Magnetic Quantum Dot Encoded Microspheres, Preparation Method Thereof and Device for High-Throughput Detection

Abstract

The present invention discloses a magnetic quantum dot-encoded microsphere, a preparation method thereof, and a device for high-throughput detection. The preparation method includes the following steps: S1. Prepare a continuous phase solution and a dispersed phase solution; the dispersed phase includes a dispersed phase doped with quantum dot particles and a dispersed phase doped with magnetic nanoparticles; S2. Prepare uniform droplets through a microfluidic chip; push the continuous phase and the dispersed phase at a set speed, and the dispersed phase doped with quantum dot particles and the dispersed phase doped with magnetic nanoparticles always maintain laminar flow. After the dispersed phase is sheared into droplets, an obvious demarcation line in the middle of the droplets can be seen; S3. Add a photoinitiator so that the droplets can be rapidly cured into microspheres with a Janus structure after being irradiated by ultraviolet light in the channel; S4. Introduce functional groups on the surface of the microspheres for specifically binding to the antigen to be detected. The microspheres prepared by the method in the present invention can be covalently coupled with specific antibodies, used for specifically binding to the antigen to be detected, and can encapsulate more magnetic materials.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical detection, and particularly to a magnetic quantum dot-encoded microsphere, a preparation method thereof, and a device for high-throughput detection. Background Art

[0002] High-throughput detection technologies include solid-phase chips and liquid-phase chip technologies. Solid-phase chips use position encoding and have a simple decoding method. However, their encoding method integrated on a plane limits the flexibility of detection schemes. In the face of different detection requirements, solid-phase chips with different analysis arrays need to be prepared. At the same time, the speed at which analytes bind to the chip substrate during the detection process is relatively slow, affecting the detection speed. Moreover, the same substrate used in solid-phase chips has different matching properties for different types of biological target molecules. Liquid-phase chips have good flexibility, high sensitivity, and high detection accuracy. However, they use non-magnetic organic dye-encoded microspheres as carriers and require a flow cytometer as a basic analysis instrument. A flow cytometer mainly consists of a liquid flow system, a light source system, and a detection and analysis system. The light source system needs to integrate a specific laser source corresponding to the encoded microspheres. The manufacturing technology threshold of the equipment is relatively high, the use cost is high, and it has a certain volume, which hinders its wide popularization in point-of-care testing and underdeveloped areas. Currently, combining suspension detection and array analysis, that is, arranging the encoded microspheres on a plane and replacing the individual decoding of traditional flow cytometry microspheres with planar batch decoding of microspheres, has become a research hotspot.

[0003] In existing technical solutions based on planar fixation of encoded microspheres, the microspheres used mainly include two types. One is a single fluorescent-encoded microsphere without magnetism, and the other is a microsphere simply mixed with magnetic nanoparticles and a fluorescent encoding material.

[0004] In existing technical solutions based on planar fixation of encoded microspheres, the fixation schemes used include the following two:

[0005] The first scheme is to use a magnetic field to fix magnetic encoded microspheres. After the microspheres capture analytes in the solution, a magnetic field is applied to make the microspheres settle from the solution to the substrate and arrange in a two-dimensional pattern. The schematic diagram is as Figure 1 shown.

[0006] The second scheme is to use non-magnetic fluorescent-encoded microspheres. The microspheres are pre-fixed in a microporous membrane and encapsulated at the top to form a semi-closed space. The sample to be detected is introduced from the inlet. The microspheres capture analytes in the solution, and then a buffer solution is introduced to wash the microspheres. The schematic diagram is as Figure 2 shown.

[0007] The disadvantages of Solution 1 include: simple hybrid magnetic nanoparticles and microspheres of coding materials are used. The magnetic materials will absorb the fluorescence of the coding materials, and the fluorescence intensity is inversely proportional to the amount of magnetic materials, resulting in difficulty in coating more magnetic materials on the microspheres. Fixing the microspheres using a magnetic field, the inevitable overlap due to the too-close distance between the microspheres and the stacking of the microspheres in the vertical direction also exist, leading to significant fluorescence interference, affecting the detection accuracy, and the uneven distribution of the microspheres on the plane.

