Microfluidic chemiluminescence immunoassay system, detection method and application

Abstract

According to the microfluidic chemiluminescence immunoassay system, the detection method and the application, the microfluidic chemiluminescence immunoassay system comprises a microfluidic immunoreactor, a pipettor and a chemiluminescence detection unit, and the microfluidic immunoreactor can be tightly attached to the pipettor and an adapter piece connected with the pipettor; the pipettor can control liquid entering and exiting from the micro-fluidic immune reactor, and the chemiluminescence detection unit can fix the pipettor and the micro-fluidic immune reactor attached to the pipettor at designated positions and collect chemiluminescence image signals; meanwhile, the chemiluminescence image signal is converted into a quantitative value signal to obtain the virus strain specific antibody binding capacity and / or the neutralizing capacity of the strain and / or the immunoreaction marker concentration in fingertip blood, and the microfluidic chemiluminescence immunoassay system provided by the embodiment of the invention is simple in structure, good in portability and high in sensitivity. Deployment and use in a field or a remote area are facilitated; the blood poisoning strain specific antibody binding capacity and / or the strain neutralizing capacity and / or the immunoreaction marker concentration are / is obtained through trace (1 microliter level) fingertip blood, and the method has great value in prevention and treatment of infectious diseases and can be popularized and popularized on a large scale.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a microfluidic chemiluminescence immunoassay system, a detection method and an application thereof. Background Art

[0002] The levels of spike protein binding antibodies and neutralizing antibodies in serum have a crucial impact on preventing reinfection with coronavirus and the risk of progression to severe illness after infection. Due to the difference in vaccination status and viral infection status, the immune background of the population varies greatly, and the immune protection against emerging mutant strains also varies significantly. Accurately measuring the body's antibody immune response to the new epidemic new coronavirus strain is of great significance for predicting an individual's risk of infection with future strains and the risk of developing severe illness after infection.

[0003] Traditional quantitative tests for antibody levels are often performed in serum, which requires a large sample volume (>1mL serum), and therefore relies on the effective collection and separation of venous serum, which requires trained professional medical staff. At the same time, the ELISA-based antibody test has certain deficiencies in quantitative ability and requires the measurement of multiple samples with different dilution multiples, which has caused great difficulties in the popularization and promotion of quantitative measurement of antibody levels related to epidemics such as the new coronavirus in peripheral blood. Summary of the invention

[0004] In view of this, it is necessary to provide a microfluidic chemiluminescence immunoassay system, detection method and application that can quickly, accurately and quantitatively detect immune response markers in trace samples to address the defects of existing technologies that make quantitative measurement of epidemic-related antibody levels difficult to popularize and promote.

[0005] To solve the above problems, this application adopts the following technical solutions:

[0006] One of the purposes of the present application is to provide a microfluidic chemiluminescent immunoassay system, comprising a microfluidic immunoreactor, a pipette and a chemiluminescent detection unit, wherein the microfluidic immunoreactor can be tightly fitted with the pipette, and the pipette can control the liquid entering and exiting the microfluidic immunoreactor, and the chemiluminescent detection unit can fix the pipette tightly fitted with the microfluidic immunoreactor, collect chemiluminescent image signals, and convert the chemiluminescent image signals into quantitative numerical signals to obtain the virus strain-specific antibody binding ability and / or the strain neutralization ability and / or the immune marker concentration in the fingertip blood.

[0007] In some of the embodiments, any of the microfluidic immunoreactors is made of high protein affinity polystyrene material by a high-precision temperature-controlled injection molding method.

[0008] In some embodiments, the total liquid volume of the microfluidic immunoreactor is 5-25 μL.

[0009] In some of the embodiments, there are multiple microfluidic immunoreactors capable of simultaneously performing chemiluminescence detection, and any one of the microfluidic immunoreactors is tightly fitted with any one of the pipettes or its adapter.

[0010] In some of the embodiments, any one of the microfluidic immunoreactors is in a "cross" tubular structure, and the middle area protruding from the tube body is a spacer area, and the spacer area divides the microfluidic immunoreactor into a docking area and a detection area. There is a groove above the spacer area and facing the docking area, and the pipette or the adapter connected to it can fit tightly in the groove.

[0011] In some embodiments, the inner diameters of the spacer, the docking zone, and the detection zone are the same, and the length of the detection zone is greater than that of the docking zone.

[0012] In some of the embodiments, any of the pipettes is multi-channel or single-channel.

[0013] In some embodiments, the pipette includes but is not limited to a multi-channel pipette or a liquid pump.

