Preparation and application methods of monodisperse gel microspheres for chemiluminescence detection
The monodispersed gel microspheres formed by the confluent focus microneedle channel array generation and photocrosslinking reaction, combined with the biotin-streptavidin reaction, solve the complexity and high consumption problems of traditional magnetic bead methods and achieve high efficiency , simple chemiluminescence detection improves detection sensitivity and efficiency.
Patent Information
- Application Number
- CN202411075953.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-07
- Publication Date
- 2024-12-13
AI Technical Summary
Traditional magnetic bead chemiluminescence detection has problems such as complex preparation process, cumbersome process, difficult to accurately regulate particle size, and large reagent consumption, and magnetic beads are the most consumed raw materials.
The oil phase and aqueous phase reagent are used to generate monodisperse microemulsion through a co-flow focusing microneedle channel array, and the photocrosslinking reaction is carried out to form gel microspheres, and antibody labeling is carried out through biotin-streptavidin reaction to achieve high efficiency, Simple preparation of monodispersed gel microspheres.
It realizes efficient and simple preparation of monodispersed gel microspheres, flexibly regulates the particle size of the microspheres, reduces reagent consumption, improves the sensitivity and detection efficiency of chemiluminescence detection, and solves the traditional method's low throughput, high cost and chip Processing difficulty issue.
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Figure CN119113949A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of monodisperse microsphere preparation, in particular to a method for preparing and applying monodisperse gel microspheres for chemiluminescence detection. Background Art
[0002] Monodisperse microspheres refer to microspheres with relatively uniform particle size, and generally require the particle size distribution variance to be less than 15%. Monodisperse gel microspheres are widely used in biochemical testing, chromatographic analysis, liquid crystal display, medical beauty filling, cell culture and other fields.
[0003] At present, the functionalized gel microspheres commonly used in chemiluminescent reagents on the market are magnetic microspheres. Magnetic bead microsphere chemiluminescence combines active substances such as antibodies / antigens with active groups on the surface of magnetic beads through a certain method and coats them on magnetic beads. During detection, the object to be tested and the magnetic beads coated with antibodies / antigens and antibodies / antigens labeled with chemiluminescent molecules are incubated under certain conditions, and combined through antigen-antibody reaction. By increasing the external magnetic field, the magnetic beads generate magnetism and gather together, which can be washed to separate the bound and unbound parts. Finally, the chemiluminescent molecules are excited and the signals are detected with photomultiplier tubes.
[0004] There are two ways to coat antibodies / antigens on magnetic beads: direct labeling and streptavidin. Generally speaking, direct coating of antibodies / antigens is affected by the isoelectric point and molecular size of the coated antibodies / antigens, and the coating scheme needs to be optimized. The streptavidin coating method can directly combine biotinylated antibodies / antigens with streptavidin-coated magnetic beads, which is more universal. A single detection of magnetic bead chemiluminescence requires about 25μg of magnetic beads, while the consumption of antibodies is 0.025-0.05μg. Therefore, magnetic beads are the raw material with the largest consumption of chemiluminescent reagents. However, magnetic bead chemiluminescence detection has shortcomings such as complex preparation process, cumbersome process, long production cycle, inability to accurately control particle size, and large reagent loss. Summary of the invention
[0005] In view of this, the embodiments of the present invention provide a method for preparing and applying monodisperse gel microspheres for chemiluminescence detection, which can efficiently and simply prepare monodisperse gel microspheres for chemiluminescence detection.
[0006] On the one hand, an embodiment of the present invention provides a method for preparing monodisperse gel microspheres for chemiluminescence detection, comprising:
[0007] Prepare an oil phase reagent and an aqueous phase reagent containing a photoinitiator;
[0008] Based on oil phase reagents and water phase reagents, a monodisperse microemulsion is generated by co-flow focusing microneedle channel array, and then gel microspheres are formed and transported inside the channel through photo-crosslinking reaction;
[0009] The gel microspheres are eluted to obtain monodisperse microspheres;
[0010] The monodisperse microspheres were labeled with the first antibody by the principle of antigen-antibody reaction between biotin-streptavidin.