[0008] The disadvantages of Solution 2 include: the speed at which the microspheres fixed in the holes bind to the analyte is slower than the speed at which the microspheres move in the solution to capture the analyte. The microspheres rely only on the confinement of the microholes and are prone to continuous movement, resulting in ghost images during imaging, affecting the image quality and detection accuracy. The microspheres are pre-fixed and encapsulated, and the detection ability is determined at the time of encapsulation, making it difficult to flexibly adjust the types and proportions of the microspheres according to the actual detection requirements, with poor flexibility. Summary of the Invention

[0009] In order to make up for the deficiencies of the above-mentioned background technology, the present invention proposes a magnetic quantum dot-coded microsphere and its preparation method and a high-throughput detection device to solve the problems that it is difficult to coat more magnetic materials on the microspheres, the detection accuracy of the detection device is low, and the flexibility is poor.

[0010] The technical problems of the present invention are solved by the following technical solutions:

[0011] The present invention discloses a preparation method of a magnetic quantum dot-coded microsphere, including the following steps:

[0012] S1. Prepare a continuous phase and a dispersed phase solution; the dispersed phase includes a dispersed phase doped with quantum dot particles and a dispersed phase doped with magnetic nanoparticles;

[0013] S2. Prepare uniform droplets through a microfluidic chip; push the continuous phase and the dispersed phase at a set speed, and the dispersed phase doped with quantum dot particles and the dispersed phase doped with magnetic nanoparticles always maintain laminar flow. After the dispersed phase is sheared into droplets, an obvious demarcation line in the middle of the droplets can be seen;

[0014] S3. Add a photoinitiator so that the droplets can be quickly solidified into microspheres maintaining the Janus structure after being irradiated by ultraviolet light in the channel;

[0015] S4. Introduce functional groups on the surface of the microspheres for specifically binding to the analyte antigen.

[0016] In some embodiments, in step S1, the main body of the dispersed phase is a photo-polymerizable polymer monomer.

[0017] In some embodiments, in step S1, the preparation methods of the dispersion phase of the doped quantum dot particles and the dispersion phase of the doped magnetic nanoparticles include: uniformly dispersing the oil-soluble quantum dot particles and magnetic nanoparticles in the main body of the dispersion phase by means of solvent evaporation.

[0018] Further, it also includes: obtaining multiple groups of quantum dot codes by changing the types and masses of the quantum dots doped in the main body of the dispersion phase.

[0019] In some embodiments, in step S3, the proportion of the photoinitiator is 1% - 5%.

[0020] In some embodiments, step S4 is specifically: adding maleic anhydride and a photopolymerizable monomer to carry out copolymerization to introduce functional groups on the surface of the microspheres; partially hydrolyzing the microspheres in an alkaline solution to hydrolyze the anhydride groups into carboxyl groups, and covalently coupling with specific antibodies for specifically binding to the antigen to be detected.

[0021] The present invention also discloses a magnetic quantum dot-encoded microsphere. The microsphere is prepared by using the method described in any one of the above. The microsphere includes a microsphere main body, and the microsphere main body includes two parts, both parts are hemispherical. One hemisphere is coated with oil-soluble quantum dot particles for encoding; the other hemisphere is coated with oil-soluble magnetic nanoparticles; the surface of the microsphere is rich in carboxyl groups, and the surface of the microsphere can covalently couple with specific antibodies for specifically binding to the antigen to be detected; the microsphere has a Janus structure for integrating quantum dot encoding and magnetism on the microsphere.

[0022] The present invention also discloses a device for high-throughput detection, including a microporous array film and the microsphere as described above. One side of the microporous array film is provided with a plurality of micropores, and at most one microsphere can be accommodated in each micropore. The microspheres are fixed in the micropores, and different microspheres are at the same horizontal plane.

[0023] In some embodiments, it further includes a magnet, and the magnet is placed below the microporous array film.

[0024] In some embodiments, the component of the microporous array film is polydimethylsiloxane.

[0025] The beneficial effects of the present invention compared with the prior art include:

[0026] The microsphere prepared by using the preparation method of the magnetic quantum dot-encoded microsphere provided by the present invention can be partially hydrolyzed in an alkaline solution to hydrolyze the anhydride groups into carboxyl groups, and covalently couple with specific antibodies under suitable reaction conditions for specifically binding to the antigen to be detected.

