Microfluidic immune reactor capable of performing multiple immunoassays, immune evaluation system and method

Abstract

According to the microfluidic immune reactor capable of performing multiple immunoassay, the community infectious disease immune evaluation system and the evaluation method provided by the invention, the microfluidic immune reactor comprises an upper-layer coating region, a lower-layer coating region and an excessive closed region, and the upper-layer coating region and the lower-layer coating region are isolated by the excessive closed region; the upper-layer coating area is provided with a first antigen or a first antibody, the lower-layer coating area is provided with a second antigen or a second antibody, and the excessive sealing area is sealed with a sealing buffer solution. According to the present invention, the multiple biomarkers and the multiple dried blood spots in the same immune reaction device can be simultaneously measured, the amount of the biological information obtained by the single sample is improved, the disease diagnosis of the patient and the protection evaluation of the immune system can be simultaneously performed, and the social health department and the hospital clinical laboratory can easily provide the multi-aspect diagnosis opinions for the patient in the short time.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a microfluidic immunoreactor, a community infectious disease immunity assessment system and an assessment method. Background Art

[0002] With the development of society, communication between people has become increasingly close, and the risk of disease transmission has also increased.

[0003] Common IgG level detection methods, such as ELISA (enzyme-linked immunosorbent assay), have been widely validated for their practicality and sensitivity. However, the ELISA method requires long experimental times and is expensive, limiting its application in large-scale community immune protection assessments. Furthermore, due to the relatively large sample volume required, ELISA typically uses venous blood samples, making blood collection difficult for specific populations. While fingertip blood is relatively simple to collect, its storage conditions are relatively demanding (frozen at -20°C or lower), necessitating cold chain transportation for fingertip blood samples, which increases transportation costs. Furthermore, a single ELISA test only provides a limited number of indicators, making it difficult to comprehensively assess the body's immune status. This single-biomarker detection model is no longer sufficient to meet the needs of targeted epidemic prevention. Emerging multiplexed immunosensor technology offers unique advantages by simultaneously detecting multiple differential biomarkers. This allows for multi-indicator qualitative and quantitative analysis in a single sample, improving diagnostic accuracy while also reducing single-indicator costs through data integration, minimizing batch errors, and increasing detection throughput. This provides a breakthrough solution for building a multi-dimensional immune assessment system.

[0004] ELISA-based IgG testing is widely used in immunology research and clinical diagnosis. The ELISA method detects specific immunoglobulins (such as IgG) through an antigen-antibody reaction. Its basic principle is to use an enzyme-labeled antibody to react with the target antibody or antigen, and ultimately perform quantitative detection through an enzyme-catalyzed substrate reaction.

[0005] The classic ELISA IgG detection method achieves quantitative determination of specific IgG through antigen-antibody reaction. The steps include plate preparation, sample addition, secondary antibody reaction, substrate color development, and final optical density measurement. By comparing with the standard curve, the concentration of IgG in the sample can be accurately calculated. The advantages of this method are high sensitivity and good specificity, which is suitable for clinical diagnosis and immune research. However, the required sample volume is large (100-200μL), and it is not possible to analyze trace samples of fingertip blood. At the same time, these platforms can only apply single antibody or antigen detection. If multiple analysis is to be achieved, multiple sensors need to be prepared for detection. The processing technology is relatively complicated and the operation threshold is relatively high, which causes great inconvenience to the test. Summary of the Invention

[0006] In view of this, it is necessary to provide a community infectious disease immunity assessment system and assessment method that can quickly, flexibly, stably and reliably detect the defects of the existing technology such as complex process, complex operation and high cost.

[0007] To solve the above problems, this application adopts the following technical solutions:

[0008] One of the purposes of the present application is to provide a microfluidic immunoreactor, comprising an upper coating area, a lower coating area and an excess blocking area, wherein the upper coating area and the lower coating area are separated by the excess blocking area, the upper coating area is provided with a first antigen or a first antibody, the lower coating area is provided with a second antigen or a second antibody, and the excess blocking area is blocked with a blocking buffer.

[0009] In some embodiments, the microfluidic immunoreactor further includes a third coating area, a fourth coating area... and an Nth coating area, and the upper coating area, the third coating area, the fourth coating area... the Nth coating area and the lower coating area are arranged in sequence and the excess closed area is arranged between each other.

[0010] The second purpose of this application is to provide a community infectious disease immunity assessment system, comprising: a microfluidic immunoreactor, a high-precision pipette, a chemiluminescent substrate, and a chemiluminescent detection unit, wherein:

[0011] The microfluidic immunoreactor comprises an upper coating area, a lower coating area and an excess blocking area, wherein the upper coating area and the lower coating area are separated by the excess blocking area, the upper coating area is provided with a first antigen or a first antibody, the lower coating area is provided with a second antigen or a second antibody, and the excess blocking area is blocked with a blocking buffer;

[0012] The pipette is used to adsorb the first antigen, the first antibody, the second antigen, the second antibody, the blocking buffer, the chemiluminescent substrate and the labeled antibody complex to the corresponding area of the microfluidic immunoreactor;

[0013] The chemiluminescence detection unit can fix the pipette that is tightly attached to the microfluidic immunoreactor, collect chemiluminescence image signals and convert the chemiluminescence image signals into quantitative numerical signals. The quantitative numerical signals are converted into equivalent standard cytokine / antibody / antigen concentrations through a standard curve to evaluate the level of immune protection against infectious diseases.

