A high-throughput rapid screening chip for allergens based on 3D microarray and reciprocating flow and its application

Abstract

The present invention discloses a high-throughput rapid screening chip for allergens based on 3D microarray and reciprocating flow and its application. The chip uses fiber materials to construct a microarray with a 3D porous structure on a solid substrate, providing more binding sites for allergen molecules than 2D planar microdots, so as to enhance the detection signal and obtain higher detection sensitivity in a shorter detection time. The preparation of the chip uses the orientation force and hydrogen bonds of the fiber material to construct an adsorption / binding system for allergen molecules, and completes the pre-adsorption of allergen molecules and fiber materials by pre-mixing the allergen molecules and the fiber material. The controllable preparation of 3D structured microdots is completed by continuous spotting technology and moisture-induced phase conversion technology. The present invention further provides a high-throughput rapid screening method for allergens based on the 3D microarray chip, which uses a reciprocating flow control strategy to promote repeated contact between the molecules to be tested in the sample solution and the allergen molecules on the solid surface, thereby achieving rapid detection of allergens.

Description

Technical Field

[0001] The present invention belongs to the field of medical diagnosis technology, and more specifically, relates to a high-throughput rapid screening chip for allergens based on 3D microarray and reciprocating flow and its application. Background Art

[0002] Allergic diseases affect people of all ages, with a global prevalence exceeding 25% and a growing trend. Severe allergic reactions can lead to systemic anaphylaxis or anaphylactic shock, potentially resulting in death within half an hour. The World Allergy Organization points out that helping patients with severe allergies quickly identify and avoid allergens is crucial for both emergency treatment and long-term health management.

[0003] Currently, there are hundreds of known allergens. Multi-index allergen detection technology is an important tool for identifying allergens. Common methods for allergen detection include skin testing and in vitro detection of allergen-specific immunoglobulin E (sIgE). In vitro sIgE allergen testing provides more objective results and more validation indicators, making it widely used in hospital allergen testing programs. Key methods include microplate-based enzyme-linked immunosorbent assay (ELISA), strip-based chromogenic chromatography / ELISA, and microarray chips. Due to the limitations of the detection platform, microplate- and strip-based methods typically can simultaneously detect fewer than 100 allergen indicators. Compared to the aforementioned two methods, high-throughput allergen screening using microarray chips is an important research tool. A typical high-throughput allergen screening chip is the ImmunoCAP ISAC system developed by Thermo Fisher Scientific, the most widely used high-throughput allergen screening method in Europe and the United States.

[0004] Specifically, allergen molecules are immobilized on amino-modified glass slides, creating 2D microdots with a diameter of 200 μm and forming a planar microdot array. After several hours of incubation, allergen sIgE is captured in the sample solution, and the results are read by fluorescence detection. However, the 2D surface provides too few binding sites, and reading the test results relies on sophisticated optical detection devices; for example, the ISAC system is equipped with a small fluorescence confocal reader. To address this problem, researchers have attempted for many years to use micro-nanoparticles to construct microarray chips with larger surface areas. For example, MacroArray, a company, couples nanoparticles and allergen extract molecules and deposits them on a solid surface to create the ALEX series of screening chips. These chips require only a CCD element to read the test results, making them one of the most advanced high-throughput allergen screening products in Europe and the United States. However, these methods still suffer from the drawback of lengthy detection times, making them unsuitable for rapid allergen screening in patients with acute allergies.

[0005] In recent years, the strategy of using 3D porous structures to provide biological probes or antibodies with more binding sites than 2D planes has begun to be applied in the field of immunoassays, but there are still technical difficulties in forming 3D micro-array structures. The main difficulty is the problem of controllable molding of the 3D structure of the micro-array (including the controllability of external dimensions and internal pores). After the 3D micro-array structure is completed, the antibodies or allergen antigens are further fixed on the micro-dots. For automated equipment, the precision requirements are very high, which makes the preparation of 3D micro-arrays difficult and greatly increases the cost of detection and screening. In addition, the current technology has achieved faster immunoassay speeds in microfluidic chips through reciprocating flow. In a microfluidic chip with 3D micro-dots, reciprocating flow control can still prompt the sample solution and the molecules to be tested carried therein to repeatedly pass through the reaction area (i.e., 3D micro-dots), which in principle can speed up the immunoassay. However, the 3D microdot array will have a significant impact on the velocity distribution and flow trajectory distribution of the liquid flow, and it is difficult to obtain direct inspiration for the application of the reciprocating flow immunoassay strategy on the 3D microdot array chip from the reciprocating flow immunoassay strategy in ordinary microfluidic chips. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a 3D microarray chip for high-throughput rapid screening of allergens and a preparation method thereof.

