High-throughput monkey pox virus microfluidic detection chip and application thereof

Abstract

The embodiment of the invention belongs to the technical field of microfluidics, and relates to a high-throughput monkey pox virus microfluidic detection chip and application thereof, the high-throughput monkey pox virus microfluidic detection chip comprises a controllable pneumatic pump, a cleaning bin, a sample adding port, a first sample application pad, a second sample application pad and a waste liquid bin which are connected in sequence; wherein the controllable pneumatic pump, the cleaning bin, the sample adding port, the first sample application pad, the second sample application pad and the waste liquid bin are arranged in a shell of the high-throughput monkey pox virus micro-fluidic detection chip; a fluorescent microsphere labeled monkey pox antibody is fixed on the first sample application pad; a monkey pox antibody coated with magnetic beads is arranged on the second sample application pad. According to the application, the sensitivity of detecting the monkey pox virus is improved.

Description

Technical Field

[0001] This application relates to the field of microfluidic technology, and particularly to a high-throughput microfluidic detection chip for monkeypox virus and its applications. Background Art

[0002] Monkeypox virus is a zoonotic virus that belongs to the genus Orthopoxvirus, along with smallpox and vaccinia virus, and is generally less severe than smallpox. Mature monkeypox virus is oval-shaped and can be cultured and grown in African green monkey kidney cells, causing cytopathic effects. Monkeypox occurs in monkeys and can also infect other animals, and occasionally infect humans. It presents similarly to smallpox but with a milder condition. Monkeypox virus can be transmitted from animals to humans through direct close contact and can also spread from person to person. The main routes of transmission include blood and body fluids. However, the infectivity of monkeypox is much lower than that of smallpox virus. Poxviruses exist in two forms: mature virus particles and lipid membranes.

[0003] There are several monkeypox antigen detection proteins as follows: A29L: an envelope protein on the surface of mature virus within cells. A35R: an envelope component of intracellular enveloped virus particles (IEV) and extracellular enveloped virus particles (EEV). B6R: located on the membrane of extracellular enveloped virus particles (EEV) and homologous to the vaccinia virus complement control protein B5. M1R: homologous to the vaccinia virus L1 protein, a transmembrane protein found on the surface of mature IMV particles. E8L: an important protein for virus invasion of host cells, which acts as a cell surface binding protein of MV and binds to chondroitin sulfate on the cell surface during the process of virus invasion of the host to complete replication, providing attachment of virus particles to target cells. H3L: a glycosyltransferase 324 AA long, located on the virus envelope, and plays a key role in the attachment to host cells and subsequent entry of poxviruses. The replication cycle of the virus is as Figure 1 shown Figure 1 is a schematic diagram of the virus replication cycle.

[0004] Currently, the main detection methods for monkeypox virus are qPCR detection, colloidal gold method, etc., which are complex in operation, long in detection time, and insufficient in sensitivity. Summary of the Invention

[0005] The purpose of the embodiments of this application is to provide a high-throughput microfluidic detection chip for monkeypox virus and its applications, and this application improves the sensitivity of monkeypox virus detection.

[0006] To solve the above technical problems, the embodiments of this application provide a high-throughput microfluidic detection chip for monkeypox virus, and adopt the following technical solutions:

[0007] A high-throughput microfluidic detection chip for monkeypox virus, comprising:

[0008] A controllable air pressure pump, a cleaning chamber, a sample loading port, a first spotting pad, a second spotting pad, and a waste liquid chamber that are connected in sequence;

[0009] Among them, the controllable air pressure pump, the cleaning chamber, the sample loading port, the first spotting pad, the second spotting pad, and the waste liquid chamber are arranged inside the housing of the high-throughput monkeypox virus microfluidic detection chip;

[0010] The first spotting pad is fixed with monkeypox antibodies labeled with fluorescent microspheres;

[0011] The second spotting pad is provided with monkeypox antibodies coated with magnetic beads.