[0014] In some embodiments, the microfluidic immunoreactor further includes a third coating area, a fourth coating area... and an Nth coating area, and the upper coating area, the third coating area, the fourth coating area... the Nth coating area and the lower coating area are arranged in sequence and the excess closed area is arranged between each other.

[0015] In some embodiments, there are multiple microfluidic immunoreactors, and any one of the microfluidic immunoreactors is tightly fitted with any one of the pipettes or its adapter.

[0016] In some embodiments, the high-precision pipette is multi-channel or single-channel, and the high-precision pipette includes but is not limited to a multi-channel pipette gun or a liquid pump.

[0017] In some embodiments, the first antibody and the second antibody include IgG, IgM or IgE or IgA and nanobody, the blocking buffer is BSA, casein or SuperBlock, and the chemiluminescent substrate is hydrogen peroxide and luminol.

[0018] In some embodiments, the chemiluminescence 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 the computer connected to the CMOS camera signal, the limiting device is fixed with the pipette tightly fitted with the microfluidic immunoreactor, the CMOS camera collects the chemiluminescence image signal, the computer converts the chemiluminescence image signal into a quantitative numerical signal, and the quantitative numerical signal is converted into an equivalent standard cytokine / antibody / antigen concentration through a standard curve to evaluate the level of immune protection against infectious diseases.

[0019] The third object of this application is to provide an assessment method using the community infectious disease immunity assessment system, comprising the following steps:

[0020] Using a high-precision pipette, the microfluidic immunoreactor is partitioned into coated detection areas for 30-60 minutes, wherein the microfluidic immunoreactor comprises at least two detection areas, including the upper coating area and the lower coating area;

[0021] aspirating the sample to be tested into the microfluidic immunoreactor for incubation;

[0022] Using the high-precision pipette, the specific detection antibody or antibody mixture is added to the microfluidic immunoreactor and incubated for 2 to 20 minutes;

[0023] Using the high-precision pipette to draw in luminol chemiluminescent substrate into the microfluidic immunoreactor;

[0024] placing a high-precision pipette with the microfluidic immunoreactor in a chemiluminescence detection unit, wherein the chemiluminescence detection unit collects chemiluminescence image signals in the microfluidic immunoreactor;

[0025] The chemiluminescent image signal is converted into a quantitative numerical signal, and the quantitative numerical signal is converted into an equivalent standard cytokine / antibody / antigen concentration through a standard curve to evaluate the level of immune protection against infectious diseases.

[0026] In some embodiments, the sample type may be finger stick blood, venous blood, finger stick blood serum, venous blood serum, and dried blood spot reconstitution solution.

[0027] In the embodiments related to dried blood spots, 5-80 μL of fingertip blood sample is dropped onto filter paper for air-drying and solidification to form a dried blood spot sample, which is then packaged and stored at room temperature or low temperature. The dried blood spot sample is cut in a standardized manner, divided, and immersed in a reconstitution solution for a dissolution reaction. After centrifugation, the insoluble matter is removed, and finally the dried blood spot reconstitution solution is extracted to obtain a test sample.

[0028] In some embodiments, the sample to be tested includes fingertip blood, venous blood, serum or dried blood spot reconstitution solution; the first antigen includes the recombinant S protein of the new coronavirus, and the first antigen includes the S protein of a mutant strain of the recombinant S protein of the new coronavirus; the molecular type of the sample to be tested includes strain-specific IgG, IgM or IgE or IgA and nanoantibodies; the specific detection antibody is a highly specific monoclonal antibody or a nanoantibody; the blocking buffer is BSA or casein or SuperBlock, and the chemiluminescent substrate is hydrogen peroxide and luminol.

[0029] This application adopts the above technical solution, and its beneficial effects are as follows:

[0030] The present application provides a microfluidic immunoreactor, a community infectious disease immune assessment system, and an assessment method. The microfluidic immunoreactor includes an upper coating area, a lower coating area, and an excess blocking area. The upper coating area and the lower coating area are separated by the excess blocking area. The upper coating area is provided with a first antigen or a first antibody, the lower coating area is provided with a second antigen or a second antibody, and the excess blocking area is blocked with a blocking buffer; the pipette is used to adsorb the first antigen, the first antibody, the second antigen, the second antibody, the blocking buffer, the chemiluminescent substrate, and the labeled antibody complex to the corresponding area of the microfluidic immunoreactor; the chemiluminescent substrate ... first antibody, the second antigen, the second antibody, the blocking buffer, the chemiluminescent substrate, and the labeled antibody complex to the corresponding area of the microfluidic immunoreactor; the chemiluminescent substrate is used to adsorb the first antigen, the first antibody, the first antibody, the second antibody, the blocking buffer, the chemiluminescent substrate, and the labeled antibody complex to the corresponding area of the microfluidic immunoreactor; the chemiluminescent substrate is used to adsorb the first antigen, the first antibody, the first antibody, the second antibody, the blocking buffer, the chemiluminescent substrate, and the labeled antibody complex to the The detection unit can fix the pipette that is tightly attached to the microfluidic immunoreactor, collect chemiluminescent image signals and convert the chemiluminescent image signals into quantitative numerical signals. The quantitative numerical signals are converted into equivalent standard cytokine / antibody / antigen concentrations through a standard curve to evaluate the level of immune protection against infectious diseases. The microfluidic immunoreactor partitioned antibody / antigen coating scheme provided in this application realizes the simultaneous measurement of multiple biomarkers in the same immune response device, improves the amount of biological information obtained from a single sample, and can simultaneously diagnose the patient's disease and evaluate the protection of the immune system, which is beneficial for community health and hospital laboratories to provide multi-faceted diagnostic opinions to patients in a short period of time. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments of the present application or the description of the prior art. 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 any creative work.