[0007] The second object of the present invention is to provide a high-throughput rapid screening method for allergens based on the 3D microarray chip and reciprocating flow control strategy.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for preparing a 3D microarray-based high-throughput rapid allergen screening chip, comprising the following steps:

[0010] S1. The cellulose or its derivative material powder is dissolved in a mixed solvent containing a solvent, a porogen, a protein protective agent, and stirred to obtain a fiber suspension;

[0011] S2. adding different allergen molecules to the fiber suspension and stirring to form a multi-allergen-fiber slurry;

[0012] S3. Extruding a plurality of allergen-fiber slurries onto the surface of a solid substrate to form a plurality of orderly arranged microdots to obtain a microdot array pattern;

[0013] S4. The micro-dot pattern is allowed to stand to form gel-like micro-dots, and steam containing a poor solvent is introduced to remove the slurry solvent of the micro-dot pattern to obtain a solid substrate having a 3D micro-dot array fixed on the surface;

[0014] S5. Assemble the solid substrate with the 3D micro array fixed on the surface to produce a 3D micro array chip.

[0015] This invention designs an allergen-fiber slurry made by mixing allergen extract molecules with a fiber suspension. This slurry is then used to create 3D microdots immobilized with allergens, forming a microdot array chip. The 3D microdots can increase the allergen loading on the solid surface, enhance the detection signal, and achieve high detection sensitivity in a shorter detection time. Specifically, the 3D microdot array chip prepared by this invention uses cellulose or its derivatives with good polarity as the fiber component of the allergen-fiber slurry. The allergen-fiber adsorption / binding system is constructed through the orientation forces and hydrogen bonds of the cellulose or its derivatives. The allergens are pre-dispersed in the cellulose or its derivative slurry, pre-adsorbed and immobilized on the fiber molecules, and then fixed to the 3D microdot structure as the fiber molecules form. Cellulose and its derivatives are generally soluble in organic solvents, which can damage proteins such as allergens or antibodies. Therefore, a protein protectant (sucrose or trehalose) is added to the slurry to reduce the organic solvent's competition for hydrogen bonds with the protein surface, thus developing a targeted allergen-fiber slurry. The controlled preparation of 3D microdot arrays on a chip's solid-phase substrate is based on moisture-induced phase transition technology. The underlying principle is that vapor composed of a poor solvent for cellulose or its derivatives promotes the thermodynamically unstable state of the solvent and solute in the allergen-fiber slurry, rapidly forming a rich phase rich in allergens and fiber molecules. This phase, under the influence of a corresponding lean phase (i.e., vapor composed of the poor solvent), precipitates and solidifies into a 3D fiber structure, thereby producing 3D microdots. Continuous spotting technology allows for the controlled preparation of 3D microdot arrays on solid-phase substrates, resulting in a chip with a 3D microdot array fixed to a microchannel substrate.

[0016] The preparation method of the 3D micro-array chip provided by the present invention is simple and fast to operate, and can be used to prepare 3D micro-array chips in large quantities, thus solving the problems of the current micro-array chip processing difficulty and high precision requirements. At the same time, the 3D structured microdots prepared by the allergen-fiber slurry provide more binding sites for allergen molecules, which can increase the solid loading capacity of allergen molecules on the microdots, thereby improving the detection sensitivity. In addition, the present invention is based on moisture-induced phase transition technology to quickly make allergen-fiber slurry into 3D structured microdots; the 3D structured microdots prepared by using cellulose or its derivative materials have the advantages of excellent mechanical properties (not easy to be brittle), controllable processing and production, resistance to dehydration cracking / water absorption swelling, stronger adhesion on solid substrates, etc.

[0017] Furthermore, the cellulose or its derivative material powder in step S1 is selected from one or a combination of cellulose, cellulose acetate (CA), nitrocellulose (NC), and carboxymethyl cellulose (CMC); the solvent is selected from one or a combination of water, acetone, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and polyethersulfone (PES); the porogen is selected from one of polyethylene glycol (PEG) and ethyl acetate; and the protein protective agent is selected from one of sucrose and trehalose.