[0012] Further, the monkeypox antibodies labeled with fluorescent microspheres include the first A35R antibody labeled with fluorescent microspheres, the first A29L antibody labeled with fluorescent microspheres, the first MIR antibody labeled with fluorescent microspheres, and the first B6R antibody labeled with fluorescent microspheres;

[0013] The monkeypox antibodies coated with magnetic beads include the second A35R antibody coated with magnetic beads, the second A29L antibody coated with magnetic beads, the second MIR antibody coated with magnetic beads, and the second B6R antibody coated with magnetic beads.

[0014] Further, the fluorescent microspheres include Eu 3+ lanthanide element fluorescent microspheres; and / or

[0015] The diameter of the fluorescent microspheres is 100 nm to 350 nm.

[0016] Further, the magnetic beads include Dynabeads TM magnetic beads; and / or

[0017] The diameter of the magnetic beads is 1 μm - 3 μm.

[0018] Further, the cleaning chamber is filled with a cleaning solution;

[0019] By mass fraction, the cleaning solution includes 10 mM PBS buffer and 0.05% Tween20.

[0020] Further, the first spotting pad is prepared by the following steps:

[0021] Each monkeypox antibody is labeled with fluorescent microspheres respectively to obtain a variety of monkeypox antibodies labeled with fluorescent microspheres, where the monkeypox antibodies include the first A35R antibody, the first A29L antibody, the first MIR antibody, and the first B6R antibody;

[0022] The various monkeypox antibodies labeled with fluorescent microspheres are mixed in an equal volume ratio to obtain a mixed labeled antibody, and the mixed labeled antibody is fixed on a pre-prepared spotting pad with a spotter to obtain the first spotting pad.

[0023] Further, the step of respectively labeling each monkeypox antibody with fluorescent microspheres to obtain a plurality of fluorescent microsphere-labeled monkeypox antibodies includes:

[0024] Take fluorescent microspheres with a solid content of 1% and add them to the labeling buffer;

[0025] Add any monkeypox antibody, mix well and then add 10 mg / ml of EDC;

[0026] Perform rotary reaction, centrifuge, discard the supernatant, and retain the precipitate;

[0027] Add TBST buffer, centrifuge, and retain the precipitate to obtain the fluorescent microsphere-labeled monkeypox antibody.

[0028] Further, the second spotting pad is prepared by the following steps:

[0029] Respectively coat each monkeypox antibody with magnetic beads to obtain a plurality of magnetic bead-coated monkeypox antibodies, wherein the monkeypox antibodies include the second A35R antibody, the second A29L antibody, the second MIR antibody, and the second B6R antibody;

[0030] Use a spotting instrument to respectively spot the plurality of magnetic bead-coated monkeypox antibodies at designated positions on a pre-prepared spotting pad to obtain the second spotting pad.

[0031] Further, the step of respectively coating each monkeypox antibody with magnetic beads to obtain a plurality of magnetic bead-coated monkeypox antibodies includes:

[0032] Take magnetic beads with a solid content of 1% in the labeling buffer and vortex to mix evenly;

[0033] Add any monkeypox antibody, mix well and then add 10 mg / ml EDC;

[0034] Perform rotary reaction, place it on a magnetic rack for magnetic separation operation, and retain the precipitate;

[0035] Add TBST buffer, mix well, perform magnetic separation operation on the magnetic rack, and retain the precipitate to obtain the magnetic bead-coated monkeypox antibody.

[0036] To solve the above technical problems, an embodiment of the present application also provides a high-throughput microfluidic detection chip for monkeypox virus, which is used for detecting monkeypox virus for non-diagnostic purposes and adopts the following technical solutions:

[0037] The application of the high-throughput microfluidic detection chip for monkeypox virus in detecting monkeypox virus for non-diagnostic purposes includes the following steps:

[0038] Add the sample to be tested into the chip through the sample loading port, and puncture the sealing film of the cleaning chamber;

[0039] Turn on the first gear of the controllable air pressure pump, and drive the sample to be tested to flow through the first spotting pad multiple times by the controllable air pressure pump;

[0040] Drive the sample to be tested to flow through the second spotting pad multiple times by the controllable air pressure pump;

[0041] Turn on the second gear of the above-mentioned controllable air pressure pump, and drive the cleaning liquid in the cleaning chamber to rinse the second spotting pad by the controllable air pressure pump, wherein the air pressure of the second gear is higher than that of the first gear;

[0042] Read the experimental results.