[0032] Figure 1 A schematic structural diagram of the microfluidic immunoreactor provided in Example 1 of the present application, including two coating areas.

[0033] Figure 2 This is a schematic diagram of the structure of each coating area provided by the microfluidic immunoreactor provided in Example 1 of the present application.

[0034] Figure 3 This is a schematic diagram of the principle of the community infectious disease immunity assessment system provided in Example 2 of this application.

[0035] Figure 4 This is a schematic diagram of the structure of the community infectious disease immunity assessment system provided in Example 2 of this application.

[0036] Figure 5 Schematic diagram of the principle of the method for assessing the level of immune protection against infectious diseases in a community provided in Example 3 of the present application.

[0037] Figure 6 This is a flowchart of the steps of the method for assessing the community infectious disease immune protection level provided in Example 3 of the present application.

[0038] Figure 7 This is a flow chart of the steps for obtaining a microfluidic immunoreactor coated with corresponding antigens and antibodies provided in Example 3 of the present application.

[0039] Figure 8 These are chemiluminescent imaging images obtained at different exposure times during the regional coating and blocking provided in Example 3 of the present application.

[0040] Figure 9 This is a schematic diagram of the quantified signal intensity in the regional coating and blocking provided in Example 3 of the present application.

[0041] Figure 10 This is a flowchart of the steps of the method for assessing the level of immune protection against infectious diseases in a community provided in Example 4 of the present application.

[0042] Figure 11 This is a flowchart of the steps of a specific embodiment of the method for assessing the community infectious disease immune protection level provided in Example 4 of the present application.

[0043] Figure 12 Schematic diagram of the stability verification of the dried blood spot sample provided in Example 4 under long-term storage at room temperature.

[0044] Figure 13 This is a schematic diagram comparing the IgG detection levels of dried blood spot samples and fresh blood at different reconstitution volumes provided in Example 4.

[0045] Figure 14 This is a schematic diagram comparing the IgG detection levels of dried blood spot samples stored at room temperature and 37°C provided in Example 4.

[0046] Figure 15 This is a schematic diagram comparing the IgG detection levels of dried blood spot samples under different sampling methods provided in Example 3. DETAILED DESCRIPTION

[0047] The following describes in detail embodiments of the present application, examples of which 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.

[0048] In the description of this 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 this 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 this application.

[0049] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0050] In order to make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below with reference to the accompanying drawings and embodiments.

[0051] Example 1

[0052] See also Figures 1 to 2 A microfluidic immunoreactor provided in this embodiment includes an upper coating area, a lower coating area and an excess blocking area. The upper coating area and the lower coating area are separated by the excess blocking area. The upper coating area is provided with a first antigen or a first antibody, the lower coating area is provided with a second antigen or a second antibody, and the excess blocking area is blocked with a blocking buffer.

[0053] In this embodiment, the microfluidic immunoreactor further includes a third coating area, a fourth coating area... and an Nth coating area, and the upper coating area, the third coating area, the fourth coating area... the Nth coating area and the lower coating area are arranged in sequence and the excess closed area is arranged between each other.

[0054] The microfluidic immunoreactor provided in this Example 1 adopts a microfluidic immunoreactor partitioned antibody / antigen coating scheme to achieve simultaneous measurement of multiple biomarkers in the same immune response device, improve the amount of biological information obtained from a single sample, and can simultaneously diagnose patients' diseases and evaluate the protection of the immune system.

[0055] Example 2

[0056] See also Figures 3 and 4 , which is a schematic diagram of the structure of the community infectious disease immunity assessment system provided in Example 2 of the present application, including: a microfluidic immunoreactor 100, a high-precision pipette 200, a chemiluminescent substrate, and a chemiluminescent detection unit 300. The specific structure of each component and its connection relationship are described in detail below.

[0057] See also Figure 3 The microfluidic immunoreactor 100 includes an upper coating area, a lower coating area and an excess blocking area. The upper coating area and the lower coating area are separated by the excess blocking area. The upper coating area is provided with a first antigen or a first antibody, the lower coating area is provided with a second antigen or a second antibody, and the excess blocking area is blocked with a blocking buffer.

[0058] 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. The pipette is used to adsorb the first antigen, the first antibody, the second antigen, the second antibody, the blocking buffer, the chemiluminescent substrate, and the labeled antibody complex to the corresponding area of the microfluidic immunoreactor;

[0059] In this embodiment, there are multiple microfluidic immunoreactors 100, and any one of the microfluidic immunoreactors 100 is tightly attached to any one of the pipettes 200 or its adapter. Since the microfluidic immunoreactor 100 can be tightly attached to the pipette 200, and after attachment, no auxiliary materials such as glue are required, an airtight seal is naturally achieved, which is conducive to batch processing and application.

[0060] In this embodiment, any of the pipettes 200 is a multi-channel or single-channel pipette, including but not limited to a multi-channel pipette gun or a liquid pump.