[0018] Preferably, the amount of cellulose or its derivative material powder used is 3-10% by weight, which can be flexibly changed according to the internal structure size requirements of the 3D micro-dots.

[0019] Preferably, the amount of the protein protective agent is 0.1 to 1% wt.

[0020] Preferably, the amount of the porogen is 2 to 6% by weight.

[0021] Preferably, the porogen is PEG, and the molecular weight of PEG can be selected from 200, 400 or 800, which can be flexibly changed according to the fiber pore size requirements of the 3D micro-dots.

[0022] Furthermore, the stirring in steps S1 and S2 is magnetic stirring for 2 hours.

[0023] Preferably, the rotation speed of the magnetic stirring is 800-1000 rpm, which can be flexibly changed according to the solubility of the cellulose or its derivative material in the solvent.

[0024] Furthermore, the types and concentrations of the allergen molecules in step S2 can be flexibly changed according to the requirements of allergen screening.

[0025] Furthermore, in step S3, the allergen-fiber slurry is extruded on the surface of the solid substrate by sucking a plurality of allergen-fiber slurries into a sampler respectively through a continuous spotting system and extruding them on the solid substrate.

[0026] Furthermore, the diameter of the micro-dots in step S3 is 100-500 μm, which can be flexibly changed according to the number of allergen screening indicators required.

[0027] Preferably, the distance between the micro-dots is 2 to 3 times the diameter of the micro-dots.

[0028] Furthermore, the standing time in step S4 is 2 to 10 minutes.

[0029] Furthermore, the poor solvent in step S4 is selected from one of water, ethanol, and isopropanol, or any combination thereof.

[0030] Furthermore, the conditions for introducing the steam containing the poor solvent in step S4 are constant temperature, constant humidity and constant airflow, and the specific condition parameters are: temperature 15-30° C., humidity 60-70% RH, and airflow speed 1.2-3.4 m / s.

[0031] The present invention provides a 3D microarray chip prepared by any one of the above methods.

[0032] The present invention provides the use of the 3D microarray chip in preparing a disease marker screening product or a biological sample detection kit.

[0033] The present invention also provides application of the 3D microarray chip in environmental analysis or food analysis.

[0034] The present invention provides a high-throughput rapid screening method for allergens based on the 3D microarray chip and reciprocating flow, comprising the following steps:

[0035] S1. The sample solution to be tested is mixed with a solution containing a labeled antibody to obtain a mixed sample solution;

[0036] S2. The mixed sample solution is added to the 3D microarray chip, so that the mixed sample solution flows back and forth in the 3D microarray chip, and the solution is discharged;

[0037] S3. Add cleaning solution to the 3D microarray chip, drain the solution after cleaning, and use the CCD element to obtain the allergen detection fluorescence image for analysis.

[0038] The high-throughput rapid allergen screening method utilizes a reciprocating flow control strategy to promote repeated contact and rapid binding between the molecules to be tested in the sample solution and the allergen molecules on the 3D microarray solid phase surface, thereby achieving rapid detection of allergens and completing rapid screening of allergens in the sample solution.

[0039] Furthermore, the labeled antibody in step S1 is an anti-human IgE antibody labeled with a fluorescent probe.

[0040] Furthermore, in step S1, the volume ratio of the sample solution to be tested to the solution containing the labeled antibody is 1:1.

[0041] Preferably, the volume of the sample solution to be tested is 35-50 μL.

[0042] Furthermore, the mixing in step S1 is followed by incubation for 1 hour to obtain a mixed sample solution.

[0043] Furthermore, the volume of the mixed sample solution in step S2 is 35 to 50 μL.

[0044] Furthermore, the reciprocating flow in step S2 is to control the reciprocating flow of the sample solution by using air pressure.

[0045] Preferably, the air pressure controls the reciprocating flow of the sample solution at a flow rate of 0.1 to 1 mm / s.

[0046] Preferably, the reciprocating flow time is 10 minutes.

[0047] Furthermore, the cleaning solution in step S3 is a phosphate buffered saline (PBS) solution containing 0.1-0.5% wt Tween-20, with a pH range of 7.0-8.0 and a concentration range of 10-100 mM.

[0048] Furthermore, the discharge of the solution after cleaning in step S3 is to discharge the solution by one-way cleaning of the micro-dot array by air pressure control.