[0014] In some of the embodiments, the chemiluminescent detection unit includes a light-shielding box, a CMOS camera arranged at the bottom of the light-shielding box, a limiting device arranged opposite to the CMOS camera, and a computer connected to the CMOS camera signal, the limiting device fixes the pipette tightly fitted with the microfluidic immunoreactor, the CMOS camera collects the chemiluminescent image signal, the computer acquires the chemiluminescent image signal and converts the chemiluminescent image signal into a quantitative numerical signal to obtain the protein antibody binding ability and / or the neutralization ability of the strain and / or the immune marker in the fingertip blood.

[0015] In some of the embodiments, the position of the limiting device relative to the CMOS camera is adjustable.

[0016] In some embodiments, the chemiluminescence detection unit further comprises a slidable protective cover, and the protective cover can seal the pipette tightly fitted with the microfluidic immunoreactor in the light-shielding box.

[0017] The second object of the present application is to provide a method for detecting the protein-antibody binding ability in fingertip blood using the microfluidic chemiluminescence immunoassay system, comprising the following steps:

[0018] Aspirate the diluted fingertip blood sample into the microfluidic immunoreactor pre-coated with the recombinant SARS-CoV-2 spike protein RBD and incubate for 2-15 minutes;

[0019] Using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding;

[0020] A monoclonal antibody coupled to horseradish peroxidase that can specifically detect human IgG is diluted to 50-1000 ng / mL using a blocking solution system and then sucked into the microfluidic immunoreactor and incubated for 1-10 minutes;

[0021] Using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding;

[0022] Use the pipette to suck in the luminol chemiluminescent substrate, and fix the pipette tightly attached to the microfluidic immunoreactor in the chemiluminescent detection unit;

[0023] The chemiluminescence detection unit collects chemiluminescence image signals and converts the chemiluminescence image signals into quantitative numerical signals. The luminol chemiluminescence signal intensity corresponding to each sample can be converted into an equivalent standard antibody concentration through a standard curve.

[0024] In some embodiments, the washing solution is 0-0.25% Tween 20 dissolved in PBS, and the blocking solution system includes 1-5% bovine serum albumin and 0.1-3% bovine casein.

[0025] The third object of the present application is to provide a method for detecting the neutralization ability of virus strains in fingertip blood using the microfluidic chemiluminescence immunoassay system, comprising the following steps:

[0026] The diluted fingerstick blood sample was mixed with biotinylated recombinant human angiotensin-converting enzyme 2 receptor;

[0027] Inhale the microfluidic immunoreactor pre-coated with the recombinant novel coronavirus spike protein trimer and incubate for about 10-20 minutes;

[0028] Use the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding;

[0029] Use the pipette to aspirate streptavidin coupled with horseradish peroxidase and incubate for 1-10 minutes;

[0030] Using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding;

[0031] Use the pipette to suck in the luminol chemiluminescent substrate, and fix the pipette tightly attached to the microfluidic immunoreactor in the chemiluminescent detection unit;

[0032] The chemiluminescent detection unit collects chemiluminescent image signals and converts the chemiluminescent image signals into quantitative numerical signals to obtain the neutralization ability of the virus strain in the fingertip blood.

[0033] In some embodiments, the cleaning solution is 0-0.25% Tween20 dissolved in PBS, the blocking solution system includes 1-3% bovine serum albumin and 0.1-5% bovine casein. The cleaning solution is 0.05% Tween20 dissolved in PBS.

[0034] The fourth object of the present application is to provide a method for detecting immune markers in fingertip blood using the microfluidic chemiluminescence immunoassay system, comprising the following steps:

[0035] Aspirate the diluted fingertip blood sample into the microfluidic immunoreactor pre-coated with capture antibody and incubate for 10-20 minutes;

[0036] Using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding;

[0037] The monoclonal detection antibody coupled with horseradish peroxidase is diluted to 100-500 ng / mL using a blocking solution system, then sucked into the microfluidic immunoreactor, and incubated for 10-20 minutes;

[0038] Use the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding;

[0039] Aspirating luminol chemiluminescent substrate, and fixing the pipette tightly attached to the microfluidic immunoreactor in the chemiluminescent detection unit;

[0040] The chemiluminescence detection unit collects chemiluminescence image signals and converts the chemiluminescence image signals into quantitative numerical signals. The intensity of the luminol chemiluminescence signal corresponding to each sample can be converted into the concentration of the immune marker through a standard curve.

[0041] In some embodiments, the washing solution is 0-0.25% Tween 20 dissolved in PBS, and the blocking solution system includes 1-5% bovine serum albumin and 0.1-3% bovine casein.

[0042] The fifth purpose of the present application is to provide an application of the microfluidic chemiluminescent immunoassay system in detecting protein antibody binding ability and / or strain neutralization ability and / or immune markers in fingertip blood.