[0061] See also Figure 4 In this embodiment, the microfluidic immunoreactor also includes a third coating area, a fourth coating area... and an Nth coating area, and the upper coating area, the third coating area, the fourth coating area... the Nth coating area and the lower coating area are arranged in sequence and the excess closed area is arranged between each other.

[0062] It is understandable that the sub-region packaging of the microfluidic immunoreactor is not limited to two regions, and multiple regions can be planned and set according to actual conditions.

[0063] In this embodiment, the first antibody and the second antibody include IgM, IgE, or IgA, the blocking buffer is BSA, casein, or SuperBlock, and the chemiluminescent substrate is hydrogen peroxide and luminol.

[0064] The chemiluminescence detection unit 300 can fix the pipette 200 tightly attached to the microfluidic immunoreactor 100 and collect chemiluminescence image signals, and at the same time convert the chemiluminescence image signals into quantitative numerical signals. The quantitative numerical signals are converted into equivalent standard cytokine / antibody / antigen concentrations through a standard curve to evaluate the level of immune protection against infectious diseases.

[0065] Please refer to 2 again. The chemiluminescence 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. The limiting device 330 is fixed with the pipette 200 tightly fitted with the microfluidic immunoreactor 100. The CMOS camera 320 collects the chemiluminescence image signal, and the computer converts the chemiluminescence image signal into a quantitative numerical signal. The quantitative numerical signal is converted into an equivalent standard cytokine / antibody / antigen concentration through a standard curve to evaluate the level of immune protection against infectious diseases.

[0066] Furthermore, the chemiluminescence detection unit 300 further includes a slidable protective cover 340 , which can seal the pipette 200 tightly attached to the microfluidic immunoreactor 100 within the light-shielding box 310 .

[0067] It should be noted that the community infectious disease immune assessment system provided in this embodiment, the immunoreactor is used as a disposable structure for performing immunoadsorption assays on fingertip blood samples; the chemiluminescence imaging station is designed to quantitatively measure the chemiluminescence signal intensity of the immunoreactor. For IgG binding detection, it provides a very large dynamic range (4.5 orders of magnitude) and high sensitivity (100pg / mL). For the assessment of SARS-CoV-2 neutralization ability, the TOI system integrates an updated version of the rapid in vitro inhibition test that can be used for rapid, cost-effective and reliable assessments using a small portion of 5-10μL fingertip blood samples.

[0068] The community infectious disease immune assessment system provided in Example 2 of this application utilizes a microfluidic immunoreactor with compartmentalized antibody / antigen coating to enable simultaneous measurement of multiple biomarkers within the same immune response device, improving the amount of biological information obtained from a single sample and enabling simultaneous diagnosis of a patient's disease and immune system protection assessment. This facilitates community health centers and hospital laboratories to provide comprehensive diagnostic advice to patients in a short period of time.

[0069] Example 3

[0070] See also Figure 5, a schematic diagram of the principle of the community infectious disease immune protection level assessment method provided in this embodiment, the assessment method provided in this embodiment, spatially divides the detection area of the microfluidic immunoreactor, and can realize the immunoassay measurement of the antibody immune protection level of multiple specific pathogens and the antigens of the corresponding pathogens in trace blood (fingertip blood) dried blood spot samples. The specific implementation method is described in detail below.

[0071] See also Figure 6 The flowchart of the method for assessing community infectious disease immunity level provided in Example 3 includes the following steps:

[0072] Step S10: The upper coating area is provided with a first antigen or a first antibody, the lower coating area is provided with a second antigen or a second antibody, and the excess blocking area is blocked with a blocking buffer, thereby obtaining a microfluidic immunoreactor coated with corresponding antigens and antibodies.

[0073] See also Figure 7 , wherein a first antigen or a first antibody is provided in the upper coating area, a second antigen or a second antibody is provided in the lower coating area, and the excess blocking area is blocked with a blocking buffer, thereby obtaining a microfluidic immunoreactor coated with the corresponding antigens and antibodies, specifically comprising:

[0074] Step S11: dissolving and diluting the first antigen, the first antibody, the second antigen, and the second antibody in phosphate buffer solution respectively.

[0075] In this example, the first antigen, the first antibody, the second antigen, and the second antibody were dissolved in ultrapure water and diluted in phosphate buffered saline (PBS), with target concentrations of 5 μg / mL (lower coating area) and 5 μg / mL (upper coating area).

[0076] Step S12: using the high-precision pipette to aspirate the second antigen or the second antibody into the microfluidic immunoreactor to form a lower coating area, and allowing it to stay and incubate in the microfluidic immunoreactor for 30-60 minutes.

[0077] Specifically, a 12-channel high-precision pipette was used to first aspirate 6 μL of the second antigen or second antibody into the microfluidic immunoreactor, and the second antigen or second antibody was allowed to stay in the reactor for incubation for 30-60 minutes.

[0078] Step S13: using the high-precision pipette to aspirate and discharge the phosphate buffer solution to remove unstable non-specific binding.

[0079] It is understood that the phosphate buffer solution can be aspirated and expelled using a multi-channel pipette to remove unstable non-specific binding without disturbing the underlying coating.

[0080] Step S14: using the high-precision pipette to aspirate the blocking buffer onto the surface of the lower coating area for blocking.

[0081] Specifically, a blocking solution system developed by the scientific research team (containing 3% bovine serum albumin and 1% bovine casein) was used for rapid blocking for about 6 minutes.