[0049] Furthermore, the washing step in step S3 is repeated three times.

[0050] The present invention also provides applications of the high-throughput rapid allergen screening method in disease marker screening, biological sample detection, environmental analysis, and food analysis.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) The present invention provides a high-throughput rapid screening chip for allergens based on a 3D microarray and reciprocating flow. The chip utilizes fiber materials to construct a microarray with a 3D porous structure on a solid substrate, providing more binding sites for allergen molecules than 2D planar microdots, thereby enhancing the detection signal and obtaining higher detection sensitivity within a shorter detection time. The preparation method utilizes the orientation force and hydrogen bonds of the fiber material to construct an adsorption / binding system for allergen molecules, pre-adsorbs the allergen molecules and the fiber material by pre-mixing them, and completes the controllable preparation of 3D structured microdots through continuous spotting technology and moisture-induced phase transition technology. The preparation method is simple and fast to operate, and can be used to prepare 3D microarray chips in large quantities, solving the current problems of difficult processing and high precision requirements for microarray chips.

[0053] (2) The present invention further provides a high-throughput rapid screening method for allergens based on the 3D microarray chip, which utilizes a reciprocating flow control strategy to promote repeated contact between the sIgE molecules to be tested in the sample solution and the allergen molecules on the solid surface, thereby achieving rapid allergen detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic diagram of the preparation of allergen-fiber slurry.

[0055] Figure 2 Schematic diagram of the preparation of 3D microarray chip.

[0056] Figure 33D microarray on the prepared solid substrate.

[0057] Figure 4 This is a process for high-throughput rapid detection of allergen sIgE model protein based on 3D microarray and reciprocating flow control.

[0058] Figure 5 This is the result of high-throughput rapid detection of allergen sIgE model protein based on the 3D microarray and reciprocating flow control. DETAILED DESCRIPTION

[0059] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0060] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0061] Example 1 Preparation of allergen-fiber slurry and 3D microarray chip

[0062] S1. Dissolve 6% wt CA powder in a mixed solution consisting of DMF as solvent, PEG200 (5% wt) as porogen, glycerol (10% wt) and trehalose (0.6% wt) as protein protectants. Stir at 1000 rpm for 2 h with the aid of magnetic stirring to obtain a CA fiber suspension, which is then divided into 25 small tubes. Figure 1 shown.

[0063] S2. The purified allergen molecules were added to the subpackaged CA fiber suspension tubes, and stirred at 1000 rpm for 2 hours with the assistance of magnetic stirring to form a variety of allergen-fiber slurries, such as Figure 1 Tubes 1 to 4 were loaded with goat anti-human IgE antibodies to a final concentration of 1 mg / mL; tubes 5 to 25 were loaded with allergen recombinant proteins, including the types listed in Table 1 below.

[0064] Table 1 Allergen composition of allergen-fiber slurry in Example 1

[0065]

[0066]

[0067] S3. A variety of allergen-fiber slurries are sucked into the sample dispenser through a continuous spotting system and extruded onto the surface of a solid substrate to form a plurality of orderly arranged microdots. The diameter of each microdot is 500 μm, and the spacing between each microdot is twice the diameter of the microdot, resulting in a 5×5 microdot array pattern.

[0068] S4. The micro-dot pattern is allowed to stand to form gel-like micro-dots, and then steam with water as a poor solvent is introduced at a constant airflow rate of 1.8 m / s at 26-27°C and 60-70% RH to continuously remove the allergen-fiber slurry solvent in the micro-dot pattern, thereby promoting the formation of the micro-dots and forming a 3D fiber structure, thereby obtaining a solid phase substrate with a 3D micro-dot array fixed on the surface. Figure 3 shown.

[0069] S5, assembling a 3D microarray chip on the solid phase substrate having the 3D microarray fixed thereon. Example 2 High-throughput rapid detection of allergen sIgE model protein based on 3D microarray and reciprocating flow control

[0070] The process of high-throughput rapid detection of allergen sIgE model protein based on 3D microarray and reciprocating flow control is as follows Figure 4 The specific steps are as follows:

[0071] S1. The sIgE model protein corresponding to the allergen recombinant protein described in Example 1 was mixed in PBS at a concentration of 1 μg / mL to prepare a sample solution, and then mixed with a mouse anti-human IgE antibody solution labeled with FITC fluorescent microspheres (concentration of 1 mg / mL) at a volume ratio of 1:1, and incubated for 1 hour to obtain a mixed sample solution.