[0027] In some embodiments, multiple sets of quantum dot codes are obtained by changing the types and masses of the quantum dots doped in the dispersed phase matrix, thereby flexibly changing the number and types of microspheres and improving the flexibility of detection.

[0028] The magnetic quantum dot-encoded microspheres provided by the present invention are integrated with quantum dot coding and magnetism, reducing the quenching of quantum dots caused by the light absorption of magnetic nanoparticles, enabling the microspheres to coat more magnetic materials, and the magnetic properties of the microspheres can simplify the washing and separation steps during subsequent immune reactions. Under the action of a magnetic field, the microspheres can move or be fixed directionally.

[0029] The device for high-throughput detection provided by the present invention uses a microporous array film to fix the microspheres. Each micropore can accommodate at most one microsphere, and the microspheres do not overlap or interfere with each other, reducing fluorescence interference and detection errors and improving the detection accuracy.

[0030] In some embodiments, the microporous array film and the magnetic field are used jointly to fix the microspheres. Under the action of the magnetic field, the microspheres can maintain a fixed position without moving, avoiding afterimages on the fluorescence image and further improving the detection accuracy. Description of the Drawings

[0031] Figure 1 is a schematic diagram of the first planar fixing scheme of the coded microspheres in the prior art of the present invention.

[0032] Figure 2 is a schematic diagram of the second planar fixing scheme of the coded microspheres in the prior art of the present invention.

[0033] Figure 3 is a flowchart of the preparation method of the magnetic quantum dot-encoded microspheres according to the embodiments of the present invention.

[0034] Figure 4 is a schematic diagram of the preparation of microspheres through a microfluidic chip according to the embodiments of the present invention.

[0035] Figure 5 is a scanning electron microscope image of the microspheres according to the embodiments of the present invention.

[0036] Figure 6 is a schematic diagram of the magnetic quantum dot-encoded microspheres with a Janus structure according to the embodiments of the present invention.

[0037] Figure 7 is a schematic diagram of a polydimethylsiloxane film with a microporous array according to the embodiments of the present invention.

[0038] Figure 8 is a top view of a polydimethylsiloxane film with a microporous array according to the embodiments of the present invention.

[0039] Figure 9It is a cross-sectional view of a polydimethylsiloxane film with a microporous array according to an embodiment of the present invention.

[0040] Figure 10 It is a scanning electron microscope image of a polydimethylsiloxane film with a microporous array according to an embodiment of the present invention.

[0041] Figure 11 It is a schematic diagram of fixing various microspheres on a microporous array film according to an embodiment of the present invention.

[0042] Figure 12 It is a scanning electron microscope image of fixing microspheres on a microporous array film according to an embodiment of the present invention.

[0043] Figure 13 It is a schematic diagram of using a device for high-throughput detection to obtain an image for detection according to an embodiment of the present invention. Detailed implementation manners

[0044] The present invention will be further described below with reference to the accompanying drawings and preferred embodiments. It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments may be combined with each other.

[0045] It should be noted that the azimuth terms such as left, right, up, down, top, bottom, etc. in this embodiment are only relative concepts to each other, or are referenced based on the normal use state of the product, and should not be considered restrictive.

[0046] As Figure 3 shown, an embodiment of the present invention provides a method for preparing magnetic quantum dot-encoded microspheres, including the following steps:

[0047] S1. Prepare continuous phase and dispersed phase solutions. The dispersed phase includes a dispersed phase doped with quantum dot particles and a dispersed phase doped with magnetic nanoparticles. The main body of the dispersed phase is a photopolymerizable polymer monomer.

[0048] Specifically, the preparation methods of the dispersed phase doped with quantum dot particles and the dispersed phase doped with magnetic nanoparticles include: uniformly dispersing oil-soluble quantum dot particles and magnetic nanoparticles in the main body of the dispersed phase by the solvent evaporation method, and obtaining multiple sets of quantum dot encodings by changing the types and masses of the quantum dots doped in the main body of the dispersed phase.

[0049] S2. Prepare uniform droplets through a microfluidic chip; push the continuous phase and the dispersed phase at a set speed, and the dispersed phase doped with quantum dot particles and the dispersed phase doped with magnetic nanoparticles always maintain laminar flow. After the dispersed phase is sheared into droplets, an obvious demarcation line can be seen in the middle of the droplets.