[0011] Optionally, an oil phase reagent is prepared, comprising:
[0012] The base oil and the surfactant are fully mixed and stirred to prepare an oil phase reagent;
[0013] Among them, the base oil includes paraffin oil, mineral oil, vegetable oil and fluorinated oil; the surfactant includes span-80, polyglycerol-2-triisostearate, polyglycerol-3-diisostearate and polyglycerol-2-isostearate.
[0014] Among them, paraffin oil, mineral oil, vegetable oil and fluorinated oil are used as base oils, which provide a rich selection of chemical and physical properties and help to adjust the stability and functionality of microemulsions. By mixing span-80 and polyglycerol series surfactants, the emulsification ability and stability of oil phase reagents can be significantly improved, which helps to form uniform microemulsion droplets.
[0015] Optionally, an aqueous phase reagent containing a photoinitiator is formulated, comprising:
[0016] The gel solution, the functional group, the enzyme and the photoinitiator are fully mixed and stirred to prepare an aqueous phase reagent;
[0017] The gel includes PEG and gelatin; the functional group includes biotin; the enzyme includes glucose oxidase and peroxidase; and the photoinitiator includes LAP.
[0018] Among them, the addition of gel solutions (such as PEG and gelatin), functional groups (such as biotin) and enzymes (such as glucose oxidase and peroxidase) to the aqueous phase provides additional biological activity and functionality for the microemulsion, enhancing its potential for application in biological detection and diagnosis. In addition, the addition of photoinitiators (such as LAP) allows the cross-linking reaction to be triggered by light, achieving rapid preparation of gel microspheres and improving preparation efficiency and controllability.
[0019] Optionally, the co-flow focusing microneedle channel array includes a first microneedle array and a second channel array, the interior of the needle tube of the first microneedle array is used to transport an aqueous phase reagent, and the exterior of the needle tube of the first microneedle array is used to transport an oil phase reagent; the needle tube outlet of the first microneedle array is a pointed tip, which is arranged inside the inlet of the second channel array, the inlet and outlet of the second channel array are both flat heads, and the needles of the second channel array and the first microneedle array are aligned to maintain coaxiality; based on the oil phase reagent and the aqueous phase reagent, a monodisperse microemulsion is generated by using the co-flow focusing microneedle channel array, and then gel microspheres are formed and transported inside the channel through a photocrosslinking reaction, including:
[0020] Based on the reagent water phase transported inside the needle tubes of the first microneedle array and the oil phase reagent transported outside the needle tubes of the first microneedle array, co-flow focusing is performed in the inlet of the second channel array to generate a monodisperse microemulsion;
[0021] The monodisperse microemulsion is transported through the interior of the second channel array and undergoes a photo-crosslinking reaction to form gel microspheres.
[0022] Among them, the design of the co-flow focusing microneedle channel array realizes the precise co-flow focusing of oil phase and water phase reagents, generates microemulsion droplets with good monodispersity, and then forms gel microspheres of uniform size through photo-cross-linking reaction.
[0023] Optionally, a UV curing lamp is arranged around the second channel array; and the monodisperse microemulsion is transported through the second channel array and subjected to a photo-crosslinking reaction to form gel microspheres, comprising:
[0024] The monodisperse microemulsion transported inside the second channel array is photo-crosslinked and cured by an ultraviolet curing lamp to form gel microspheres.
[0025] Among them, the use of UV curing lamp makes the photocross-linking reaction more rapid and uniform, which helps to quickly form stable gel microspheres and improves the preparation efficiency.
[0026] Optionally, the method further comprises:
[0027] The gel microspheres output from the outlet of the second channel array are received by a collecting container.