[0043] Compared with the prior art, the embodiments of the present application mainly have the following beneficial effects:

[0044] The present application provides a high-throughput microfluidic detection chip for monkeypox virus. The high-throughput microfluidic detection chip for monkeypox virus uses a method combining microfluidic technology and fluorescence labeling technology to detect monkeypox virus in a sample. The concentration of monkeypox virus is positively correlated with the relative luminescence intensity. At the same time, four proteins of monkeypox virus are detected, effectively reducing the risk of missed detection; and the detection method is simple and fast, effectively improving the sensitivity of monkeypox virus detection. Description of the Drawings

[0045] In order to more clearly illustrate the solutions in the present application, the following will briefly introduce the drawings required for the description of the embodiments of the present application. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0046] Figure 1 It is a schematic diagram of the replication cycle of the virus in the present application;

[0047] Figure 2 It is a schematic structural diagram of an embodiment of a high-throughput microfluidic detection chip for monkeypox virus according to the present application;

[0048] Figure 3 It is a schematic structural diagram of another embodiment of a high-throughput microfluidic detection chip for monkeypox virus according to the present application;

[0049] Figure 4 It is a schematic flow diagram of an embodiment of the application of a high-throughput microfluidic detection chip for monkeypox virus in detecting monkeypox virus for non-diagnostic purposes;

[0050] Figure 5It is a schematic diagram of the operation of another embodiment of the application of the high-throughput monkeypox virus microfluidic detection chip according to the present application for the detection of monkeypox virus for non-diagnostic purposes.

[0051] Reference numerals: 101, controllable air pressure pump; 102, cleaning chamber; 103, sample addition port; 104, first spotting pad; 105, second spotting pad; 106, waste liquid chamber; 107, monkeypox antibody labeled with fluorescent microspheres; 108, monkeypox antibody coated with magnetic beads. Detailed implementation manners

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs; the terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above drawings are intended to cover non-exclusive inclusion. The terms "first", "second", etc. in the specification and claims of this application or the above drawings are used to distinguish different objects and not to describe a specific order.

[0053] Reference to "embodiment" herein means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of this application. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0054] The following embodiments facilitate a better understanding of this application, but do not limit this application. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The experimental materials used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0055] In order to enable those skilled in the art to better understand the solution of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the drawings.

[0056] This application provides a high-throughput monkeypox virus microfluidic detection chip, as Figure 2 and Figure 3 shown, Figure 2 is a schematic structural diagram of an embodiment of the high-throughput monkeypox virus microfluidic detection chip of this application;

[0057] Figure 3 is a schematic structural diagram of another embodiment of the high-throughput monkeypox virus microfluidic detection chip according to this application.

[0058] The chip includes a controllable air pressure pump 101, a cleaning chamber 102, a sample adding port 103, a first spotting pad 104, a second spotting pad 105, and a waste liquid chamber 106 connected in sequence;

[0059] Among them, the controllable air pressure pump 101, the cleaning chamber 102, the sample adding port 103, the first spotting pad 104, the second spotting pad 105, and the waste liquid chamber 106 are arranged in a housing;

[0060] The first spotting pad 104 is fixed with a monkeypox antibody 107 labeled with fluorescent microspheres;

[0061] The second spotting pad 105 is provided with a monkeypox antibody 108 coated with magnetic beads.

[0062] In this embodiment, the housing includes a first housing and a second housing connected by a buckle; the materials of the first housing and the second housing are both colorless and transparent polystyrene.

[0063] The specific structure of the high-throughput monkeypox virus microfluidic detection chip is shown in the following table:

[0064] Table 1 High-throughput monkeypox virus microfluidic detection chip

[0065]

[0066] As an option of the present application, the monkeypox antibody labeled with fluorescent microspheres includes a first A35R antibody labeled with fluorescent microspheres, a first A29L antibody labeled with fluorescent microspheres, a first MIR antibody labeled with fluorescent microspheres, and a first B6R antibody labeled with fluorescent microspheres;

[0067] The monkeypox antibody coated with magnetic beads includes a second A35R antibody coated with magnetic beads, a second A29L antibody coated with magnetic beads, a second MIR antibody coated with magnetic beads, and a second B6R antibody coated with magnetic beads.