[0050] S3. Add a photoinitiator so that the droplets can be quickly cured into microspheres maintaining the Janus structure after being irradiated by ultraviolet light in the channel. The proportion of the photoinitiator is 1%-5%.

[0051] S4. Introduce functional groups on the surface of the microspheres for specifically binding to the antigen to be detected.

[0052] Specifically, maleic anhydride and a photopolymerizable monomer are added for copolymerization to introduce functional groups on the surface of the microspheres. The microspheres are partially hydrolyzed in an alkaline solution to hydrolyze the anhydride groups into carboxyl groups, which are covalently coupled with specific antibodies for specifically binding to the antigen to be detected.

[0053] In some embodiments, the preparation process of the magnetic quantum dot-encoded microspheres is as follows:

[0054] As Figure 4 shown, it is a schematic diagram of preparing microspheres through a microfluidic chip. Highly uniform droplets are prepared through a PDMS (polydimethylsiloxane)-based microfluidic chip, and the droplets are cured into firm spheres under ultraviolet light irradiation. The microfluidic chip is fabricated using a mature process, and the channels inside the chip are modified to be hydrophilic. Preparing microspheres requires a continuous phase and a dispersed phase solution. The continuous phase is an aqueous solution added with a surfactant, and the main body of the dispersed phase is a photopolymerizable polymer monomer. The oil-soluble quantum dots and magnetic nanoparticles can be uniformly dispersed in the polymer monomer through the solvent evaporation method. By changing the types and masses of the quantum dot fluorescent materials doped in the polymer monomer, multiple sets of quantum dot fluorescence coding can be obtained. 1%-5% of a photoinitiator is added to enable the droplets to be rapidly cured after being irradiated by ultraviolet light in the channels. Maleic anhydride and a photopolymerizable monomer are added for copolymerization to introduce functional groups on the surface of the microspheres. An injection pump is used to push the continuous phase and the dispersed phase at a set speed. The dispersed phase doped with quantum dot particles and the dispersed phase doped with magnetic nanoparticles always maintain laminar flow. After the dispersed phase is sheared into droplets, an obvious demarcation line in the middle of the droplets can be seen. After the droplets pass through the ultraviolet light region, they are cured into spheres and maintain the Janus structure. The prepared microspheres are uniform in size and have a small coefficient of variation. They can move or be fixed directionally under the action of a magnetic field, showing good magnetic responsiveness. The fluorescence emitted by the coated fluorescent material is bright. The prepared microspheres can be partially hydrolyzed in an alkaline solution to hydrolyze the anhydride groups into carboxyl groups, and are covalently coupled with specific antibodies under suitable reaction conditions for specifically binding to the antigen to be detected. To simultaneously detect the concentrations of multiple antigens to be detected, multiple types of encoded microspheres need to be prepared, and different antibodies are connected to different types of encoded microspheres. The scanning electron microscope image of the microspheres is as Figure 5 shown.

[0055] In one embodiment, the continuous phase is an aqueous solution of 2% polyvinyl alcohol - 3% poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol). In the dispersed phase, an acrylate compound is used as the polymerization monomer, maleic anhydride is used as the comonomer, and diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide is used as the photoinitiator. The quantum dots used are two kinds of oil-soluble CdSe / CdS / ZnS quantum dots with emission peaks at 450 nm and 640 nm respectively, and the magnetic nanoparticles are oil-soluble magnetite particles with an average particle size of 10 nm. The quantum dots and magnetic nanoparticles in the toluene solution are dispersed into the monomer by the solvent evaporation method, 4% of the photoinitiator is added, and a variety of encoded microspheres are prepared by changing the ratio of the two kinds of quantum dots doped in the monomer. The completely cured microspheres are soaked in a sodium hydroxide solution, and the anhydride groups in the copolymer of the microsphere spheres are slowly hydrolyzed into carboxyl groups in the strong base solution, and the carboxyl groups on the surface of the microspheres are convenient for connecting different types of target molecules.