[0028] The gel microspheres are received by a collection container, thereby realizing batch collection and subsequent processing of the microspheres.
[0029] Optionally, the gel microspheres are subjected to an elution treatment to obtain monodisperse microspheres, comprising:
[0030] The gel microspheres are sequentially eluted with an eluent and deionized water to obtain monodisperse microspheres;
[0031] Wherein, the eluent includes at least one of n-hexadecane, n-undecane, acetone, Tween, ethanol and deionized water.
[0032] Among them, the elution treatment removes impurities and unreacted substances on the surface of the microspheres, improves the purity and stability of the microspheres, and provides high-quality microsphere materials for subsequent applications.
[0033] On the other hand, an embodiment of the present invention provides a method for applying monodisperse gel microspheres, comprising:
[0034] Obtaining monodisperse microspheres labeled with a first antibody; wherein the monodisperse microspheres are prepared by the above-mentioned method for preparing monodisperse gel microspheres for chemiluminescence detection;
[0035] The monodisperse microspheres are mixed with the antigen to be detected and the second antibody to form a sandwich immune mixture;
[0036] Chemiluminescent immunoassay was performed based on sandwich immunoassay mixture using chemiluminescent excitation solution.
[0037] Optionally, the monodisperse microspheres are mixed with the antigen to be detected and the second antibody to form a sandwich immune mixture, comprising:
[0038] Adding a sample reagent containing a predetermined concentration of the antigen to be detected to the monodisperse microspheres;
[0039] adding an antibody reagent of an acridinium ester-labeled secondary antibody to the monodisperse microspheres;
[0040] The monodisperse microspheres of the mixed sample reagent and antibody reagent are placed in a metal bath at a preset temperature and for a preset time to form a sandwich immune mixture.
[0041] Optionally, before the step of performing chemiluminescent immunoassay using a chemiluminescent excitation solution, the method further comprises:
[0042] Remove the liquid reagents soaked in the sandwich immune mixture; the liquid reagents include sample reagents and antibody reagents.
[0043] Among them, the use of monodisperse microspheres labeled with the first antibody combined with the formation of sandwich immune complexes improves the sensitivity of chemiluminescence detection and can accurately detect low-concentration antigens. The operation process is simplified and the detection efficiency is improved through the preset steps of adding sample reagents and antibody reagents, metal bath treatment, etc. After removing the liquid reagent, the chemiluminescence excitation liquid is used for detection, which reduces background interference and improves the accuracy and reliability of the test results.
[0044] The embodiment of the present invention proposes a method for preparing monodisperse gel microspheres for chemiluminescent detection, by configuring an oil phase reagent and an aqueous phase reagent containing a photoinitiator; based on the oil phase reagent and the aqueous phase reagent, a monodisperse microemulsion is generated by using a co-flow focusing microneedle channel array, and then a gel microsphere is formed and transported inside the channel by a photocrosslinking reaction; the gel microsphere is eluted to obtain a monodisperse microsphere; and the monodisperse microsphere is firstly labeled with an antibody by the antigen-antibody reaction principle between biotin-streptavidin. The embodiment of the present invention can flexibly control the particle size of the microspheres and the preparation flux, and provide raw material mass production conditions for new chemiluminescent reagents, mainly based on an oil-in-water microfluidic emulsification system, and then a large amount of monodisperse emulsions are generated in a microfluidic device based on the co-flow focusing principle by using the oil phase and the aqueous phase, which can be used for coupling antibodies for chemiluminescent detection applications, and can effectively solve the problems of low flux, high cost and high chip processing difficulty of traditional microfluidic methods. BRIEF DESCRIPTION OF THE DRAWINGS
[0045] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.