[0082] Step S15: using the high-precision pipette to aspirate and discharge the phosphate buffer solution to remove unstable non-specific binding.

[0083] It is understood that the PBS is aspirated and discharged using a multi-channel pipette to remove unstable non-specific binding without disturbing the coating area on the upper layer.

[0084] Step S16: Use the high-precision pipette to aspirate the first antigen or the first antibody into the microfluidic immunoreactor, aspirate the first antigen or the first antibody solution corresponding to the volume of the upper coating area, lift the microfluidic immunoreactor out of the liquid surface, and then when the microfluidic immunoreactor aspirates 1 to 2 μL of air, quickly extend the microfluidic immunoreactor into the blocking solution and aspirate the blocking solution of the lower coating area, so that all the solutions stay in the reactor and incubate for 30-60 minutes.

[0085] It can be understood that the secondary aspiration of the blocking solution is beneficial to preventing nonspecific adsorption of the lower layer.

[0086] It is understandable that after completing step S16, the following steps may also be included: using a multi-channel pipette to aspirate and discharge the cleaning solution (using PBS containing 0.05% Tween20) to remove unstable non-specific binding, and then using the blocking solution system developed by the scientific research team (containing 3% bovine serum albumin and 1% bovine casein) to perform rapid blocking for about 6 minutes, and using a multi-channel pipette to aspirate and discharge the cleaning solution (using PBS containing 0.05% Tween20).

[0087] It can be understood that according to the above steps S11 to S16, a microfluidic immunoreactor coated with antigens and antibodies can be obtained. Since this technology needs to avoid cross-interference between partitions, this embodiment develops a corresponding partition sealing solution to efficiently seal the partitions, with a high signal-to-noise ratio, and can perform highly sensitive and reliable detection.

[0088] Step S20: The microfluidic immunoreactor pre-coated with antigens and antibodies absorbs the labeled antibody and antigen mixed solution and incubates for 2-20 minutes.

[0089] It can be understood that this step can allow the antigen-antibody to specifically recognize the capture antibody or antigen on the surface of the immunoreactor.

[0090] Step S30: using the high-precision pipette to aspirate and discharge the cleaning solution to remove unstable non-specific binding.

[0091] It is understood that the washing solution (PBS containing 0.05% Tween 20) was aspirated and discharged using a multi-channel pipette to remove unstable non-specific binding.

[0092] Step S40: The monoclonal antibody coupled with horseradish peroxidase was diluted to 100-200 ng / mL using 1% casein buffer, and was aspirated into the microfluidic immunoreactor using the high-precision pipette and incubated for 3 minutes.

[0093] Step S50: using the high-precision pipette to aspirate and discharge the washing solution to remove unstable non-specific binding.

[0094] It is understood that a multichannel pipette is used to aspirate and expel the wash solution to remove unstable non-specific binding. This wash process is repeated three times to achieve a good signal-to-noise ratio.

[0095] Step S60: using the high-precision pipette to aspirate the chemiluminescent substrate, and placing the high-precision pipette with the microfluidic immunoreactor in the chemiluminescent detection unit.

[0096] Step S70: The chemiluminescence detection unit collects chemiluminescence image signals in the microfluidic immunoreactor.

[0097] Step S80: converting the chemiluminescent image signal into a quantitative numerical signal, and converting the quantitative numerical signal into an equivalent standard cytokine / antibody / antigen concentration through a standard curve to evaluate the level of immune protection against infectious diseases.

[0098] The above methods can also be used to detect the levels of different antibodies such as IgG, IgA, IgM, IgE, or antibodies / antigens of different infectious diseases.

[0099] See also Figure 8 These are chemiluminescent images obtained at different exposure times during the regional coating and blocking process provided in Example 2. The left image shows the second antigen coated at the lower end of the immunoreactor, while the first antigen coated at the upper end. The right image shows the first antigen coated at the upper end of the immunoreactor, while the second antigen 2 coated at the lower layer of the immunoreactor.

[0100] See also Figure 9, which shows the quantitative signal intensity during the regional coating and blocking provided in Example 2. Red indicates that the second antigen is coated at the lower end of the immunoreactor and the first antigen is coated at the upper end of the immunoreactor. Yellow indicates that the first antigen is coated at the upper end of the immunoreactor and the second antigen is coated at the lower end of the immunoreactor.

[0101] The method for assessing the immune protection level of infectious diseases in the community provided in Example 3 of the present application adopts a microfluidic immunoreactor partitioned antibody / antigen coating scheme to achieve simultaneous measurement of multiple biomarkers in the same immune response device, improve the amount of biological information obtained from a single sample, and can simultaneously perform disease diagnosis and immune system protection assessment on patients, which is beneficial for community health centers and hospital laboratories to provide multi-faceted diagnostic opinions to patients in a short period of time.