[0056] The microspheres prepared by the method for preparing magnetic quantum dot-encoded microspheres provided by the embodiment of the present invention can partially hydrolyze the anhydride groups into carboxyl groups in an alkaline solution, and covalently couple with specific antibodies under suitable reaction conditions for specifically binding to the antigen to be detected. Further, by changing the types and masses of the quantum dots doped in the main body of the dispersed phase, multiple groups of quantum dot encodings are obtained, so as to flexibly change the number and types of the microspheres and improve the flexibility of detection.

[0057] The embodiment of the present invention also provides a magnetic quantum dot-encoded microsphere, which is prepared by using the method for preparing magnetic quantum dot-encoded microspheres according to any one of the above. The magnetic quantum dot-encoded microsphere is integrated with quantum dot encoding and magnetism, reducing the fluorescence quenching caused by the light absorption of the magnetic nanoparticles. The magnetic properties of the microspheres can simplify the cleaning and separation steps during subsequent immune reactions, and the microspheres can move or be fixed directionally under the action of a magnetic field.

[0058] Such as Figure 6As shown, the magnetic quantum dot-encoded microspheres provided by the embodiments of the present invention are spherical, with a diameter that can be adjusted within 50-100 microns. The microspheres are of uniform size, smooth on the outside, and have a certain mechanical strength. It includes a microsphere body, which consists of two parts, both of which are roughly hemispherical. One hemisphere is coated with oil-soluble quantum dot particles for encoding. By combining quantum dots with different emission wavelengths in different proportions and different masses, multiple encodings can be obtained. The other hemisphere is coated with oil-soluble magnetic nanoparticles, which endow the microspheres with superparamagnetism. Under the action of a magnetic field, the microspheres can move or be fixed directionally, facilitating washing and separation. After the microspheres are soaked in an alkaline solution, the anhydride groups in the copolymer are hydrolyzed, making the surface of the microspheres rich in carboxyl groups. Under appropriate reaction conditions, specific antibodies can be covalently coupled to the surface of the microspheres for specifically binding to the antigen to be detected. The microspheres have a Janus structure, which is used to integrate quantum dot encoding and magnetism on the microspheres.

[0059] The embodiments of the present invention also provide a device for high-throughput detection, including a microporous array film and the microspheres as above. One side of the microporous array film is provided with a plurality of micropores, and each micropore contains one microsphere. The microspheres are fixed in the micropores, and different microspheres are at the same horizontal plane.

[0060] In some embodiments, the device for high-throughput detection further includes a magnet, which is placed below the microporous array film.

[0061] Specifically, the component of the microporous array film is polydimethylsiloxane. One side of the microporous array film has micropores arranged periodically. Assuming the diameter of the prepared microspheres is d, when the diameter D1 of the micropores is between 1d and 1.5d, the interval D2 between the micropores is between 1 / 3d and 1 / 2d, and the depth H2 of the micropores is between 0.5d and 1d, the effect of fixing the microspheres in the micropores is better. The sizes L1 and L2 of the microporous array film can be flexibly adjusted according to the number of microspheres to be fixed. The more microspheres to be fixed, the larger L1 and L2 should be. When the number of micropores is close to the number of microspheres to be fixed, the fixing efficiency is higher. The height H1 of the microporous array film has a relatively small impact on the fixing effect, generally between 0.5 mm and 3 mm. The micropores are arranged in a honeycomb shape. The polydimethylsiloxane film with microporous arrays needs to be surface-modified to be hydrophilic before use so that the solution rich in microspheres can better spread on the surface of the microporous array film.

[0062] Furthermore, the size of the micropores is designed to just accommodate one microsphere, with a diameter slightly larger than that of the microsphere and a depth of about half of the microsphere diameter, avoiding the occurrence of multiple microspheres in one pore. The micropore spacing is small to reduce the possibility of microspheres being distributed outside the micropores. The microspheres are magnetic, and under the action of a magnetic field, the microspheres enter the micropores from the solution faster and are fixed. At the same time, the magnetic field force ensures the fixation of the microsphere position during the shooting process. When dropping the microsphere solution, the side of the microporous array film with the microporous array faces upward, and a magnet is used to assist in fixing the microspheres. The magnet is placed under the microporous array film, and the microspheres move from the solution to the microporous array film faster under the action of the magnetic field. After the microspheres are fixed by the micropores, the magnetic field further restricts the movement of the microspheres.

