be detected containing the antigen and mixing and culturing the sample with 10-90% of the microsphere solution modified with the antibody for reaction;
3) adding the rest of the antibody-modified microsphere solution in the step 2) again, reacting, adding antigen-modified magnetic particles with the same volume as the microsphere solution, and incubating;
4) finally, removing the immune microspheres combined with the magnetic particles and the free nano magnetic particles from the solution obtained in the step 3) of magnetic separation.
Preferably, the incubation reaction time in the step 2) is 45-60 minutes; the reaction time in the step 3) is 45-60 minutes, and the incubation time is 1.5-2 hours.
Preferably, the application of the microfluidic chip in the detection of the C-reactive protein comprises the following detection steps:
step A, microsphere modified antibody and magnetic particle modified antigen: uniformly mixing the microspheres with carboxyl with MES buffer solution, performing activation reaction with EDC and NHS, washing with PBS buffer solution, re-suspending, adding antibody for reaction, washing with PBS buffer solution again, adding blocking solution, sealing at 4 ℃ overnight, and re-suspending and dispersing in PBS buffer solution;
step B, magnetic particle modified antigen: fe having a carboxyl group 3 O 4 Mixing the nano magnetic particles with MES buffer solution, reacting with EDC and NHS, washing with PBS buffer solution for three times, and performing magnetic separation to remove supernatant; resuspending, adding antigen for reaction in a dark place, washing with PBS buffer solution for three times, performing magnetic separation to remove supernatant, and then resuspending and dispersing in the PBS buffer solution;
c, incubating the antibody modified microspheres, the antigen modified magnetic particles and the solution to be detected according to the two-step immune competition strategy;
step d. capture and stacking of microsphere complexes bound with free antigen: after the step A is finished, sucking a detection sample, mixing and incubating the detection sample with the microspheres and the antibody, injecting the uniformly mixed sample into a solution inlet of the microfluidic chip, discharging the solution from an outlet after the solution flows through the microchannel, and stacking the microspheres from the tail end of the channel to form a visible strip;
and E, data reading: and D, after the step B is finished, reading the stacking length of the microspheres by naked eyes, and calculating the concentration of the combined antigen to obtain a detection result.
The specific parameters in the above steps can be set according to actual requirements.
The advantages of the invention over the prior art are as follows:
(1) the processing method is non-photoetching, and has the advantages of good economy, simple preparation, low production threshold, low cost, high detection precision, and high practicability and universality;
(2) the microspheres are intercepted by the film and the solution is allowed to flow out, so that not only is the accumulation of the microspheres in the chip realized, but also the plugging of the chip is avoided, and the practicability is high;
(3) the quantification of the quantity of the microspheres with different volumes and sizes can be conveniently realized by controlling the thickness of the film, and the applicability is wide;
(4) other complex equipment is not needed during detection, naked eye quantification of the CRP concentration in a sample to be detected can be realized, and the result can be directly observed by naked eyes;
(5) according to the invention, by adopting a two-step immune competition strategy, a plurality of channels can be arranged in the chip simultaneously to inject solution so as to realize the detection of a plurality of biomarkers, and the integration level is high.
Drawings
FIG. 1 is a schematic diagram of a microfluidic microsphere stacked chip in example 1, wherein the chip comprises a PDMS chip-1, a solution inlet-2, a microchannel-3, a solution outlet-4, a circular hole-5, a PDMS film-6, a substrate-7 and a visible strip-8;
FIG. 2 is a schematic cross-sectional view of the outlet of the microfluidic microsphere stacking chip of example 1;
FIG. 3 is a two-step immuno-competitive strategy comparison at different volume ratios;
FIG. 4 is a calibration curve of the chip for CRP detection.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1: micro-fluidic chip
Fig. 1 shows a microfluidic chip of this embodiment, which includes a PDMS chip 1 located at the top, a PDMS film 6 in the middle, and a substrate 7 at the bottom, where the PDMS chip 1 is provided with microchannels 3 or a microchannel array, a solution outlet 4, and a solution inlet 2 in different shapes, the solution inlet 2 is located at the beginning of the microchannel 3 or the microchannel array, and the solution outlet 4 is located at the end of the microchannel 3 or the microchannel array; the thickness of the PDMS film is smaller than the diameter of the microspheres, and a round hole is formed in the PDMS film; the micro-channel 3 or the micro-channel array and the PDMS film form a closed channel, and the tail end of the channel covers the round hole to form an opening; the solution outlet 4 is located in the circular hole part of the PDMS membrane.
In the working process of the micro-fluidic chip, after the solution is injected from the solution inlet 2, the solution can only flow from the opening to the outlet of the round hole through the micro-channel 3 or the micro-channel array, and in the process, the diameter of the microsphere is larger than the thickness of the PDMS film and cannot be discharged, so that the microsphere is accumulated from the tail end of the channel to form a visual accumulation line; the substrate is made of glass or silicon wafers and the like.