[0046] Figure 1 A schematic flow chart of a method for preparing monodisperse gel microspheres for chemiluminescence detection provided in an embodiment of the present invention;
[0047] Figure 2 A schematic flow chart of a method for applying monodisperse gel microspheres provided in an embodiment of the present invention;
[0048] Figure 3 A flow chart of high-throughput preparation of functionalized gel microspheres for chemiluminescent detection reagents provided in an embodiment of the present invention;
[0049] Figure 4 A schematic diagram of the process of emulsion generation and solidification based on a co-flow focusing microneedle channel array provided in an embodiment of the present invention. DETAILED DESCRIPTION
[0050] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.
[0051] On the one hand, if Figure 1 As shown, an embodiment of the present invention provides a method for preparing monodisperse gel microspheres for chemiluminescence detection, comprising:
[0052] S100, preparing an oil phase reagent and an aqueous phase reagent containing a photoinitiator;
[0053] It should be noted that, in some embodiments, preparing the oil phase reagent may include: fully mixing and stirring the base oil and the surfactant to prepare the oil phase reagent; wherein the base oil includes paraffin oil, mineral oil, vegetable oil and fluorinated oil; the surfactant includes span-80, polyglycerol-2-triisostearate, polyglycerol-3-diisostearate and polyglycerol-2-isostearate.
[0054] In some embodiments, preparing an aqueous phase reagent containing a photoinitiator may include:
[0055] The gel solution, the functional group, the enzyme and the photoinitiator are fully mixed and stirred to prepare an aqueous phase reagent;
[0056] The gel includes PEG and gelatin; the functional group includes biotin; the enzyme includes glucose oxidase and peroxidase; and the photoinitiator includes LAP.
[0057] For example, in some specific embodiments, the reagent preparation includes oil phase reagent preparation and water phase reagent preparation, the oil phase reagent is used as the continuous phase in the microfluidic system, and the water phase reagent is used as the dispersed phase in the microfluidic system.
[0058] The preparation method of the oil phase reagent is to fully mix and stir the base oil and the surfactant, the base oil includes paraffin oil, mineral oil, vegetable oil, fluorinated oil, etc.; the surfactant includes span-80, polyglycerol-2-triisostearate, polyglycerol-3-diisostearate, polyglycerol-2-isostearate, etc.;
[0059] The method for configuring the aqueous phase reagent is to fully mix and stir the gel solution, functional groups, enzymes, and photoinitiators. The gel includes PEG, gelatin, etc.; the functional groups include Biotin, etc.; the enzymes include glucose oxidase (GOX) enzyme, peroxidase (peroxidase) enzyme, etc.; the photoinitiators include LAP, etc.
[0060] S200, based on oil phase reagents and water phase reagents, a monodisperse microemulsion is generated by using a co-flow focusing microneedle channel array, and then gel microspheres are formed and transported inside the channel through a photo-crosslinking reaction;
[0061] It should be noted that the co-flow focusing microneedle channel array includes a first microneedle array and a second channel array, the inside of the needle tube of the first microneedle array is used to transport aqueous phase reagents, and the outside of the needle tube of the first microneedle array is used to transport oil phase reagents; the needle tube outlet of the first microneedle array is a pointed tip, which is arranged inside the inlet of the second channel array, and the inlet and outlet of the second channel array are both flat heads, and the needles of the second channel array and the first microneedle array are kept coaxial by alignment; in some embodiments, based on the oil phase reagent and the aqueous phase reagent, a monodisperse microemulsion is generated by using a co-flow focusing microneedle channel array, and then gel microspheres are formed and transported inside the channel through a photocrosslinking reaction, which may include: based on the reagent aqueous phase transported inside the needle tube of the first microneedle array and the oil phase reagent transported outside the needle tube of the first microneedle array, co-flow focusing is performed in the inlet of the second channel array to generate a monodisperse microemulsion; the monodisperse microemulsion is transported through the inside of the needle tube of the second channel array and a photocrosslinking reaction is performed to form gel microspheres.
[0062] In some embodiments, a UV curing lamp is provided around the second channel array; transporting the monodisperse microemulsion inside the second channel array and performing a photo-crosslinking reaction to form gel microspheres may include: performing photo-crosslinking and curing of the monodisperse microemulsion transported inside the second channel array by a UV curing lamp to form gel microspheres.