[0068] It should be noted that: the antibody labeled with fluorescent microspheres and the antibody coated with magnetic beads of the present application are different. Specifically, the antibody labeled with fluorescent microspheres is the first A35R antibody, the first A29L antibody, the first MIR antibody, and the first B6R antibody; the antibody coated with magnetic beads is the second A35R antibody, the second A29L antibody, the second MIR antibody, and the second B6R antibody.

[0069] The monkeypox antibody labeled with fluorescent microspheres is different from the monkeypox antibody coated with magnetic beads, which can improve the accuracy of the present application. When there is a corresponding antigen protein, it can be realized that both the antibody labeled with fluorescent microspheres and the antibody coated with magnetic beads can bind to different sites of the antigen protein, improving the accuracy of subsequent detection.

[0070] As an option of the present application, the fluorescent microspheres include Eu 3+Lanthanide fluorescent microspheres. The diameter of the fluorescent microspheres is 290 nm to 350 nm.

[0071] Optionally, in the present application, the magnetic beads include Dynabeads TM Magnetic beads; the diameter of the magnetic beads is 1 μm - 3 μm.

[0072] Optionally, in the present application, the cleaning chamber is encapsulated with a cleaning solution. By mass fraction, the cleaning solution includes 10 mM PBS buffer and 0.05% Tween 20.

[0073] Optionally, in the present application, the second spotting pad includes a first region and a second region. The monkeypox antibody-coated magnetic beads are disposed in the first region, and the thickness of the second spotting pad corresponding to the first region is less than the thickness of the second spotting pad corresponding to the second region.

[0074] Optionally, in the present application, the first spotting pad is prepared by the following steps:

[0075] Each monkeypox antibody is labeled with fluorescent microspheres respectively to obtain a variety of fluorescent microsphere-labeled monkeypox antibodies. Among them, the monkeypox antibodies include A35R antibody, A29L antibody, MIR antibody and B6R antibody;

[0076] The various fluorescent microsphere-labeled monkeypox antibodies are mixed in an equal volume ratio to obtain a mixed labeled antibody, and the mixed labeled antibody is fixed on a pre-prepared spotting pad with a spotter to obtain the first spotting pad.

[0077] Optionally, in the present application, the step of respectively labeling each monkeypox antibody with fluorescent microspheres to obtain a variety of fluorescent microsphere-labeled monkeypox antibodies includes:

[0078] Take fluorescent microspheres with a solid content of 1% and add them to the labeling buffer;

[0079] Add any monkeypox antibody, mix well and then add 10 mg / ml of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide);

[0080] Rotate the reaction, centrifuge, discard the supernatant, and retain the precipitate;

[0081] Add TBST buffer, centrifuge, and retain the precipitate to obtain the fluorescent microsphere-labeled monkeypox antibody.

[0082] In this embodiment, 100 μg of monkeypox antibody is added to 100 μL of fluorescent microspheres with a solid content of 1%. The labeling buffer includes MES. The rotation reaction is to place the centrifuge tube on a rotary mixer and rotate for 120 min; the centrifugation conditions are 12,000 rpm for 30 min. The final concentration of the monkeypox antibody labeled with fluorescent microspheres is 1 mg / ml. The TBST buffer, that is, TBS with Tween-20, is an isotonic buffered salt solution, which consists of Tris-HCl to form a stable pH buffer system, NaCl to provide isotonic conditions, and Tween-20 as a detergent to increase the elution ability of the buffer.

[0083] As an option of the present application, the second spotting pad is prepared through the following steps:

[0084] Each monkeypox antibody is coated with magnetic beads respectively to obtain a variety of monkeypox antibodies coated with magnetic beads, wherein the monkeypox antibodies include the second A35R antibody, the second A29L antibody, the second MIR antibody, and the second B6R antibody;

[0085] The various monkeypox antibodies coated with magnetic beads are respectively spotted at designated positions on a pre-prepared spotting pad using a spotter to obtain the second spotting pad.