[0063] Among them, the preparation method of the polydimethylsiloxane film with a microporous array is as follows:

[0064] A silicon mold with a microcolumn array structure is prepared by photolithography. The surface of the silicon mold is fluorinated. The monomers and crosslinking agents of dimethylsiloxane are mixed in a mass ratio of 10:1 and poured on the silicon mold, heated and cured. After casting, a polydimethylsiloxane film with a microporous array is obtained. The surface of the film is modified to be hydrophilic.

[0065] In one embodiment, a honeycomb-like densely arranged microcolumn array is obtained on a silicon wafer by photolithography using a negative photoresist of the SU 82000 series. The diameter of the microcylinders is 60 microns, the height is 30 microns, and the microcolumn spacing is 20 microns. After the silicon wafer is fluorinated with 1H, 1H, 2H, 2H-perfluorodecyltrichlorosilane, a small amount of a mixture of monomers and crosslinking agents of Dow Corning 184 is poured, and after heating and curing, the polydimethylsiloxane film with a microporous array is peeled off to complete the casting. The polydimethylsiloxane film with a microporous array needs to be treated in oxygen plasma for 2 minutes before being used to fix microspheres. The hydrophilic surface of the film is conducive to the entry of microspheres into the micropores. The prepared polydimethylsiloxane film with a microporous array is as Figures 7 to 10 shown.

[0066] When using this device for high-throughput detection, the solution containing microspheres is dropped on the microporous array film treated with oxygen plasma, and a magnet is placed under the microporous array film. The microspheres enter the micropores under the action of gravity and magnetic field force. Due to the structural design of the micropores, at most one microsphere is accommodated in each micropore, and the microspheres are fixed in the micropores. Different microspheres are on the same horizontal plane, which is easy to focus.

[0067] As Figures 11 to 12As shown, microspheres A, B, and C are three different microspheres. The three microspheres have the same structure, each having a hemisphere coated with a quantum dot encoding material and a hemisphere coated with magnetic nanoparticles. The difference lies in the type and quality of the coated quantum dot encoding materials. Due to the Janus structure of the microspheres, during the fixation process, the hemisphere coated with magnetic nanoparticles is attracted by the magnet and flipped to face the magnet, while the hemisphere coated with the quantum dot encoding material (fluorescent encoding material) faces upward. The microspheres are irradiated with ultraviolet light and the excitation light of the reporter molecule respectively, and the fluorescence images of the microspheres are taken with a microscope. The taken images can simultaneously obtain the information of all the microspheres within the field of view.

[0068] In one embodiment, as Figure 13 shown, after the microspheres are fixed on the film by micropores and magnetic fields in a regular arrangement, the same area is irradiated with ultraviolet light and the excitation light corresponding to the fluorescently labeled antibody to obtain two fluorescence images. The microspheres are regularly arranged in a two-dimensional plane and do not overlap with each other, greatly reducing fluorescence interference. Compared with the existing method of simply aggregating microspheres by magnetic fields, the imaging quality is higher, and there is no need to perform image segmentation processing on overlapping microspheres, greatly simplifying the subsequent algorithm. After obtaining two sets of fluorescence images of the microspheres arranged in a plane, the first set of quantum dot fluorescence images obtained by ultraviolet light excitation is used to decode the microspheres. The hues and intensities of the light emitted after mixing different ratios and amounts of quantum dots are different. Analyze the color and intensity of the emitted light to decode. The second set of fluorescence images is used to calculate the concentration of each analyte. In the two sets of fluorescence images taken at the same position, the positions of the microspheres remain unchanged. First, the second set of fluorescence images is classified according to the position distribution of the microspheres decoded from the first set of images. The fluorescence images of the microspheres with the same encoding in the second set of images are summarized, and the average fluorescence intensity of the reporter molecule on the microspheres with the same encoding is used to calculate the concentration of the analyte.

[0069] The high-throughput detection device of the embodiment of the present invention uses magnetic quantum dot-encoded microspheres with a Janus structure as carriers, and uses a polydimethylsiloxane film with a microporous array to fix the microspheres under the action of a magnetic field for high-throughput detection and analysis, and takes pictures of the microspheres arranged in a plane under a fluorescence microscope for analysis.