[0063] In some embodiments, the method may further include: receiving the gel microspheres output from the outlet of the second channel array through a collection container.
[0064] Exemplarily, in some specific embodiments, a water-in-oil emulsion containing gel microspheres is simultaneously generated by a multi-channel emulsion generation method using a co-flow focusing microneedle channel array; the co-flow focusing microneedle channel array includes a first microneedle array and a second channel array;
[0065] The first microneedle arrays are made of non-transparent materials, including stainless steel, aluminum, copper, and polyetheretherketone, with an inner diameter ranging from 30-60 μm;
[0066] The second channel array is made of transparent materials, including quartz, plexiglass and polypropylene, with an inner diameter ranging from 100-120μm;
[0067] The inside of the needle tube of the first microneedle array is used to transport the water phase, and the outside of the needle tube of the first microneedle array is used to transport the oil phase; the inside of the inlet of the second channel array is used for co-flow focusing to generate a monodisperse microemulsion; the outlet of the second channel array is used to transport the solidified gel microspheres;
[0068] Ultraviolet curing lamps are arranged around the second channel array. The wavelength and light intensity of the ultraviolet curing lamps are adjustable and are used to cure microemulsions of different sizes. Under the action of ultraviolet rays, the microemulsion undergoes a photo-crosslinking reaction in the second channel array to form solid particles, which are transported from the outlet of the second channel array to a collection container.
[0069] The microsphere collection method is to use a collection container filled with an oil phase to receive the monodisperse microgel microspheres output from the second channel array.
[0070] Among them, in some preferred embodiments, high-throughput preparation can be achieved by adjusting the number of microneedle channel arrays and parameters such as tube diameter, and the preparation throughput is greater than 2000 per second, and the gel particle size is adjustable, with a particle size range of 20-100 microns.
[0071] S300, eluting the gel microspheres to obtain monodisperse microspheres;
[0072] It should be noted that, in some embodiments, eluting the gel microspheres to obtain monodisperse microspheres may include: eluting the gel microspheres sequentially through an eluent and deionized water to obtain monodisperse microspheres; wherein the eluent includes at least one of n-hexadecane, n-undecane, acetone, Tween, ethanol and deionized water.
[0073] For example, in some specific embodiments, the elution method is a method of eluting the oil on the surface of the microspheres, the eluent is selected from one or more of n-hexadecane, n-undecane, acetone, Tween, ethanol and deionized water, and the collected microspheres are eluted sequentially by the eluent and deionized water to finally obtain monodisperse microspheres.
[0074] S400, labeling the monodisperse microspheres with the first antibody.
[0075] Exemplarily, in some specific embodiments, the chemiluminescent detection application is to label the first antibody on functionalized gel microspheres prepared based on a high-throughput method to prepare immune gel microspheres. Because the microspheres are coated with a chemiluminescent initiator, when the antigen to be tested combines with the second antibody to form a sandwich immune complex, a photon signal can be generated on the surface of the microspheres under the action of a chemiluminescent excitation solution, thereby realizing chemiluminescent immunoassay.
[0076] On the other hand, Figure 2 As shown, an embodiment of the present invention provides a method for applying monodisperse gel microspheres, comprising:
[0077] T100, obtaining monodisperse microspheres labeled with the first antibody; wherein the monodisperse microspheres are prepared by the above-mentioned method for preparing monodisperse gel microspheres for chemiluminescence detection;
[0078] T200, mixing the monodisperse microspheres with the antigen to be tested and the second antibody to form a sandwich immune mixture;
[0079] It should be noted that, in some embodiments, the monodisperse microspheres are mixed with the antigen to be tested and the second antibody to form a sandwich immune mixture, which may include: adding a sample reagent of the antigen to be tested at a preset concentration to the monodisperse microspheres; adding an antibody reagent of a second antibody labeled with an acridinium ester to the monodisperse microspheres; and subjecting the monodisperse microspheres mixed with the sample reagent and the antibody reagent to a metal bath at a preset temperature and for a preset period of time to form a sandwich immune mixture.