[0043] This application adopts the above technical solution, and its beneficial effects are as follows:

[0044] The microfluidic chemiluminescent immunoassay system, detection method and application provided by the present application include a microfluidic immunoreactor, a pipette and a chemiluminescent detection unit. The microfluidic immunoreactor can be tightly fitted with the pipette, and the pipette can control the liquid entering and leaving the microfluidic immunoreactor. The chemiluminescent detection unit can fix the pipette tightly fitted with the microfluidic immunoreactor and collect chemiluminescent image signals, and at the same time convert the chemiluminescent image signals into quantitative numerical signals to obtain the protein antibody binding ability and / or the neutralizing ability and / or immune markers of the strain in the fingertip blood. The microfluidic chemiluminescent immunoassay system provided by the above embodiments of the present application has a simple structure and good portability, which is conducive to deployment and use in the wild or remote areas; by using a trace amount (1 μL level) of fingertip blood to obtain the protein antibody binding ability and / or the neutralizing ability and / or immune markers of the strain in the fingertip blood, it can quickly, accurately and quantitatively detect immune response markers in trace samples, and can be popularized and promoted on a large scale.

[0045] In addition, the microfluidic chemiluminescent immunoassay system provided in this application can be used in addition to testing the antibody binding ability / neutralization ability in fingertip blood of the new coronavirus, and can also be used in a variety of clinical and scientific research applications including testing of antibody immune responses to other infectious diseases, testing of neutralization ability of infectious diseases in peripheral blood, and testing of antibody immune responses to autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application or the description of the prior art 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 paying any creative work.

[0047] Figure 1 This is a schematic diagram of the structure of the microfluidic immunoreactor and pipette provided in Example 1 of the present application.

[0048] Figure 2 This is a schematic diagram of the structure of the microfluidic immunoreactor provided in Example 1 of the present application.

[0049] Figure 3 This is a schematic diagram of the structure of the chemiluminescence detection unit provided in Example 1 of the present application.

[0050] Figure 4 Another structural schematic diagram of the chemiluminescence detection unit provided in Example 1 of the present application.

[0051] Figure 5 Another structural schematic diagram of the chemiluminescence detection unit provided in Example 1 of the present application.

[0052] Figure 6 This is a flow chart of the steps of the method for detecting the protein-antibody binding ability in fingertip blood provided in Example 2 of the present application.

[0053] Figure 7 The chemiluminescent imaging images at different exposure times and their corresponding quantitative signal intensities provided in Example 4 of the present application.

[0054] Figure 8 This is a flow chart of the steps of the method for detecting the neutralizing ability of virus strains in fingertip blood provided in Example 3 of the present application.

[0055] Fig. 9 This is a flow chart of the steps of the method for detecting immune markers in fingertip blood provided in Example 4 of the present application. DETAILED DESCRIPTION

[0056] Embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0057] In the description of the present application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0058] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0059] In order to make the objectives, technical solutions 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.

[0060] Example 1

[0061] See also Figures 1 to 5, is a schematic diagram of the structure of the microfluidic chemiluminescent immunoassay system provided in Example 1 of the present application, comprising: a microfluidic immunoreactor 100, a pipette 200 and a chemiluminescent detection unit 300. The specific structure of each component and its connection relationship are described in detail below.

[0062] The microfluidic immunoreactor 100 is tightly fitted with the pipette 200 , and the pipette 200 can control the liquid entering and exiting the microfluidic immunoreactor 100 .

[0063] In this embodiment, there are multiple microfluidic immunoreactors 100, and any one of the microfluidic immunoreactors 100 is closely attached to any one of the pipettes 200 or its adapter. Since the microfluidic immunoreactor 100 can be closely attached to the pipette 200, and no auxiliary materials such as glue are needed after attachment, airtightness can be naturally achieved, which is conducive to batch processing and application.

[0064] In this embodiment, any of the microfluidic immunoreactors 100 is made of high protein affinity polystyrene material by high precision temperature controlled injection molding. Due to the use of polystyrene polymer material injection molding process with high protein affinity, the microfluidic immunoreactor does not require any surface chemical treatment when in use, greatly reducing the complexity of detection.

[0065] Any of the microfluidic immunoreactors 100 is a "cross" tubular structure, and the area protruding from the tube body in the middle is the spacer 110, and the spacer 110 divides the microfluidic immunoreactor 100 into a docking area 120 and a detection area 130. There is a groove (not shown) above the spacer 110 and facing the docking area 120, and the pipette 200 can fit tightly with the groove. It should be noted that: since the pipette 200 often has a certain elastic deformation space, it can naturally achieve airtightness after fitting without the use of auxiliary materials such as glue.

[0066] Further, the diameters of the spacing zone 110 , the docking zone 120 and the detection zone 130 are the same, and the length of the detection zone 130 is greater than that of the docking zone 120 .