Example 2: micro-fluidic chip processing method
The embodiment provides a method for processing a microfluidic chip. Fig. 1 shows a microfluidic chip according to this embodiment. The microfluidic chip comprises a PDMS chip 1, a PDMS film 6 and a substrate glass slide 7 as shown in embodiment 1; a solution inlet 2, a micro-channel 3 and a solution outlet 4 are arranged in the PDMS chip 1; a round hole 5 is arranged in the PDMS film 6; the microspheres are stacked to form a visible strip 8.
The steps of the preparation of the microfluidic chip in this example are as follows:
(1) processing by a traditional machining method to obtain a PMMA female die, wherein the thickness of the die plate is 0.5cm, and a micro-channel 3 with the side length of the cross section of 100 mu m and the length of 2cm is arranged in the die plate;
(2) PDMS (prepolymer: cross-linking agent: 10: 1; Sylgard-184; Dow Corning) is poured on a PMMA female die, the PMMA female die is placed into a vacuum pump, vacuumized for 15-30 min until no bubbles exist, and then placed into a 70 ℃ oven to be dried for 8 h; peeling off the dried product to obtain a PDMS male mold with a raised micro-channel, wherein the thickness of the template is 0.5 cm; treating the PDMS male mold for 5-10 min by using a plasma cleaner, and dropwise adding a small amount of 1H,1H,2H, 2H-perfluorooctyltrichlorosilane (Sigma-Aldrich) beside the template in a chemical fume hood overnight to uniformly deposit the PDMS male mold on the surface of the template;
(3) pouring PDMS on a silanized PDMS male mold, putting the PDMS on a vacuum pump, vacuumizing for 15-30 min until no bubbles exist, and putting the PDMS in a 70 ℃ drying oven for drying for 8 h; drying and peeling off to obtain a PDMS chip 1 with a microchannel, wherein the thickness of the chip is 0.5cm, the length of the chip is 2.5cm, and the width of the chip is 0.8 cm; punching a hole at the starting end of a channel of the PDMS chip to be used as a solution inlet 2, punching a hole beside the tail end of the channel to be used as a solution outlet 4, wherein the diameters of the inlet 2 and the outlet 4 are both 0.5 mm;
(4) placing a smooth disc in the center of a high-speed spin coater, dropwise adding a small amount of PDMS in the center of the disc, setting the rotation speed at 5800rpm for 10 min; uniformly coating PDMS on the surface of the disc after the glue is homogenized, putting the disc into an oven to be dried for 10 hours at 70 ℃, and drying to obtain a PDMS film 6 with the thickness less than 5 mu m; arranging a round hole 5 on the PDMS film, wherein the diameter of the round hole 5 is 3.5 mm;
(5) and (3) placing the PDMS chip 1 and the PDMS film 6 with the inlet and the outlet arranged in a plasma cleaning machine for treatment for 1min, wherein the channel surface of the PDMS chip 1 is the adhesion surface with the PDMS film 6. After bonding, the micro-channel 3 and the PDMS film 6 form a closed channel, and the tail end of the channel covers the round hole 5 to form an opening; then separating the PDMS chip 1 adhered with the film 6 from the disc, treating the PDMS chip 1 with the substrate slide 7 by using a plasma cleaning machine for 1min, and adhering the PDMS chip 1 with the substrate slide 7, wherein the film surface of the PDMS chip 1 adhered with the PDMS film 6 is the adhesion surface with the substrate slide 7; and obtaining the microfluidic microsphere stacking chip capable of quantifying the number of microspheres by naked eyes.
Example 3:
the invention further provides a two-step immune microsphere competition strategy capable of improving the detection precision and the detection limit, which specifically comprises the following steps:
1) firstly, preparing microspheres for modifying antibodies and magnetic particles for modifying antigens;
2) then sucking a sample to be detected (such as 200 mu L) containing antigen (free state) and mixing and culturing the sample with 20 percent of volume (such as 2 mu L) of the microsphere solution modified with the antibody (such as 45-60 minutes) (the first step);
3) adding the rest 80% volume (such as 8 mu L) of the microsphere solution modified with the antibody in the step 2) again, reacting (such as 45-60 minutes) (step two), adding magnetic particles (such as 10 mu L) with the same volume as the microsphere solution, and incubating (such as 1.5-2 hours);
4) finally, removing the immune microspheres combined with the magnetic particles and the free nano magnetic particles from the solution obtained in the step 3) of magnetic separation.
Wherein, the volumes of the microspheres modified with the antibody added in the step 2) and the step 3) can be respectively 30% and 70% or 10% and 90% or 40% and 60%. The specific optimal parameters can be optimized according to actual requirements.