[0080] T300, based on sandwich immunoassay, performs chemiluminescent immunoassay using chemiluminescent excitation solution.
[0081] In some embodiments, before the step of performing chemiluminescent immunoassay using chemiluminescent excitation solution, the method may further include: removing the liquid reagent soaked in the sandwich immunoassay mixture; the liquid reagent includes a sample reagent and an antibody reagent.
[0082] For example, in some specific embodiments, the process of chemiluminescence detection application is described in combination with specific application examples, and the experimental steps of the chemiluminescence method based on GOX functionalized microspheres are as follows:
[0083] (1) Take 50 μL of functionalized microspheres labeled with anti-cTn I primary antibody.
[0084] (2) Add 50 μL of cTn I antigen sample with a known concentration of 19.05 ng / mL.
[0085] (3) Add 50 μL of acridinium ester-labeled cTn I secondary antibody and mix well.
[0086] (4) Incubate in metal bath at 37°C for 10 min.
[0087] (5) Remove liquid reagents.
[0088] (6) Add 300 μL of excitation solution and use PMT to instantly test the luminescence signal.
[0089] The following is a detailed description of the process steps of the method for preparing monodisperse gel microspheres for chemiluminescence detection and the structure and function of the co-flow focusing microneedle channel array for preparing monodisperse gel microspheres for chemiluminescence detection in conjunction with the accompanying drawings of the specification:
[0090] First of all, it should be noted that, at present, monodisperse gel microspheres are mainly prepared in the laboratory through the microfluidic chip method. The traditional microfluidic method relies on high-precision microfluidic chips, which are mainly processed and prepared by soft lithography or injection molding. However, traditional microfluidic chips are difficult to process, expensive equipment, and low integration, making it difficult to achieve high-throughput preparation requirements.
[0091] like Figure 3As shown, the present invention provides a high-throughput preparation and application method of monodisperse gel microspheres, including reagent preparation, multi-channel emulsion generation and solidification, elution and chemiluminescence detection application. In some specific embodiments, the process steps include:
[0092] 1. Reagent preparation: Mix 20 mL of paraffin oil and 2 mL of Span-80 surfactant, shake for 2 minutes, and then stir with a magnetic stirrer for 5 minutes to fully mix them as the oil phase; weigh 5 mg of GOX and 8.5 mg of Biotin-PEG-SH and add them to centrifuge tube 1, add 250 μL of ultrapure water and shake well; then weigh 250 mg of PEGDA and add it to centrifuge tube 2, add 250 μL of ultrapure water and shake well; then weigh 3 mg of LAP under light-proof conditions, add it to centrifuge tube 3, add 250 μL of ultrapure water and shake well; finally, transfer the solutions in centrifuge tube 1 and centrifuge tube 3 to centrifuge tube 2 and mix well to complete the preparation of the aqueous phase;
[0093] 2. Multi-channel emulsion generation and curing: Figure 4 As shown, the prepared aqueous phase solution 4 and oil phase solution 5 are added respectively Figure 4 In the multi-channel microfluidic device shown, pressures of 10 kPa and 12 kPa are applied through the first pressurizing port 6 and the second pressurizing port 7, respectively, to drive the aqueous solution 4 into the first microneedle array 1, and to drive the oil phase solution 5 into the second channel array 2, and to form a monodisperse emulsion in the inlet of the second channel array 2, and to be transported to the outlet through the second channel array 2, and during the transportation process, the microemulsion passes through the irradiation area of the ultraviolet light source 3, so that the microemulsion completes cross-linking and solidification to form gel microspheres and is collected in a container;
[0094] 3. Elution: Transfer the microspheres in the collector to a new glass bottle, remove the oil phase as much as possible, then add an eluent such as n-hexadecane or n-undecane, and wash repeatedly for 3 times; then wash away the eluent with deionized water, and finally resuspend the microspheres in water to obtain monodisperse microspheres;
[0095] 4. Chemiluminescence detection application:
[0096] Experimental steps of chemiluminescence method based on GOX functionalized microspheres:
[0097] (1) Take 50 μL of functionalized microspheres labeled with anti-cTn I primary antibody.