[0070] The microspheres integrate quantum dot encoding and magnetism on the microspheres by using the Janus structure. The Janus structure reduces the fluorescence quenching caused by the light absorption of magnetic nanoparticles. The magnetic properties of the microspheres simplify the cleaning and separation steps during subsequent immune reactions, and the microspheres can move or be fixed directionally under the action of a magnetic field.

[0071] After the microspheres capture the analyte in the solution, they are then fixed on the plane for analysis. The speed of capturing the analyte is faster than that of Scheme 2. At the same time, the number and type of microspheres can be flexibly changed according to the detection requirements, and the flexibility is good.

[0072] Use a polydimethylsiloxane film with a micropore array and a magnetic field to jointly fix microspheres. Each micropore can accommodate at most one microsphere, and the microspheres do not overlap or interfere with each other. Under the action of the magnetic field, the microspheres can maintain a fixed position without moving, avoiding afterimages on the fluorescence image.

[0073] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those skilled in the technical field to which the present invention belongs, without departing from the concept of the present invention, several equivalent substitutions or obvious modifications can be made, and if the performance or use is the same, they should all be regarded as belonging to the protection scope of the present invention.

Claims

1. A preparation method of magnetic quantum dot-encoded microspheres, characterized in that, It includes the following steps: S1. Prepare the continuous phase and the dispersed phase solutions; the dispersed phase includes the dispersed phase doped with quantum dot particles and the dispersed phase doped with magnetic nanoparticles; S2. Prepare uniform droplets through a microfluidic chip; push the continuous phase and the dispersed phase at a set speed, and the dispersed phase doped with quantum dot particles and the dispersed phase doped with magnetic nanoparticles always maintain laminar flow. After the dispersed phase is sheared into droplets, an obvious demarcation line can be seen in the middle of the droplets; S3. Add a photoinitiator so that the droplets can be rapidly cured into microspheres with a Janus structure after being irradiated by ultraviolet light in the channel; S4. Introduce functional groups on the surface of the microspheres for specifically binding to the antigen to be detected; In step S1, the main body of the dispersed phase is a photopolymerizable polymer monomer.

2. The preparation method of the microspheres according to claim 1, characterized in that, In step S1, the preparation methods of the dispersed phase doped with quantum dot particles and the dispersed phase doped with magnetic nanoparticles include: uniformly dispersing oil-soluble quantum dot particles and magnetic nanoparticles in the main body of the dispersed phase through a solvent evaporation method.

3. The preparation method of the microspheres according to claim 2, characterized in that, It also includes: Obtain multiple groups of quantum dot encodings by changing the types and masses of the quantum dots doped in the main body of the dispersed phase.

4. The preparation method of the microspheres according to claim 1, characterized in that, In step S3, the proportion of the photoinitiator is 1%-5%.

5. The preparation method of the microspheres according to claim 1, characterized in that, Step S4 is specifically: add maleic anhydride and a photopolymerizable monomer to copolymerize to introduce functional groups on the surface of the microspheres; partially hydrolyze the microspheres in an alkaline solution to hydrolyze the anhydride groups into carboxyl groups, and covalently couple with specific antibodies for specifically binding to the antigen to be detected.

6. A magnetic quantum dot-encoded microsphere, characterized in that, The microspheres are prepared by using the preparation method according to any one of claims 1-5. The microspheres include a microsphere main body, the microsphere main body includes two parts, both parts are hemispherical, one hemisphere is coated with oil-soluble quantum dot particles for encoding; the other hemisphere is coated with oil-soluble magnetic nanoparticles; the surface of the microspheres is rich in carboxyl groups, and the surface of the microspheres can covalently couple with specific antibodies for specifically binding to the antigen to be detected; the microspheres have a Janus structure for integrating quantum dot encoding and magnetism on the microspheres.

7. A device for high-throughput detection, characterized in that, It includes a microporous array film and the microspheres according to claim 6. One side of the microporous array film is provided with a plurality of micropores, and at most one microsphere can be accommodated in each micropore. The microspheres are fixed in the micropores, and different microspheres are at the same horizontal plane.

8. The device for high-throughput detection according to claim 7, wherein, It also includes a magnet, and the magnet is placed under the microporous array film.

9. The device for high-throughput detection according to claim 7, characterized in that The component of the microporous array film is polydimethylsiloxane.

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