Example 4: detection of CRP by micro-fluidic chip
The embodiment provides a detection method for CRP based on the microfluidic chip described in embodiment 1, which specifically includes the following steps:
step A. microsphere modified antibody (PMPs @ Anti-CRP): 400 μ L of polystyrene microspheres with carboxyl groups (12 μ M diameter; 250 mg/mL; seoul) were mixed with 600 μ L of MES buffer (2- (N-Morpholino) ethanesulfonic acid buffer; 0.5M; PH 6.5; Macklin), reacted with 50mmol of EDC (1-Ethyl-3- (3-dimethylamino) carbodiimide; aladin) and NHS (N-hydroxysuccinimide; aladin) for 30min, washed three times with PBS buffer (phosphate-buffered saline; 1X; PH 7.4; Cytiva), and centrifuged (5000rpm, 1 min); resuspending, adding 20. mu.L CRP antibody (Cloud-Clone Corp), reacting in the dark for 4h, washing with PBS buffer solution for three times, centrifuging to remove supernatant, adding 1% BSA solution (Clone albubin; jetway), sealing at 4 deg.C overnight, and resuspending in 200. mu.L PBS buffer;
step B, magnetic particle synthesis and magnetic particle modified antigens (MMPs @ CRP): anhydrous sodium acetate (1.2 g), trisodium citrate (0.2g) and FeCl 3 6H 2 Dissolving O (0.65g) in 20mL of glycol solution (all the materials are purchased from Aladdin), stirring for 30min, and reacting at 200 ℃ for 10 h; cooling to room temperature, washing with ethanol and distilled water to obtain Fe 3 O 4 The block mass is dried in vacuum to obtain Fe with carboxyl 3 O 4 (MMPs). 0.5mg of Fe having a carboxyl group 3 O 4 Dispersing the mixture evenly in 1mL MES buffer solution, carrying out activation reaction with 50mmol EDC and NHS for 30min, washing the mixture with PBS buffer solution for three times, and carrying out magnetic separation to remove supernatant; resuspending, adding 40. mu.L CRP (Cloud-Clone Corp), reacting for 4h in dark, washing with PBS buffer again for three times, removing supernatant by magnetic separation, and resuspending and dispersing in 200. mu.L PBS buffer;
step c, two-step immuno-competitive incubation: after step A, B was completed, the assay sample was first pipetted at 200. mu.L and reacted with 2. mu.L of PMPs @ Anti-CRP for 1h (first step), and then 8. mu.L of PMPs @ Anti-CRP was added again for 1h (second step). After completion of the reaction, 10. mu.L of MMPs @ CRP was added and incubated for 2 h. Removing MMPs by magnetic separation, centrifuging 210 μ L of supernatant (5000rpm, 1min), discarding 150 μ L of supernatant, and mixing the rest 60 μ L of solution;
step d. capture of antigen-bound microsphere-antibody complexes: after the step C is finished, injecting the uniformly mixed sample into a solution inlet of the microfluidic chip, discharging the solution from an outlet after the solution flows through the microchannel, and accumulating the microspheres from the tail end of the channel to form a visible strip;
and E, data reading: and C, after the step B is finished, reading the stacking length of the microspheres by naked eyes, and calculating the concentration of the combined antigen to obtain a detection result.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. A micro-fluidic chip is characterized by comprising a PDMS chip positioned at the top, a PDMS film positioned in the middle and a substrate positioned at the bottom, wherein micro-channels or micro-channel arrays with different shapes, a solution outlet and a solution inlet are arranged in the PDMS chip, the solution inlet is positioned at the starting end of the micro-channel or the micro-channel array, and the solution outlet is positioned at the tail end of the micro-channel or the micro-channel array;
the thickness of the PDMS film is smaller than the diameter of the microspheres, and a round hole is formed in the PDMS film; the micro-channel or the micro-channel array and the PDMS film form a closed channel, and the tail end of the channel covers the round hole to form an opening; the solution outlet is positioned in the circular hole part of the PDMS film;
in the working process of the microfluidic chip, after the solution is injected from the solution inlet, the solution can only flow from the opening to the outlet of the round hole through the microchannel or the microchannel array, and the microspheres are accumulated from the tail end of the channel to form a visual accumulation line.