[0102] Example 4

[0103] See also Figure 10 , which is a flowchart of the steps of the multi-dimensional assessment method for community infectious disease immune protection level provided in Example 4 of the present application, can assess the levels of different pathogen antibodies such as IgG or antigens of different infectious diseases based on fingertip blood spot samples. The specific implementation method is as follows:

[0104] Step S40: using a high-precision pipette to coat the detection area in the microfluidic immunoreactor for 30-60 minutes, wherein the microfluidic immunoreactor comprises at least two detection areas, including the upper coating area and the lower coating area;

[0105] Step S41: aspirating a sample to be tested into the detector of the microfluidic immunoreactor for incubation, wherein the sample to be tested includes fingertip blood, venous blood, serum, or a dried blood spot complex solution; the molecular types of the sample to be tested include antibodies such as IgG, IgA, and IgE, and disease-related antigen proteins;

[0106] Step S42: using the high-precision pipette, adding the specific detection antibody or antibody mixture into the microfluidic immunoreactor and incubating for 2 to 20 minutes;

[0107] Step S43: using the high-precision pipette to aspirate luminol chemiluminescent substrate into the microfluidic immunoreactor;

[0108] Step S44: placing a high-precision pipette with the microfluidic immunoreactor in a chemiluminescence detection unit, and the chemiluminescence detection unit collects chemiluminescence image signals in the microfluidic immunoreactor;

[0109] Step S45: converting the chemiluminescent image signal into a quantitative numerical signal, and converting the quantitative numerical signal into an equivalent standard cytokine / antibody / antigen concentration through a standard curve to evaluate the level of immune protection against infectious diseases.

[0110] The microfluidic immunoreactor partitioned antibody / antigen coating scheme provided in this application realizes the simultaneous measurement of multiple biomarkers and multiple dried blood spots in the same immune response device, improves the amount of biological information obtained from a single sample, and can simultaneously diagnose patients' diseases and evaluate the protection of the immune system, which is beneficial for community health and hospital laboratories to provide multi-faceted diagnostic opinions to patients in a short period of time.

[0111] In order to further describe the above technical solution, the above solution is described in detail below in conjunction with embodiments.

[0112] Step S31: using the high-precision pipette to aspirate the second antigen into the microfluidic immunoreactor, and allowing it to stay and incubate in the microfluidic immunoreactor for 30-60 minutes.

[0113] In this embodiment, a 12-channel high-precision pipette is used to first aspirate 15 μL of the second antigen into the microfluidic immunoreactor, and the second antigen is allowed to remain in the reactor for incubation for 30-60 minutes.

[0114] Step S32: using the high-precision pipette to aspirate and discharge the phosphate buffer solution to remove unstable non-specific binding.

[0115] In this example, a multi-channel pipette was used to aspirate and expel phosphate buffer to remove unstable non-specific binding without interfering with the coating of the second antigen.

[0116] Step S33: using the high-precision pipette to aspirate the blocking solution system onto the surface of the lower coating area for blocking.

[0117] In this embodiment, a blocking solution system (containing 3% bovine serum albumin and 1% casein) was used for blocking for about 30 minutes.

[0118] Step S34: using the high-precision pipette to aspirate and discharge the phosphate buffer solution to remove unstable non-specific binding.

[0119] In this example, a multi-channel pipette was used to aspirate and expel a washing solution (PBS containing 0.05% Tween 20) to remove unstable non-specific binding without interfering with antigen coating.

[0120] Step S35: using the high-precision pipette to aspirate the first antigen into the microfluidic immunoreactor to form an upper coating area.

[0121] Step S36: Use the high-precision pipette to aspirate the first antigen or the first antibody into the microfluidic immunoreactor, aspirate the first antigen or the first antibody solution corresponding to the volume of the upper coating area, lift the microfluidic immunoreactor out of the liquid surface, and then when the microfluidic immunoreactor aspirates 1 to 2 μL of air, quickly extend the microfluidic immunoreactor into the blocking solution and aspirate the blocking solution of the lower coating area, allowing all the solutions to stay in the reactor and incubate for 30-60 minutes.

[0122] It can be understood that the secondary aspiration of the blocking solution is beneficial to preventing nonspecific adsorption of the lower layer.

[0123] Step S37: Use the high-precision pipette to aspirate and discharge the washing solution to remove unstable non-specific binding.

[0124] In this example, a multi-channel pipette was used to aspirate and expel the washing solution (PBS containing 0.05% Tween 20) to remove unstable non-specific binding without interfering with antigen coating.

[0125] Step S38: Use the high-precision pipette to aspirate the blocking solution system onto the surface of the upper coating area for blocking.

[0126] Step S39: Use the high-precision pipette to aspirate and discharge the washing solution to remove unstable non-specific binding.

[0127] Step S310: aspirating the substance to be tested into the microfluidic immunoreactor for incubation.

[0128] Specifically, the test substance provided in this embodiment is first diluted in a gradient manner and then aspirated into the microfluidic immunoreactor for incubation.

[0129] In this embodiment, the preparation method of the dried blood spot reconstituted blood sample is as follows: 5-80 μL of the collected fingertip blood sample is dropped onto filter paper for air-drying and solidification to form a dried blood spot sample, which is then packaged and stored at room temperature or low temperature; the dried dried blood spot sample is cut in a standardized manner, divided, and immersed in a reconstituted solution for a dissolution reaction; the non-dissolved matter is removed after centrifugation, and finally the dried blood spot reconstituted solution is extracted to obtain a test sample.

[0130] Specifically, the collected fingertip blood is dropped onto prepared filter paper and air-dried, and then packaged and stored at room temperature to obtain a dried blood spot sample; the dried blood spot sample that has been stored for 1-72 days is cut using a round hole paper cutter, and the cut disc is cut into two halves; the cut disc is placed in a reaction solution for re-dissolution; then, excess paper in the solution is removed, and the re-dissolved liquid is centrifuged; the sample after centrifugation is extracted to obtain the dried blood spot re-dissolved blood sample.