[0067] In this embodiment, the length of the detection area is 22 mm, and the inner wall diameters of the spacing area 110, the docking area 120 and the detection area 130 are all 0.9 mm.

[0068] It can be understood that the microfluidic immunoreactor 100 greatly shortens the time required for immune response and improves the sensitivity of detection due to its high surface area to volume ratio.

[0069] It can be understood that the length of the detection area 110 and the inner wall diameters of the spacing area 110, the docking area 120 and the detection area 130 are not limited to the above values ​​and can be designed according to application conditions in practice.

[0070] In this embodiment, any of the pipettes 200 is multi-channel or single-channel. The pipette 200 includes but is not limited to a multi-channel pipette gun or a liquid pump. The sample capacity of each microfluidic immunoreactor is about 15 microliters, and a multi-channel or single-channel mechanical / electronic pipette can be used to control the liquid in and out, and the waste liquid after the reaction can be discharged into a water-absorbing tool such as a napkin, kitchen paper, or a sponge.

[0071] The chemiluminescent detection unit 300 can fix the pipette 200 that is tightly attached to the microfluidic immunoreactor 100 and collect chemiluminescent image signals, and at the same time convert the chemiluminescent image signals into quantitative numerical signals to obtain the protein antibody binding ability and / or the neutralizing ability of the strain and / or immune markers in the fingertip blood.

[0072] See also Figure 3-Figure 5 The chemiluminescent detection unit 300 includes a light-shielding box 310, a CMOS camera 320 arranged at the bottom of the light-shielding box 310, a limiting device 330 arranged opposite to the CMOS camera 320, and a computer (not shown) connected to the signal of the CMOS camera 320, wherein the limiting device 330 is fixed with the pipette 200 tightly fitted with the microfluidic immunoreactor 100, the CMOS camera 320 collects the chemiluminescent image signal, and the computer obtains the chemiluminescent image signal and converts the chemiluminescent image signal into a quantitative numerical signal to obtain the protein antibody binding ability and / or the neutralization ability of the strain and / or the immune marker in the fingertip blood.

[0073] In this embodiment, the position of the limiting device 330 relative to the CMOS camera 320 is adjustable.

[0074] It can be understood that the CMOS camera 320 and the corresponding ultra-wide-angle lens are arranged at one end of the light shielding box 310, which can strictly isolate the interference of external light. Considering the heat dissipation factor, the tail end of the CMOS camera 320 is fixed outside the light shielding box 310; the limit device is fixed at a position 10-15cm away from the front end of the camera lens to isolate the interference of the luminous signal between the microfluidic immunoreactor 110, and can be fine-tuned according to specific needs.

[0075] In this embodiment, the chemiluminescence detection unit 300 further includes a slidable protective cover 340 , and the protective cover 340 can seal the pipette 200 tightly attached to the microfluidic immunoreactor 100 in the light shielding box 310 .

[0076] It can be understood that since the chemiluminescent signal is extremely weak and is extremely sensitive to interference from external ambient light, each part of the light-shielding box 310 has a strict light-shielding design. Since the protective cover 340 for placing the pipette 200 adopts a sliding design, it has an interlaced structure with the light-shielding box 310 itself on both sides, which greatly reduces the possibility of external background light entering the light-shielding box 310.

[0077] The microfluidic chemiluminescence immunoassay system provided in the above-mentioned Example 1 of the present application has a simple structure and good portability, which is conducive to deployment and use in the wild or remote areas. By using a trace amount (1 μL level) of fingertip blood to obtain the protein antibody binding ability and / or the neutralization ability of the strain and / or immune markers in the fingertip blood, it can quickly, accurately and quantitatively detect immune response markers in trace samples, and can be popularized and promoted on a large scale.

[0078] Example 2

[0079] This embodiment 2 provides a method for detecting the protein-antibody binding ability in fingertip blood using the microfluidic chemiluminescence immunoassay system. The implementation method is described in detail below.

[0080] In this embodiment, fingertip blood samples are collected using a conventional blood glucose collection needle. The sample size collected each time is about 1 μL, and the maximum sample size that can be collected when necessary is 10 μL, which is collected by a small-range pipette. The trace fingertip whole blood sample will be diluted 50 times and stored in a 1% casein-based preservation solution developed and optimized by researchers. If the test date is the same as the collection date, the sample only needs to be diluted to 1 / 250 of the original blood concentration (using a 1% casein preservation solution system) and the subsequent immunoassay test is performed directly. If it needs to be stored for a long time after collection, red blood cells, white blood cells and lymphocytes in the sample can be removed by medium and low speed centrifugation. After removing blood cells, the sample can be stored in a 1% casein buffer system at 4 degrees Celsius for at least one month, or stored longer at -20 degrees Celsius.