[0072] S2. 50 μL of the mixed sample solution was added to the 3D microarray chip. The sample solution was controlled to flow back and forth in the 3D microarray chip by air pressure for 10 minutes, and then the mixed sample solution was discharged. The flow rate of the solution was controlled to be 0.2 mm / s.

[0073] S3. Add 50 μL of PBS containing 0.5% wt Tween-20 to the 3D microarray chip, and clean the microarray in one direction using air pressure control, then drain the solution, repeating this process three times. The solution flow rate is controlled at 0.2 mm / s.

[0074] S4. Remove the solid substrate of the 3D micro-dot array chip, illuminate the 3D micro-dot array on the solid substrate with a laser of 485 nm in a dark box, and use a CCD camera to obtain the fluorescence image results of the allergen detection. The fluorescence image is as follows: Figure 5 The test results show that the 3D microarray chip can correctly provide clear allergen positive and negative results.

[0075] Currently, existing micro-dot array-based allergen detection takes more than 2 hours. The detection method provided by the present invention takes significantly less time and achieves high-throughput rapid detection of multiple allergens and their allergenic proteins.

[0076] The above comparative examples and embodiments are preferred implementation modes of the present invention, but the implementation modes and implementation effects of the present invention are not limited to the above comparative examples and embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a high-throughput rapid screening chip for allergens based on a 3D microarray, characterized in that: The following steps are involved: S1. The cellulose or its derivative material powder is dissolved in a mixed solvent containing a solvent, a porogen, and a protein protective agent, and stirred to obtain a fiber suspension; S2. adding different allergen molecules to the fiber suspension and stirring to form a multi-allergen-fiber slurry; S3. Extruding a plurality of allergen-fiber slurries onto the surface of a solid substrate to form a plurality of orderly arranged microdots to obtain a microdot array pattern; S4. The micro-dot pattern is allowed to stand to form gel-like micro-dots, and steam containing a poor solvent is introduced to remove the slurry solvent of the micro-dot pattern to obtain a solid substrate having a 3D micro-dot array fixed on the surface; S5. The solid substrate having a 3D microarray fixed on the surface is assembled into a 3D microarray chip; In step S1, the cellulose or its derivative material powder is selected from one or a combination of cellulose, cellulose acetate, cellulose nitrate, and carboxymethyl cellulose; the solvent is selected from one or a combination of water, acetone, N,N-dimethylformamide, dimethyl sulfoxide, and polyethersulfone; the porogen is selected from one of polyethylene glycol and ethyl acetate; and the protein protective agent is selected from one of sucrose and trehalose. The poor solvent in step S4 is selected from one of water, ethanol, and isopropanol, or any combination thereof.

2. The preparation method according to claim 1, characterized in that The diameter of the micro-dots in step S3 is 100-500 μm, and the distance between the micro-dots is 2-3 times the diameter of the micro-dots.

3. The preparation method according to claim 1, characterized in that The standing time in step S4 is 2 to 10 minutes.

4. The preparation method according to claim 1, characterized in that The conditions for introducing the steam containing the poor solvent in step S4 are: temperature 15-30° C., humidity 60-70% RH, and air flow velocity 1.2-3.4 m / s.

5. A 3D microarray-based high-throughput rapid allergen screening chip prepared by the preparation method according to any one of claims 1 to 4.

6. A high-throughput rapid screening method for allergens based on a 3D microarray chip and reciprocating flow, characterized in that: The following steps are involved: S1. The sample solution to be tested is mixed with a solution containing a labeled antibody to obtain a mixed sample solution; S2. The mixed sample solution is added to the 3D microarray-based allergen high-throughput rapid screening chip according to claim 5, so that the mixed sample solution flows back and forth in the 3D microarray chip and the solution is discharged; S3. Add cleaning solution to the 3D microarray chip, drain the solution after cleaning, and use the CCD element to obtain the allergen detection fluorescence image for analysis.

7. The method for rapid high-throughput screening of allergens according to claim 6, characterized in that: The reciprocating flow in step S2 is to control the reciprocating flow of the sample solution by using air pressure.

8. The method for high-throughput rapid screening of allergens according to claim 7, characterized in that: The air pressure controls the reciprocating flow rate of the sample solution to be 0.1-1 mm / s.

Images