2. The microfluidic chip according to claim 1, wherein the substrate is made of glass or silicon wafer.
3. A method for preparing a microfluidic chip according to any one of claims 1 or 2, comprising the steps of:
(1) preparation of a PMMA female die: preparing a PMMA female die of a micro-channel or a micro-channel array by a traditional machining mode;
(2) preparing a PDMS male mold: pouring PDMS onto the PMMA female die obtained in the step (1), and vacuumizing to enable the PDMS to fill the pores without bubbles; drying, curing and molding the PDMS poured into the PMMA female mold, and removing the PDMS from the PMMA female mold to obtain a PDMS male mold with a convex microchannel or microchannel array;
(3) treating the PDMS male mold obtained in the step (2) by using a plasma cleaning machine, and silanizing the PDMS male mold by using 1H,1H,2H, 2H-perfluoro octyl trichlorosilane for chemical vapor deposition so as to facilitate the separation of the subsequent PDMS;
(4) preparing a microsphere stacking chip: pouring PDMS onto the silanized PDMS male mold in the step (3), and vacuumizing to fill the PDMS with the pores without leaving air bubbles; drying, curing and molding PDMS poured on the silanized PDMS male mold, and removing the PDMS to obtain a PDMS chip with a microchannel or a microchannel array;
(5) preparation of PDMS film: placing a smooth disc in the center of a high-speed spin coater, dripping a small amount of PDMS (polydimethylsiloxane) in the center of the disc, setting centrifugal parameters according to the size of the accumulated microspheres, uniformly coating the PDMS on the surface of the disc after spin coating, and drying to obtain a PDMS film;
(6) perforating the PDMS chip with the micro-channel or the micro-channel array obtained in the step (4), and setting a solution inlet and a solution outlet;
(7) punching the PDMS film obtained in the step (5) to obtain a PDMS film with a round hole;
(8) bonding the PDMS chip provided with the inlet and outlet and the micro-channel or the micro-channel array in the step (6) and the PDMS film with the round hole obtained in the step (7) after being processed by a plasma cleaner;
(9) and (5) bonding the PDMS chip bonded with the PDMS film in the step (8) with a substrate slide after treatment by using a plasma cleaning machine to obtain the microfluidic microsphere stacking chip capable of quantifying the number of microspheres by naked eyes.
4. The method for preparing a microfluidic chip according to claim 3, wherein the microchannel or microchannel array in step (1) is in any shape and in any number.
5. The method for preparing a microfluidic chip according to claim 3, wherein the drying temperature in the steps (2), (4) and (5) is 70 ℃; the drying time in the step (2) is 8h, the drying time in the step (4) is 8h, and the drying time in the step (5) is 10 h.
6. The method for preparing a microfluidic chip according to claim 3, wherein the treatment time of the plasma cleaning machine in the step (3), the step (8) and the step (9) is 5-10 min, 1min and 1min respectively.
7. Use of the microfluidic chip according to any of claims 1 or 2 in combination with a two-step microsphere competition method in the detection of C-reactive protein.
8. The application of the microfluidic chip combined with the two-step microsphere competition method in the detection of C-reactive protein according to claim 7, wherein the two-step immune microsphere competition strategy specifically comprises the following steps:
1) firstly, preparing microspheres modified with antibodies and magnetic particles modified with antigens;
2) then, a sample to be detected containing the antigen is sucked to be mixed with 10-90% of the microsphere solution modified with the antibody for cultivation reaction;
3) adding the rest of the antibody-modified microsphere solution in the step 2) again, reacting, adding antigen-modified magnetic particles with the same volume as the microsphere solution, and incubating;
4) finally, removing the immune microspheres combined with the magnetic particles and the free nano magnetic particles from the solution obtained in the step 3) of magnetic separation.
9. The application of the microfluidic chip combined with the two-step microsphere competition method in the detection of C-reactive protein according to claim 8 is characterized in that the specific detection steps are as follows:
step a. microsphere-modified antibody: uniformly mixing the microspheres with carboxyl with MES buffer solution, performing activation reaction with EDC and NHS, washing with PBS buffer solution, re-suspending, adding antibody for reaction, washing with PBS buffer solution again, adding blocking solution, sealing at 4 ℃ overnight, and re-suspending and dispersing in PBS buffer solution;
step B, magnetic particle modified antigen: fe having a carboxyl group 3 O 4 Mixing the nano magnetic particles with MES buffer solution, reacting with EDC and NHS, washing with PBS buffer solution for three times, and performing magnetic separation to remove supernatant; resuspending, adding antigen for reaction in a dark place, washing with PBS buffer solution for three times, performing magnetic separation to remove supernatant, and then resuspending and dispersing in the PBS buffer solution;
c, incubating the antibody modified microspheres, the antigen modified magnetic particles and the solution to be detected according to a two-step immune microsphere competition strategy;
step d. capture and stacking of microsphere complexes bound with free antigen: after the step C is finished, sucking a detection sample, mixing and incubating the detection sample with the microspheres and the antibody, injecting the uniformly mixed sample into a solution inlet of the microfluidic chip, discharging the solution from an outlet after the solution flows through the microchannel, and stacking the microspheres from the tail end of the channel to form a visible strip;
and E, data reading: and D, reading the stacking length of the microspheres by naked eyes after the step D is finished, and calculating the concentration of the combined antigen to obtain a detection result.
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