[0086] In this embodiment, first use a spotter to spot the monkeypox antibody coated with magnetic beads at a fixed position and dry it. The drying temperature is 37 °C and the drying time is 18 h, so as to facilitate the fixation of the monkeypox antibody coated with magnetic beads by the magnetic field of the electromagnetic coil during the reaction.

[0087] As an option of the present application, the step of respectively coating each monkeypox antibody with magnetic beads to obtain a variety of monkeypox antibodies coated with magnetic beads includes:

[0088] Take magnetic beads with a solid content of 1% in the labeling buffer and vortex to mix evenly;

[0089] Add any monkeypox antibody, mix evenly and then add 10 mg / ml EDC;

[0090] Perform a rotation reaction, place it on a magnetic rack for magnetic separation operation, and retain the precipitate;

[0091] Add TBST buffer, mix evenly, perform a magnetic separation operation on a magnetic rack, and retain the precipitate to obtain the monkeypox antibody coated with magnetic beads.

[0092] In this embodiment, 10 μg of monkeypox antibody is added to 100 μL of magnetic beads with a solid content of 1%. The labeling buffer includes MES; the rotation reaction is to place the centrifuge tube on a rotary mixer and rotate for 120 min; the final concentration of the monkeypox antibody coated with magnetic beads is 2 mg / ml.

[0093] This application provides a high-throughput microfluidic detection chip for monkeypox virus. This high-throughput microfluidic detection chip for monkeypox virus uses a method that combines microfluidic technology and fluorescence labeling technology to detect monkeypox virus in a sample. The concentration of monkeypox virus is positively correlated with the relative luminescence intensity. At the same time, four proteins of monkeypox virus are detected, effectively reducing the risk of missed detection.

[0094] This application also provides the above-mentioned high-throughput microfluidic detection chip for monkeypox virus in the application of detecting monkeypox virus for non-diagnostic purposes, such as Figure 4 shown Figure 4 is a schematic flow chart of an embodiment of the application of the high-throughput microfluidic detection chip for monkeypox virus according to this application in detecting monkeypox virus for non-diagnostic purposes; specifically includes the following steps:

[0095] S1: Add the sample to be tested into the chip through the sample addition port, and pierce the sealing film of the cleaning chamber;

[0096] S2: Turn on the first gear of the controllable air pressure pump, and drive the sample to be tested to flow through the first spotting pad multiple times through the controllable air pressure pump;

[0097] S3: Drive the sample to be tested to flow through the second spotting pad multiple times through the controllable air pressure pump;

[0098] S4: Turn on the second gear of the above-mentioned controllable air pressure pump, and drive the cleaning liquid in the cleaning chamber to wash the second spotting pad through the controllable air pressure pump, wherein the air pressure of the second gear is higher than that of the first gear;

[0099] S5: Read the experimental results.

[0100] In this embodiment, the sample to be tested is serum or a sample treated with sample diluent. The amount of the sample to be tested added is 30 μL;

[0101] As an option of this application, the sample to be tested includes serum.

[0102] such as Figure 5 shown Figure 5 is an operation schematic diagram of another embodiment of the application of the high-throughput microfluidic detection chip for monkeypox virus according to this application in detecting monkeypox virus for non-diagnostic purposes; during the reaction, the magnetic field generated by the electromagnetic coil can fix the magnetic beads, so the monkeypox antibody coated on the magnetic beads can be fixed during the reaction. This application uses a magnetic field to fix the magnetic beads, and the antibody is fixed stably without displacement and loss, further increasing the stability of the test.

[0103] This application controls the light intensity for the CCD camera to capture the antibody-coated position (i.e., the light intensity for fixing the position of the monkeypox antibody coated on magnetic beads) through a PLC control system. A programmable logic controller (PLC) uses a type of programmable memory to store programs internally, execute user-oriented instructions such as logical operations, sequential control, timing, counting, and arithmetic operations, and controls various types of machinery or production processes through digital or analog input / output.

[0104] This application combines microfluidic technology with fluorescence immunoassay technology to detect monkeypox virus in a test sample. Specifically, it uses high-throughput multi-protein dot matrix microfluidics to detect the proteins of monkeypox virus. The detection operation is simple, the detection time is short, it is fast, accurate, has high detection sensitivity and good specificity, and is stable within and between batches. The concentration of monkeypox virus is positively correlated with the relative luminescence intensity.