[0081] All the preparatory processes before the experiment and the test of immune response markers in fingertip blood are controlled by a multi-channel pipette (a 12-channel mechanical pipette in this embodiment) and carried out in a microfluidic immunoreactor, and 12 samples can be measured in each batch. After the immunoadsorption reaction is completed, the luminol chemiluminescent substrate will be sucked into the tube and placed in the limit device.

[0082] In this embodiment, for the antibody binding ability test, the preparation method before the experiment is: 1. Dissolve and dilute the recombinant new coronavirus spike protein in phosphate buffered saline (PBS) with a target concentration of 10μg / mL. 2. Use a multichannel pipette to aspirate 15μL of the diluted spike protein solution into the microfluidic immunoreactor and allow it to stay in the reactor for incubation for 30-60 minutes. 3. Use a multichannel pipette to aspirate and discharge the cleaning solution (0.05% Tween20 dissolved in PBS) to remove unstable non-specific binding. 4. Use the blocking solution system developed by the scientific research team (containing 3% bovine serum albumin and 1% bovine casein) for about 20 minutes of blocking. The blocking process can be repeated once to further improve the signal-to-noise ratio. 5. Use a multichannel pipette to aspirate and discharge the cleaning solution.

[0083] See also Figure 6 The method for detecting the protein antibody binding ability in fingertip blood provided in Example 2 comprises the following steps:

[0084] Step S110: The diluted fingertip blood sample is sucked into the microfluidic immunoreactor pre-coated with the recombinant novel coronavirus spike protein RBD and incubated for 2-8 minutes. The incubation time depends on the situation.

[0085] Step S120: using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding.

[0086] In this embodiment, the cleaning solution is 0.05% Tween 20 dissolved in PBS.

[0087] Step S130: A monoclonal antibody coupled to horseradish peroxidase that can specifically detect human IgG is diluted to 100-200 ng / mL using a blocking solution system and then sucked into the microfluidic immunoreactor and incubated for 3 minutes.

[0088] In this embodiment, the blocking solution system includes 3% bovine serum albumin and 1% bovine casein.

[0089] Step S140: using the pipette to aspirate the cleaning solution and discharge the cleaning solution to remove unstable non-specific binding.

[0090] It can be understood that a better signal-to-noise ratio can be achieved by using the pipette to aspirate the cleaning solution and discharge the cleaning solution three or more times.

[0091] Step S150: using the pipette to suck in the luminol chemiluminescent substrate, and fixing the pipette tightly attached to the microfluidic immunoreactor in the chemiluminescent detection unit.

[0092] Step S160: The chemiluminescence detection unit collects chemiluminescence image signals and converts the chemiluminescence image signals into quantitative numerical signals. The luminol chemiluminescence signal intensity corresponding to each sample can be converted into an equivalent standard antibody concentration through a standard curve.

[0093] Specifically, the exposure time is set using a computer program, and the CMOS camera collects chemiluminescent image signals. The chemiluminescent imaging photos are converted into quantitative numerical signals by computer open source software for subsequent data analysis, and the luminol chemiluminescent signal intensity corresponding to each sample can be converted into an equivalent standard antibody concentration by a standard curve.

[0094] See also Figure 7 , are the chemiluminescent imaging images obtained in this embodiment at different exposure times and their corresponding quantitative signal intensities, and the detection sensitivity can reach up to 0.1 ng / mL.

[0095] Example 2 of the present application provides a method for detecting the protein antibody binding ability in fingertip blood using the microfluidic chemiluminescent immunoassay system. A trace amount (1-10 μL level) of fingertip blood can be used to detect the protein antibody binding ability in fingertip blood, and a highly sensitive and reliable quantitative detection can be performed. Because the technology has a large dynamic range of molecular response (3-4 concentration magnitudes), the sample processing only requires single-point dilution, and the anti-interference ability is extremely strong. Fingertip blood does not require additional cell separation, and antibody binding and neutralization ability tests can be directly performed after dilution, which does not constitute a significant interference to the results. It can significantly improve the novel coronavirus spike protein antibody binding ability in fingertip blood, which is conducive to the disease control department to carry out large-scale antibody level research and vaccine research and development institutions to carry out vaccine protection research based on antibody levels.

[0096] Example 3

[0097] This embodiment 3 provides a method for detecting the neutralization ability of virus strains in fingertip blood using the microfluidic chemiluminescence immunoassay system. The specific implementation method is described in detail below.