[0131] The dried blood spot sample preparation process is as follows: 1. Collect blood from the patient's fingertip using a microtube. 2. Place the collected blood on a prepared filter paper. 3. Air-dry the blood spot sample at room temperature, then seal it in a plastic bag and store it at room temperature. Testing has shown that as little as 5 μL of whole blood from the fingertip can produce an effective dried blood spot.

[0132] See also Figure 11 , which verifies the stability of the dried blood spot sample provided in this embodiment under long-term storage at room temperature. Figure 11 It can be seen from the figure that the dried blood spot sample provided in this example has good stability when stored for a long time at room temperature.

[0133] Furthermore, for the multiplex test protocol for fingertip blood spot samples, the pre-experimental preparation method is as follows: 1. Take out the dried blood spot sample that has been placed for a period of time (1-72 days), cut it with a circular paper cutter, and cut the cut disc in half with clean scissors. 2. Place the cut disc into the reaction solution to re-dissolve it. 3. Use a clean gun tip to remove the excess paper in the solution and place the re-dissolved liquid into a centrifuge for centrifugation. 4. Finally, extract an appropriate amount of the sample after centrifugation for later use.

[0134] Step S311: using the high-precision pipette to discharge the substance to be tested and then washing with a cleaning solution.

[0135] Step S312: using the high-precision pipette, adding the labeled antibody into the microfluidic immunoreactor and incubating for 2 to 20 minutes.

[0136] In this embodiment, the labeled antibody is a monoclonal antibody conjugated with horseradish peroxidase (HRP).

[0137] Step S313: using the high-precision pipette to discharge the labeled antibody and washing it with a cleaning solution, wherein the labeled antibody includes a monoclonal antibody coupled with horseradish peroxidase.

[0138] Step S314: using the high-precision pipette to draw luminol chemiluminescent substrate into the microfluidic immunoreactor.

[0139] Step S315: placing the high-precision pipette with the microfluidic immunoreactor in a chemiluminescence detection unit, and the chemiluminescence detection unit collects chemiluminescence image signals in the microfluidic immunoreactor.

[0140] Step S316: converting the chemiluminescent image signal into a quantitative numerical signal, and converting the quantitative numerical signal into an equivalent standard cytokine / antibody / antigen concentration through a standard curve to evaluate the level of immune protection against infectious diseases.

[0141] Please refer to Figure 12, which shows the comparison of IgG detection levels of dried blood spot samples and fresh blood at different reconstitution volumes provided in this example. As can be seen from the figure, the chemiluminescence signal of the dried blood spot samples of most volunteers is close to the detection signal of fresh blood when the reconstitution volume is 300 μL.

[0142] Please refer to Figure 13, which shows a comparison of IgG detection levels of dry blood spot samples stored at room temperature and 37°C provided in this embodiment. As can be seen from the figure, when the storage time is 1, 7, and 14 days, the signal of the sample stored at 37°C is equivalent to that of the sample stored at 25°C, indicating that the sample can be stored at 37°C for 14 days.

[0143] Please refer to 14, which is a comparison of IgG detection levels of dry blood spot samples under different sampling methods provided in this embodiment (123 are: 10μL blood collection tube, 80μL capillary tube, and direct thumb pressure). It can be seen from the figure that the chemiluminescence detection signals obtained by the three sampling methods are relatively close.

[0144] In this embodiment, the sample to be tested includes fingertip blood, venous blood, serum or dried blood spot reconstitution solution; the first antigen includes the recombinant S protein of the new coronavirus, the first antigen includes the recombinant S protein of the new coronavirus mutant strain or the HA protein of the influenza virus; the molecular types of the sample to be tested include strain-specific IgG, IgM or IgE or IgA and nanoantibodies; the specific detection antibody is a highly specific monoclonal antibody or a nanoantibody; the blocking buffer is BSA or casein or SuperBlock, and the chemiluminescent substrate is hydrogen peroxide and luminol.

[0145] The method for assessing the level of immune protection against infectious diseases in the community provided in Example 3 of the present application can achieve accurate diagnosis of the patient's IgG level even when dried blood spot samples are made from fingertip blood. The development of this method has significantly reduced the clinical demand for the amount of patient blood, allowing some special populations (the elderly and children) to also undergo multi-index IgG level testing. For example, the lower layer is the S protein of the new coronavirus, and the upper layer is the HA protein of H1N1. This method can achieve long-term storage of fingertip blood samples at room temperature to achieve remote and accurate multi-index IgG immunity level testing.

[0146] The community infectious disease immunity assessment system and method provided in the above embodiments of the present application use dried blood spot samples from fingertips to not only assess the IgG immune level, but also can use its trace characteristics to detect multiple indicators to assess the patient's overall antibody immune protection ability. Detectable targets include but are not limited to antibodies such as IgM, IgE, IgA in fingertip blood or antigen biomarkers of pathogens. In addition, protein markers in trace samples such as saliva can also be analyzed and preserved using this method. In addition to antibody concentration analysis, dried blood spot samples from fingertips are also suitable for antibody neutralization ability analysis.

[0147] It can be understood that the various technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the various 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.

[0148] The above are merely preferred embodiments of the present application and only specifically describe the technical principles of the present application. These descriptions are intended only to explain the principles of the present application and should not be construed in any way as limiting the scope of protection of the present application. Based on the explanations herein, any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present application, as well as other specific implementations of the present application that can be conceived by those skilled in the art without inventive effort, shall be included within the scope of protection of the present application.