[0105] This application can precisely control the liquid flow through microfluidics, reduce reagent errors, and increase reagent sensitivity and reproducibility.

[0106] The above scheme is further described below in conjunction with specific implementation examples, but these examples are by no means a limitation to this application. The preferred implementation examples of this application are described in detail as follows:

[0107] Example 1

[0108] Labeling monkeypox antibodies with fluorescent microspheres:

[0109] Label four monkeypox antibodies with fluorescent microspheres: the first A35R antibody, the first A29L antibody, the first MIR antibody, and the first B6R antibody, spray them on the microsphere labeling site, and dry. Taking 100 μL of fluorescent microspheres (1% solid content) as an example:

[0110] 1) Take four clean 2 mL centrifuge tubes, and use a pipette to respectively pipette and add 1 mL of labeling buffer; vortex and mix well. Take 100 μL of fluorescent microspheres with a solid content of 1% into the labeling buffer, and vortex and mix well again. Among them, the labeling buffer includes MES.

[0111] 2) Add 100 μg of the corresponding type of monkeypox antibody to each of the above centrifuge tubes, tighten the lids, and slowly invert and mix 10 - 20 times;

[0112] 3) Add 10 μL of 10 mg / ml EDC, tighten the lid, and slowly invert and mix 10 - 20 times;

[0113] 4) After mixing is completed, place the centrifuge tubes on a rotary mixer and rotate for reaction for 120 min;

[0114] 5) After the reaction is completed, centrifuge at 12000 rpm for 30 min, and slowly suck the supernatant using a pipette to retain the precipitate;

[0115] 6) Add 1 ml of TBST buffer, mix well, centrifuge at 12,000 rpm for 30 min, slowly aspirate the supernatant using a pipette, and retain the precipitate;

[0116] 7) Repeat step 6);

[0117] 8) Finally, add 1 mL of TBST, mix well to obtain the monkey pox antibody labeled with fluorescent microspheres, and the final concentration of the monkey pox antibody labeled with fluorescent microspheres is 1 mg / ml;

[0118] 9) Mix the four kinds of monkey pox antibodies labeled with fluorescent microspheres in a volume ratio of 1:1:1:1 to obtain a mixed solution;

[0119] 10) Take 5 μL of the mixed solution, use a spotter to spot the microspheres at the corresponding position, and dry at 37°C for 18 h.

[0120] Example 2

[0121] Magnetic beads coated with monkey pox antibody:

[0122] Use thermo scientific Dynabeads TM Coat each kind of magnetic bead with the monkey pox antibody. First, use a spotter to spot the magnetic beads coated with the monkey pox antibody at the fixed position on the second spotting pad, and dry it to facilitate the fixation of the magnetic beads coated with the monkey pox antibody by the electromagnetic coil magnetic field during the reaction.

[0123] Among them, the second spotting pad includes a first region and a second region. The magnetic beads coated with the monkey pox antibody are arranged in the first region, and the thickness of the second spotting pad corresponding to the first region is less than the thickness of the second spotting pad corresponding to the second region. Therefore, the position of the second spotting pad corresponding to the magnetic beads in this application is thinner, which is convenient for the fixation of the magnetic beads coated with the monkey pox antibody by the magnetic field.

[0124] Taking 100 μL of magnetic beads (1% solid content) as an example

[0125] 1) Take four clean 2 mL centrifuge tubes, and use a pipette to add 1 mL of labeling buffer to each tube respectively; vortex and mix well. Take 100 μL of magnetic beads with a solid content of 1% into the labeling buffer, and vortex and mix well again. Among them, the labeling buffer includes MES;

[0126] 2) Add 10 μg of the corresponding kind of monkey pox antibody to each centrifuge tube. The added monkey pox antibodies include: the second A35R antibody, the second A29L antibody, the second MIR antibody, and the second B6R antibody. Tighten the lid and slowly invert and mix 10 - 20 times;

[0127] 3) Add 5 μL of 10 mg / ml EDC to each centrifuge tube, tighten the lid, and gently invert the tube 10 - 20 times up and down slowly;

[0128] 4) After mixing, place the centrifuge tube on a rotary mixer and rotate for a reaction for 120 min;