[0098] In this embodiment, for the strain neutralization ability test, the preparation method before the experiment is: 1. Dissolve and dilute the recombinant new coronavirus spike protein in phosphate buffered saline (PBS) with a target concentration of 10μg / mL. 2. Use a multichannel pipette to aspirate 15μL of the diluted spike protein solution into the microfluidic immunoreactor and allow it to stay in the reactor for incubation for 30-60 minutes. 3. Use a multichannel pipette to aspirate and discharge the cleaning solution (0.05% Tween20 dissolved in PBS) to remove unstable non-specific binding. 4. Use the blocking solution system developed by the scientific research team (containing 3% bovine serum albumin and 1% bovine casein) for about 20 minutes of blocking. The blocking process can be repeated once to further improve the signal-to-noise ratio. 5. Use a multichannel pipette to aspirate and discharge the cleaning solution (0.05% Tween20 dissolved in PBS).

[0099] See also Figure 8 The method for detecting the neutralization ability of the virus strain in the fingertip blood provided in this embodiment specifically comprises the following steps:

[0100] Step S210: Mixing the diluted fingertip blood sample with the biotinylated recombinant human angiotensin converting enzyme 2 receptor.

[0101] Specifically, the final concentration of biotinylated recombinant human angiotensin converting enzyme 2 (ACE2) is about 100 ng / mL.

[0102] Step S220: Inhale the microfluidic immunoreactor pre-coated with the recombinant SARS-CoV-2 spike protein trimer and incubate for about 10-20 minutes.

[0103] It can be understood that in the above process, the antibody of the sample can fully compete with the biotinylated ACE2. If the sample has a strong neutralizing ability against a certain strain of the new coronavirus, it can block the binding of the biotinylated ACE2 with the spike protein trimer on the surface of the microfluidic immunoreactor, and the signal intensity will be lower. On the contrary, if the neutralizing ability is weak, the signal intensity will be higher.

[0104] Step S230: using the pipette to suck in the cleaning solution and discharge the cleaning solution to remove unstable non-specific binding.

[0105] In this embodiment, the cleaning solution is 0.05% Tween 20 dissolved in PBS.

[0106] Step S240: Use the pipette to aspirate streptavidin coupled with horseradish peroxidase, and incubate for 3 minutes.

[0107] In this example, the concentration of streptavidin-HRP coupled with horseradish peroxidase is about 200 ng / mL.

[0108] Step S250: using the pipette to aspirate the cleaning solution and discharge the cleaning solution to remove unstable non-specific binding.

[0109] Step S260: using the pipette to aspirate the luminol chemiluminescent substrate, and fixing the pipette tightly attached to the microfluidic immunoreactor in the chemiluminescent detection unit.

[0110] Step S270: The chemiluminescent detection unit collects chemiluminescent image signals and converts the chemiluminescent image signals into quantitative numerical signals to obtain the neutralization ability of the virus strain in the fingertip blood.

[0111] Specifically, the exposure time is set using a computer program, and the CMOS camera collects chemiluminescent image signals. The chemiluminescent imaging photos are converted into quantitative numerical signals by computer open source software for subsequent data analysis.

[0112] Example 3 of the present application provides a method for detecting the neutralization ability of virus strains in fingertip blood using the microfluidic chemiluminescent immunoassay system, and a trace amount (1 μL level) of fingertip blood can be used to detect the neutralization ability of fingertip blood to different strains of coronavirus. Because the technology has a large dynamic range of molecular response (3-4 concentration magnitudes), the processing of the sample only requires single-point dilution, and the anti-interference ability is extremely strong. Fingertip blood does not require additional cell separation, and the neutralization ability test of virus strains in fingertip blood can be directly performed after dilution, which does not constitute a significant interference to the results. This method significantly reduces the time used for strain neutralization ability testing, which is conducive to the disease control department to carry out large-scale antibody level research and vaccine research and development institutions to carry out vaccine protection research based on antibody levels.

[0113] Example 4

[0114] See also Fig. 9 , which is a method for detecting immune markers in fingertip blood using the microfluidic chemiluminescence immunoassay system provided in Example 4 of the present application, specifically comprises the following steps:

[0115] Step S310: Aspirate the diluted fingertip blood sample into the microfluidic immunoreactor pre-coated with the capture antibody and incubate for 10-20 minutes. The incubation time depends on the situation.

[0116] Step S320: using the pipette to aspirate the cleaning solution and discharge the cleaning solution to remove unstable non-specific binding.

[0117] In this embodiment, the cleaning solution is 0.05% Tween 20 dissolved in PBS.

[0118] Step S330: The monoclonal detection antibody coupled with horseradish peroxidase is diluted to 100-500 ng / mL using a blocking solution system, then sucked into the microfluidic immunoreactor and incubated for 10-20 minutes.

[0119] In this embodiment, the blocking solution system includes 3% bovine serum albumin and 1% bovine casein.