[0098] (2) Add 50 μL of cTn I antigen sample with a known concentration of 19.05 ng / mL.
[0099] (3) Add 50 μL of acridinium ester-labeled cTn I secondary antibody and mix well.
[0100] (4) Incubate in metal bath at 37°C for 10 min.
[0101] (5) Remove liquid reagents.
[0102] (6) Add 300 μL of excitation solution and use PMT to instantly test the luminescence signal.
[0103] The present invention provides a high-throughput preparation method for functionalized microspheres that can realize no-cleaning chemiluminescent detection. The method can flexibly adjust the particle size of microspheres and the preparation flux, provide raw material mass production conditions for new chemiluminescent reagents, and is mainly based on an oil-in-water microfluidic emulsification system. Functionalized groups, enzymes, photoinitiators and other reagents are coated in a gel solution, and then a pneumatic device is used to drive the oil phase and the water phase in a multi-channel microfluidic device based on the co-flow focusing principle to generate a large amount of monodisperse emulsions and transport them to an exposure area. The gel emulsion is cross-linked and solidified into microsphere particles under the action of ultraviolet optical fiber, and finally the oil phase on the surface of the microsphere is eluted, which can be used for coupling antibodies for chemiluminescent detection applications. The process of this scheme is simple, the number of channels can be freely expanded, all processes can be highly integrated and automated, and it does not rely on expensive equipment and consumables. It can effectively solve the problems of low flux, high cost and high difficulty in chip processing of traditional microfluidic methods.
[0104] In some selectable embodiments, the function / operation mentioned in the block diagram may not occur in the order mentioned in the operation diagram. For example, depending on the function / operation involved, the two boxes shown in succession can actually be executed substantially simultaneously or the boxes can sometimes be executed in reverse order. In addition, the embodiment presented and described in the flow chart of the present invention is provided by way of example, for the purpose of providing a more comprehensive understanding of technology. The disclosed method is not limited to the operation and logic flow presented herein. Selectable embodiments are expected, wherein the order of various operations is changed and the sub-operation of a part for which is described as a larger operation is performed independently.
[0105] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.
[0106] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.
[0107] The above is a specific description of the preferred implementation of the present invention, but the present invention is not limited to the embodiments. Those skilled in the art can make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.
Claims
1. A method for preparing monodisperse gel microspheres for chemiluminescence detection, characterized in that: include: Prepare an oil phase reagent and an aqueous phase reagent containing a photoinitiator; Based on the oil phase reagent and the water phase reagent, a monodisperse microemulsion is generated by using a co-flow focusing microneedle channel array, and then gel microspheres are formed and transported inside the channel through a photo-crosslinking reaction; performing an elution treatment on the gel microspheres to obtain monodisperse microspheres; The monodisperse microspheres are labeled with the first antibody by the antigen-antibody reaction principle between biotin and streptavidin.
2. The method for preparing monodisperse gel microspheres for chemiluminescence detection according to claim 1, characterized in that: The preparation of the oil phase reagent comprises: The base oil and the surfactant are fully mixed and stirred to prepare the oil phase reagent; The base oil includes paraffin oil, mineral oil, vegetable oil and fluorinated oil; the surfactant includes span-80, polyglycerol-2-triisostearate, polyglycerol-3-diisostearate and polyglycerol-2-isostearate.