Claims

1. A microfluidic immunoreactor capable of performing multiple immunoassays, characterized in that: The method comprises an upper coating area, a lower coating area and an excess blocking area, wherein the upper coating area and the lower coating area are separated by the excess blocking area, the upper coating area is provided with a first antigen or a first antibody, the lower coating area is provided with a second antigen or a second antibody, and the excess blocking area is blocked with a blocking buffer.

2. The microfluidic immunoreactor according to claim 1, characterized in that The microfluidic immunoreactor also includes a third coating area, a fourth coating area... and an Nth coating area. The upper coating area, the third coating area, the fourth coating area... the Nth coating area and the lower coating area are arranged in sequence and the excess closed area is arranged between them.

3. A community infectious disease immunity assessment system, characterized in that: include: Microfluidic immunoreactor, high-precision pipette, chemiluminescent substrate and chemiluminescent detection unit, including: The microfluidic immunoreactor comprises an upper coating area, a lower coating area and an excess blocking area, wherein the upper coating area and the lower coating area are separated by the excess blocking area, the upper coating area is provided with a first antigen or a first antibody, the lower coating area is provided with a second antigen or a second antibody, and the excess blocking area is blocked with a blocking buffer; The high-precision pipette is used to adsorb the first antigen, the first antibody, the second antigen, the second antibody, the blocking buffer, the chemiluminescent substrate and the labeled antibody complex to the corresponding area of the microfluidic immunoreactor; The chemiluminescence detection unit can fix the pipette that is tightly attached to the microfluidic immunoreactor, collect chemiluminescence image signals and convert the chemiluminescence image signals into quantitative numerical signals. The quantitative numerical signals are converted into equivalent standard cytokine / antibody / antigen concentrations through a standard curve to evaluate the level of immune protection against infectious diseases.

4. The community infectious disease immunity assessment system according to claim 3, characterized in that: There are multiple microfluidic immunoreactors, and any one of the microfluidic immunoreactors is tightly fitted with any one of the high-precision pipettes or its adapter.

5. The community infectious disease immunity assessment system according to claim 3, characterized in that: The high-precision pipette is multi-channel or single-channel, and includes but is not limited to a multi-channel pipette gun or a liquid pump.

6. The community infectious disease immunity assessment system according to claim 3, characterized in that: The first antibody and the second antibody include IgG, IgM, IgE, IgA or nanobody, the blocking buffer is BSA or casein or SuperBlock, and the chemiluminescent substrate is hydrogen peroxide and luminol.

7. The community infectious disease immunity assessment system according to claim 1, wherein: The chemiluminescence 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 the computer connected to the CMOS camera signal. The limiting device is fixed with the pipette that is tightly fitted with the microfluidic immunoreactor. The CMOS camera collects the chemiluminescence image signal, and the computer converts the chemiluminescence image signal into a quantitative numerical signal. The quantitative numerical signal is converted into an equivalent standard cytokine / antibody / antigen concentration through a standard curve to evaluate the level of immune protection against infectious diseases.

8. An assessment method using the community infectious disease immunity assessment system according to any one of claims 3 to 7, characterized in that: The steps include: Using a high-precision pipette, the microfluidic immunoreactor is partitioned into coated detection areas for 30-60 minutes, wherein the microfluidic immunoreactor comprises at least two detection areas, the detection areas including the upper coating area and the lower coating area; aspirating the sample to be tested into the microfluidic immunoreactor for incubation; Using the high-precision pipette, the specific detection antibody or antibody mixture is added to the microfluidic immunoreactor and incubated for 2 to 20 minutes; Using the high-precision pipette to draw in luminol chemiluminescent substrate into the microfluidic immunoreactor; placing a high-precision pipette with the microfluidic immunoreactor in a chemiluminescence detection unit, wherein the chemiluminescence detection unit collects chemiluminescence image signals in the microfluidic immunoreactor; The chemiluminescent image signal is converted into a quantitative numerical signal, and the quantitative numerical signal is converted into an equivalent standard cytokine / antibody / antigen concentration through a standard curve to evaluate the level of immune protection against infectious diseases.

9. The evaluation method of the community infectious disease immunity evaluation system according to claim 8, characterized in that: The preparation method of the dried blood spot reconstituted blood sample is as follows: 5-80 μL of fingertip blood sample is dropped onto filter paper for air-drying and solidification to form a dried blood spot sample, which is then packaged and stored at room temperature or low temperature; the dried blood spot sample is cut into pieces in a standardized manner, divided, and immersed in a reconstituted solution for a dissolution reaction; After centrifugation, the insoluble matter was removed and the dried blood spot complex solution was finally extracted and used as the test sample.

10. The evaluation method of the community infectious disease immunity evaluation system according to claim 8, characterized in that: The sample to be tested includes fingertip blood, venous blood, serum or dried blood spot reconstitution solution; the first antigen includes the recombinant S protein of the new coronavirus, the first antigen includes the recombinant S protein of the new coronavirus mutant strain or the HA protein of the influenza virus; the molecular types of the sample to be tested include strain-specific IgG, IgM or IgE or IgA and nanoantibodies; the specific detection antibody is a highly specific monoclonal antibody or a nanoantibody; the blocking buffer is BSA or casein or SuperBlock, and the chemiluminescent substrate is hydrogen peroxide and luminol.

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