[0120] Step S340: Use the pipette to suck in the cleaning solution and discharge the cleaning solution to remove unstable non-specific binding.

[0121] It can be understood that a better signal-to-noise ratio can be achieved by using the pipette to aspirate the cleaning solution and discharge the cleaning solution three or more times.

[0122] Step S350: Aspirate the luminol chemiluminescent substrate, and fix the pipette tightly attached to the microfluidic immunoreactor in the chemiluminescent detection unit.

[0123] Step S360: The chemiluminescence detection unit collects chemiluminescence image signals and converts the chemiluminescence image signals into quantitative numerical signals. The luminol chemiluminescence signal intensity corresponding to each sample can be converted into an immune marker concentration through a standard curve.

[0124] Specifically, the exposure time is set using a computer program, and the CMOS camera collects chemiluminescent image signals. The chemiluminescent imaging photos are converted into quantitative numerical signals by computer open source software for subsequent data analysis, and the luminol chemiluminescent signal intensity corresponding to each sample can be converted into the concentration of the immune marker by the standard curve.

[0125] Example 4 of the present application provides a method for detecting immune markers in fingertip blood using the microfluidic chemiluminescent immunoassay system, which can use a trace amount (1-10 μL level) of fingertip blood to detect the concentration of specific immune response markers in the fingertip blood. Highly sensitive and highly reliable quantitative detection can be performed. This method has a strong anti-interference ability, and fingertip blood does not require additional cell separation, and can be directly diluted to perform quantitative tests on immune response markers.

[0126] The above-mentioned Examples 2-4 of the present application have proved through preliminary experiments that the concentration of immune response markers in fingertip blood can be accurately and quantitatively detected by using this scheme. The sensitivity of detecting the immune response marker interferon-gamma in fingertip blood can reach 0.01 ng / mL.

[0127] In addition, the microfluidic chemiluminescence immunoassay system provided in this application can be used in addition to testing the antibody binding ability / neutralization ability of the new coronavirus in fingertip blood, and can also be used in a variety of clinical and scientific research applications including antibody immune response detection for other infectious diseases such as influenza, infectious disease peripheral blood neutralization ability detection, and autoimmune disease antibody immune response detection.

[0128] It can be understood that the technical features of the above-described embodiments can be arbitrarily combined. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0129] The above are only preferred embodiments of the present application, and only specifically describe the technical principles of the present application. These descriptions are only for explaining the principles of the present application and cannot be interpreted as limiting the scope of protection of the present application in any way. Based on the explanation here, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application, and other specific implementation methods of the present application that can be associated with the technicians in this field without creative work, should be included in the scope of protection of the present application.

Claims

1. A microfluidic chemiluminescent immunoassay system, characterized in that: It includes a microfluidic immunoreactor, a pipette and a chemiluminescence detection unit. The microfluidic immunoreactor can be tightly fitted with the pipette, and the pipette can control the liquid entering and leaving the microfluidic immunoreactor. The chemiluminescence detection unit can fix the pipette tightly fitted with the microfluidic immunoreactor, collect chemiluminescence image signals, and convert the chemiluminescence image signals into quantitative numerical signals to obtain the virus strain-specific antibody binding ability and / or the strain neutralization ability and / or the immune marker concentration in the fingertip blood.

2. The microfluidic chemiluminescent immunoassay system according to claim 1, characterized in that: There are one or more microfluidic immunoreactors capable of simultaneously performing chemiluminescence detection, and any one of the microfluidic immunoreactors is tightly fitted with any one of the pipettes or its adapter.

3. The microfluidic chemiluminescent immunoassay system according to claim 1, characterized in that: Any of the microfluidic immunoreactors is made of high protein affinity polystyrene material by temperature-controlled injection molding.

4. The microfluidic chemiluminescent immunoassay system according to claim 1, 2 or 3, characterized in that: Any of the microfluidic immunoreactors is a "cross" tubular structure when viewed from the side, and the area protruding from the tube body in the middle is a spacer area, which divides the microfluidic immunoreactor into a docking area and a detection area. A groove is provided above the spacer area and facing the docking area, and the pipette can fit tightly in the groove.

5. The microfluidic chemiluminescent immunoassay system according to claim 4, characterized in that: The inner diameters of the spacing zone, the docking zone and the detection zone are the same, and the length of the detection zone is greater than that of the docking zone.

6. The microfluidic chemiluminescent immunoassay system according to claim 4, characterized in that: Any of the pipettes described herein may be multi-channel or single-channel.

7. The microfluidic chemiluminescent immunoassay system according to claim 3, characterized in that: The pipette includes but is not limited to a multi-channel pipette or a mechanical / electric liquid pump.