[0129] 5) After the reaction is completed, perform magnetic separation on a magnetic stand, and slowly aspirate the supernatant using a pipette, leaving the precipitate;

[0130] 6) Add 1 ml of TBST buffer, mix well, perform magnetic separation on a magnetic stand, and slowly aspirate the supernatant using a pipette, leaving the precipitate

[0131] 7) Repeat step 6);

[0132] 8) Finally, add 0.5 mL of TBST, mix well to obtain the magnetic beads coated with monkeypox antibodies, and the final concentration of the magnetic beads coated with monkeypox antibodies is 2 mg / ml;

[0133] 9) Spot 2 μL of the magnetic beads at the corresponding position using a spotter, and dry at 37 °C for 18 h. The specific spotting positions are as Figure 3 shown.

[0134] Example 3

[0135] The specific application steps of the above high-throughput monkeypox virus microfluidic detection chip:

[0136] 1) Add 30 μL of serum or the sample treated with sample diluent to the sample inlet, and cover it with a soft rubber cap.

[0137] 2) Pierce the seal of the cleaning solution, turn on the air pump at gear 1, and drive the liquid to flow.

[0138] 3) When the sample comes into contact with the position of the fluorescent microspheres, control the sample to flow through the fluorescent position repeatedly, and the reaction time is 120 s.

[0139] 4) Control the air pump to make the sample flow through the position of the magnetic beads repeatedly, and the reaction time is 120 s.

[0140] 5) Turn on the air pump at gear 2 to rinse the position of the magnetic beads with the cleaning solution. The rinsed solution flows into the waste liquid tank.

[0141] 6) Read and interpret the results: The excitation light source emits light of the excitation wavelength, the CCD takes a photo and collects the light intensity of the emission wavelength at the position of the magnetic beads coated with monkeypox antibodies set on the second spotting pad. The light intensity is converted into a digital signal and the result is read. When the light intensity at the coated position exceeds the threshold, it is judged as positive.

[0142] This application also provides the experimental data of the above high-throughput monkeypox virus microfluidic detection chip as follows:

[0143]

[0144]

[0145] When testing monkeypox virus samples, the light intensity at at least one position of the coated protein magnetic beads is much higher than that of the negative serum samples, indicating that this test method can not only detect whether a person is infected with the monkeypox virus, but also classify different mutant strains, which has guiding significance for laboratory experiments and scientific and technological progress.

[0146] It should be understood that although the steps in the flowchart of the accompanying drawings are shown in sequence according to the arrows, these steps are not necessarily executed in the order indicated by the arrows. Unless there is a clear indication in this article, there is no strict order restriction for the execution of these steps, and they can be executed in other orders. Moreover, at least a part of the steps in the flowchart of the accompanying drawings may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed alternately or in turn with at least a part of other steps or sub-steps or stages of other steps.

[0147] Obviously, the above-described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosed content of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing specific embodiments, or perform equivalent replacements on some of the technical features. Any equivalent structure made by using the specification and drawings of the present application and directly or indirectly applied to other related technical fields shall be equally within the scope of the patent protection of the present application.

Claims

1. A high-throughput monkeypox virus microfluidic detection chip, characterized in that: include: A controllable air pressure pump, a cleaning chamber, a sample adding port, a first sample spotting pad, a second sample spotting pad and a waste liquid chamber are connected in sequence; Wherein, the controllable air pressure pump, the cleaning chamber, the sample adding port, the first sample spotting pad, the second sample spotting pad and the waste liquid chamber are arranged in the housing of the high-throughput monkeypox virus microfluidic detection chip; The first spotting pad is immobilized with monkeypox antibodies labeled with fluorescent microspheres; Monkeypox antibodies coated with magnetic beads are arranged on the second spotting pad.

2. The high-throughput monkeypox virus microfluidic detection chip according to claim 1, characterized in that: The monkeypox antibodies labeled with fluorescent microspheres include a first A35R antibody labeled with fluorescent microspheres, a first A29L antibody labeled with fluorescent microspheres, a first MIR antibody labeled with fluorescent microspheres, and a first B6R antibody labeled with fluorescent microspheres; The monkeypox antibodies coated with magnetic beads include a second A35R antibody coated with magnetic beads, a second A29L antibody coated with magnetic beads, a second MIR antibody coated with magnetic beads, and a second B6R antibody coated with magnetic beads.