3. The method for preparing monodisperse gel microspheres for chemiluminescence detection according to claim 1, characterized in that: The aqueous phase reagent containing a photoinitiator is prepared, comprising: The gel solution, the functionalized group, the enzyme and the photoinitiator are fully mixed and stirred to prepare the aqueous phase reagent; Wherein, the gel comprises PEG and gelatin; the functionalized group comprises biotin; the enzyme comprises glucose oxidase and peroxidase; and the photoinitiator comprises LAP.
4. The method for preparing monodisperse gel microspheres for chemiluminescence detection according to claim 1, characterized in that: The co-flow focusing microneedle channel array comprises a first microneedle array and a second channel array, wherein the interior of the needle tube of the first microneedle array is used to transport the aqueous phase reagent, and the exterior of the needle tube of the first microneedle array is used to transport the oil phase reagent; the needle tube outlet of the first microneedle array is a pointed tip, which is arranged inside the inlet of the second channel array, the inlet and outlet of the second channel array are both flat heads, and the second channel array and the first microneedle array are aligned to maintain coaxiality; Based on the oil phase reagent and the water phase reagent, a monodisperse microemulsion is generated by using a co-flow focusing microneedle channel array, and then gel microspheres are formed by a cross-linking reaction, including: Based on the reagent water phase transported inside the needle tubes of the first microneedle array and the reagent oil phase transported outside the needle tubes of the first microneedle array, microfluidic co-flow focusing is performed in the inlet of the second channel array to generate the monodisperse microemulsion; The monodisperse microemulsion is transported through the interior of the second channel array and undergoes a cross-linking reaction to form gel microspheres.
5. The method for preparing monodisperse gel microspheres for chemiluminescence detection according to claim 4, characterized in that: The second channel array is surrounded by an ultraviolet curing lamp; the monodisperse microemulsion is transported through the second channel array and cross-linked to form gel microspheres, including: The monodisperse microemulsion transported inside the second channel array is cross-linked and cured by the ultraviolet curing lamp to form gel microspheres.
6. The method for preparing monodisperse gel microspheres for chemiluminescence detection according to any one of claims 4 or 5, characterized in that: The method further comprises: The gel microspheres outputted from the outlet of the second channel array are received by a collecting container.
7. The method for preparing monodisperse gel microspheres for chemiluminescence detection according to claim 1, characterized in that: The step of eluting the gel microspheres to obtain monodisperse microspheres comprises: The gel microspheres are sequentially eluted with an eluent and deionized water to obtain the monodisperse microspheres; Wherein, the eluent includes at least one of n-hexadecane, n-undecane, acetone, Tween, ethanol and deionized water.
8. A method for applying monodisperse gel microspheres, characterized in that: include: Obtaining monodisperse microspheres labeled with a first antibody; wherein the monodisperse microspheres are prepared by the method for preparing monodisperse gel microspheres for chemiluminescence detection according to any one of claims 1 to 7; The monodisperse microspheres are mixed with an antigen to be detected and a second antibody to form a sandwich immune mixture; Based on the sandwich immune mixture, chemiluminescent immunoassay is performed by using chemiluminescent excitation solution.
9. The method for applying monodisperse gel microspheres according to claim 8, characterized in that: The step of mixing the monodisperse microspheres with the antigen to be detected and the second antibody to form a sandwich immune mixture comprises: Adding a sample reagent of the antigen to be detected at a preset concentration to the monodisperse microspheres; adding an antibody reagent of the second antibody labeled with an acridinium ester to the monodisperse microspheres; The monodisperse microspheres mixed with the sample reagent and the antibody reagent are placed in a metal bath at a preset temperature and for a preset time to form the sandwich immune mixture.
10. The method for applying monodisperse gel microspheres according to claim 9, characterized in that: Before the step of performing chemiluminescent immunoassay using a chemiluminescent excitation solution, the method further comprises: The liquid reagent in which the sandwich immune mixture is soaked is removed; the liquid reagent includes the sample reagent and the antibody reagent.