8. The microfluidic chemiluminescent immunoassay system according to claim 1, characterized in that: The chemiluminescence detection unit includes a light-shielding box that can strictly isolate external light interference, a CMOS camera arranged at the bottom of the light-shielding box, a limiting device arranged opposite to the CMOS camera, and a computer connected to the CMOS camera signal, the limiting device fixes the pipette that is tightly fitted with the microfluidic immunoreactor, the CMOS camera collects the chemiluminescent image signal, the computer acquires the chemiluminescent image signal and converts the chemiluminescent image signal into a quantitative numerical signal to obtain the protein antibody binding ability and / or the neutralization ability of the strain and / or the immune marker in the fingertip blood.

9. The microfluidic chemiluminescent immunoassay system according to claim 8, characterized in that: The position of the limiting device relative to the CMOS camera is adjustable.

10. The microfluidic chemiluminescent immunoassay system according to claim 9, characterized in that: The chemiluminescence detection unit further comprises a slidably arranged protective cover, and the protective cover can seal the pipette tightly fitted with the microfluidic immunoreactor in the light shielding box.

11. A method for detecting protein-antibody binding ability in fingertip blood using the microfluidic chemiluminescence immunoassay system as claimed in claim 1, characterized in that: The steps include: Aspirate the diluted fingertip blood sample into the microfluidic immunoreactor pre-coated with the recombinant SARS-CoV-2 spike protein RBD and incubate for 2-15 minutes; Using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding; A monoclonal antibody coupled to horseradish peroxidase that can specifically detect human IgG is diluted to 50-1000 ng / mL using a blocking solution system and then sucked into the microfluidic immunoreactor and incubated for 1-10 minutes; Using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding; Use the pipette to suck in the luminol chemiluminescent substrate, and fix the pipette tightly attached to the microfluidic immunoreactor in the chemiluminescent detection unit; The chemiluminescence detection unit collects chemiluminescence image signals and converts the chemiluminescence image signals into quantitative numerical signals. The luminol chemiluminescence signal intensity corresponding to each sample can be converted into an equivalent standard antibody concentration through a standard curve.

12. The method for detecting the protein-antibody binding ability in fingertip blood according to claim 11, characterized in that: The cleaning solution is 0-0.25% Tween20 dissolved in PBS, and the blocking solution system includes 1-5% bovine serum albumin and 0.1-3% bovine casein.

13. A method for detecting the neutralization ability of virus strains in fingertip blood using the microfluidic chemiluminescence immunoassay system as claimed in claim 1, characterized in that: The steps include: The diluted fingerstick blood sample was mixed with biotinylated recombinant human angiotensin-converting enzyme 2 receptor; Inhale the microfluidic immunoreactor pre-coated with the recombinant novel coronavirus spike protein trimer and incubate for about 5-30 minutes; Use the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding; Use the pipette to aspirate streptavidin coupled with horseradish peroxidase and incubate for 1-10 minutes; Using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding; Use the pipette to suck in the luminol chemiluminescent substrate, and fix the pipette tightly attached to the microfluidic immunoreactor in the chemiluminescent detection unit; The chemiluminescent detection unit collects chemiluminescent image signals and converts the chemiluminescent image signals into quantitative numerical signals to obtain the neutralization ability of the virus strain in the fingertip blood.

14. The method for detecting the neutralization ability of virus strains in fingertip blood according to claim 13, characterized in that: The washing solution is 0-0.25% Tween20 dissolved in PBS.

15. A method for detecting immune markers in fingertip blood using the microfluidic chemiluminescence immunoassay system of claim 1, characterized in that: The steps include: Aspirating the diluted fingertip blood sample into the microfluidic immunoreactor pre-coated with immune response marker capture antibodies and incubating for 10-20 minutes; Using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding; The monoclonal detection antibody coupled with horseradish peroxidase is diluted to 100-1000 ng / mL using a blocking solution system, then sucked into the microfluidic immunoreactor, and incubated for 10-30 minutes; Use the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding; Aspirating luminol chemiluminescent substrate, and fixing the pipette tightly attached to the microfluidic immunoreactor in the chemiluminescent detection unit; The chemiluminescence detection unit collects chemiluminescence image signals and converts the chemiluminescence image signals into quantitative numerical signals. The intensity of the luminol chemiluminescence signal corresponding to each sample can be converted into the concentration of the immune marker through a standard curve.

16. The method for detecting immune markers in fingertip blood according to claim 11, characterized in that: The cleaning solution is 0-0.25% Tween20 dissolved in PBS, and the blocking solution system includes 1-5% bovine serum albumin and 0.1-3% bovine casein.

17. Use of the microfluidic chemiluminescent immunoassay system as claimed in claim 1 in detecting protein antibody binding ability and / or strain neutralization ability and / or immune markers in fingertip blood.

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