3. The high-throughput monkeypox virus microfluidic detection chip according to claim 1, characterized in that: The fluorescent microspheres include Eu 3+ Lanthanide fluorescent microspheres; and / or The diameter of the fluorescent microsphere is 290nm-350nm.

4. The high-throughput monkeypox virus microfluidic detection chip according to claim 1, characterized in that: The magnetic beads include Dynabeads TM Magnetic beads; and / or The diameter of the magnetic beads is 1 μm-3 μm.

5. The high-throughput monkeypox virus microfluidic detection chip according to claim 1, characterized in that: The cleaning chamber is encapsulated with a cleaning liquid; The cleaning solution includes 10 mM PBS buffer and 0.05% Tween20 by mass fraction.

6. The high-throughput monkeypox virus microfluidic detection chip according to claim 1, characterized in that: The first spotting pad is prepared by the following steps: Labeling each monkeypox antibody with fluorescent microspheres to obtain multiple monkeypox antibodies labeled with fluorescent microspheres, wherein the monkeypox antibodies include a first A35R antibody, a first A29L antibody, a first MIR antibody and a first B6R antibody; The plurality of monkeypox antibodies labeled with fluorescent microspheres are mixed in equal volume ratios to obtain mixed labeled antibodies, and the mixed labeled antibodies are fixed on a pre-prepared sample pad using a spotter to obtain the first spotting pad.

7. The high-throughput monkeypox virus microfluidic detection chip according to claim 6, characterized in that: The step of labeling each monkeypox antibody with fluorescent microspheres to obtain multiple monkeypox antibodies labeled with fluorescent microspheres comprises: Take fluorescent microspheres with a solid content of 1% and add them into the labeling buffer; Add any monkeypox antibody, mix well, and then add 10 mg / ml EDC; Spin the reaction, centrifuge, discard the supernatant, and retain the precipitate; TBST buffer was added, the mixture was centrifuged, and the precipitate was retained to obtain the monkeypox antibody labeled with the fluorescent microspheres.

8. The high-throughput monkeypox virus microfluidic detection chip according to claim 1, characterized in that: The second spotting pad is prepared by the following steps: Coating each monkeypox antibody with magnetic beads respectively to obtain a plurality of monkeypox antibodies coated with magnetic beads, wherein the monkeypox antibodies include a second A35R antibody, a second A29L antibody, a second MIR antibody and a second B6R antibody; The monkeypox antibodies coated with the plurality of magnetic beads are spotted respectively at designated positions on the pre-prepared sample pad using a spotter to obtain the second spot pad.

9. The high-throughput monkeypox virus microfluidic detection chip according to claim 8, characterized in that: The step of coating each monkeypox antibody with magnetic beads to obtain multiple monkeypox antibodies coated with magnetic beads comprises: Take magnetic beads with a solid content of 1% in labeling buffer and vortex to mix; Add any monkeypox antibody, mix well, and then add 10 mg / ml EDC; Spin the reaction, place it on a magnetic stand for magnetic separation, and retain the precipitate; TBST buffer was added, mixed, and magnetic separation was performed on a magnetic stand to retain the precipitate to obtain the monkeypox antibody coated with magnetic beads.

10. The high-throughput monkeypox virus microfluidic detection chip according to any one of claims 1 to 9, for use in detecting monkeypox virus for non-diagnostic purposes, characterized in that: The steps include: Add the sample to be tested into the chip through the sample adding port, and puncture the sealing film of the cleaning chamber; Turning on the first gear of the controllable air pressure pump, and driving the sample to be tested to flow through the first spotting pad multiple times by the controllable air pressure pump; The controllable air pressure pump is used to drive the sample to be tested to flow through the second spotting pad multiple times; Turning on the second gear of the controllable air pressure pump, and driving the cleaning liquid in the cleaning chamber to flush the second spotting pad through the controllable air pressure pump, wherein the air pressure of the second gear is higher than the air pressure of the first gear; Read the experimental results.

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