Micro-fluidic fluorescence immunoassay chip and device for evaluating risk of suffering from Alzheimer disease and application of micro-fluidic fluorescence immunoassay chip and device

Abstract

The invention provides a microfluidic fluorescence immunoassay chip and device for evaluating the risk of having an Alzheimer disease and application of the microfluidic fluorescence immunoassay chip and device, and belongs to the technical field of microfluidic fluorescence immunoassay chip preparation. The microfluidic fluorescence immunoassay chip provided by the invention comprises a chip body, the chip body is provided with a closed micro-channel for a sample to flow, and the micro-channel comprises a sample adding hole, a reaction bin, a capturing bin and a waste liquid pool which are communicated in sequence; the interior of the reaction bin is coated with a mixture of a fluorescent microsphere labeled antibody A beta 42, p-tau-181 and p-tau-217; according to the position of the reaction bin, the capture bin is sequentially coated with an A beta 42 paired antibody, a p-tau-181 paired antibody, a p-tau-217 paired antibody and a second antibody. The micro-fluidic fluorescence immunoassay chip provided by the invention can accurately evaluate the risk of an individual to be detected suffering from Alzheimer's disease or the risk of the individual to be detected suffering from AD-derived MCI.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic fluorescent immunoassay chip preparation, and specifically relates to a microfluidic fluorescent immunoassay chip, a device and applications thereof for assessing the risk of suffering from Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a common and devastating neurodegenerative disorder. With the increasing aging of the global population, its incidence is increasing year by year, placing a heavy burden on society and families. Early detection, early intervention, and early treatment are crucial for improving patients' quality of life and slowing disease progression. Mild cognitive impairment (MCI) due to Alzheimer's disease (AD) is the earliest stage of AD to show clinical symptoms, and this stage is the most important window for early diagnosis and prevention of AD. Therefore, accurate and efficient in vitro diagnostic technologies have become a research focus in this field.

[0003] Currently, in vitro diagnosis of AD primarily relies on the detection of biomarkers in blood or cerebrospinal fluid. While cerebrospinal fluid testing is highly accurate, it requires lumbar puncture, an invasive procedure that is not only painful for the patient but also carries a certain risk of infection and complications. Patient acceptance is low, and repeated testing is difficult, hindering long-term dynamic monitoring of the patient's condition. Compared to cerebrospinal fluid testing, blood testing offers advantages such as ease of sampling and minimal trauma, making it a more commonly used testing method.

[0004] In blood testing technology, many existing methods have obvious limitations. On the one hand, most detection technologies only detect a single biomarker, but single marker detection is easily interfered with by multiple factors, such as the individual's physiological state, the influence of other diseases, etc., resulting in a high false negative rate in the test results, which cannot fully and accurately reflect the condition of AD patients. On the other hand, even if some technologies attempt to detect multiple markers, the detection process is cumbersome and complicated. For example, some detection methods require the addition of multiple reagents in sequence, including a mixture of plasma and magnetic beads, washing buffer, lysate, eluate, a mixture of immunomagnetic beads with different antibodies, different antibodies, enzyme-linked secondary antibodies, fluorescent enzyme substrates, etc. This not only increases the detection cost, but also prolongs the detection time. It also has high technical requirements for operators, which is not conducive to large-scale promotion and application in clinical practice.

[0005] In addition, although there are also schemes in the art for simultaneously immobilizing multiple proteins on the same detection medium for detection, the following problems exist when immobilizing multiple proteins on the same detection medium for detection: (1) Cross-reaction: There may be a certain degree of sequence similarity or structural homology between different protein antibodies and antigens, resulting in non-specific binding, cross-reaction, and false positive or false negative detection results, which seriously affect the accuracy of the detection. (2) Non-specific adsorption: The simultaneous presence of multiple proteins on the detection medium will increase the probability of non-specific adsorption between proteins and the surface of the medium and between proteins. Non-specific adsorption will lead to increased background signals, reducing the sensitivity and signal-to-noise ratio of the detection. At the same time, non-specific adsorption may also change the spatial conformation of the protein, affecting its binding ability with specific antibodies, further affecting the reliability of the detection results. (3) Mutual interference between proteins: Multiple proteins in the same detection system may undergo physical or chemical interactions. For example, the presence of certain proteins may change the environmental conditions such as the ionic strength and pH value of the solution, affecting the activity and stability of other proteins; or proteins may directly interact with each other to form complexes, hindering the normal binding of the target protein with the antibody, and interfering with the detection process. (4) Difficulty in optimization: The fixation conditions (such as fixative concentration, time, temperature, etc.) and detection conditions (such as antibody concentration, reaction time, washing conditions, etc.) for each protein may be different. When multiple proteins are fixed on the same detection medium for simultaneous detection, it is difficult to find a set of optimal conditions that are applicable to all proteins. It takes a lot of time and effort to optimize the conditions, which increases the difficulty and cost of developing the detection method. (5) Signal interference: In detection methods such as immunofluorescence, when different proteins are labeled with multiple fluorescent markers, overlap and interference of fluorescent signals may occur. The emission spectra of different fluorescent dyes may partially overlap, making it difficult to accurately distinguish the signals of different proteins during detection, affecting the analysis and interpretation of the test results.

[0006] In summary, existing in vitro diagnostic technologies for AD have shortcomings in terms of accuracy, detection efficiency, and ease of use, failing to meet the clinical needs for early, accurate diagnosis of AD and dynamic disease monitoring. Therefore, developing an in vitro diagnostic product or method that can simultaneously detect multiple AD-related biomarkers, is simple to operate, and performs rapidly and accurately has important practical significance and clinical application value. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a microfluidic fluorescent immunoassay chip for assessing the risk of Alzheimer's disease, which can simultaneously detect the concentrations of β-amyloid protein (Aβ42), phosphorylated Tau protein-181 (p-Tau-181) and phosphorylated Tau protein-217 (p-Tau-217) in the sample to be tested, and thus accurately assess the risk of Alzheimer's disease in the individual to be tested.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a microfluidic fluorescent immunoassay chip for assessing the risk of Alzheimer's disease, comprising a chip body, wherein the chip body is provided with a closed microchannel for sample flow, wherein the microchannel comprises a sample addition well, a reaction chamber, a capture chamber, and a waste liquid pool connected in sequence; the reaction chamber is coated with a mixture of fluorescent microsphere-labeled antibodies, wherein the mixture includes fluorescent microsphere-labeled Aβ42 antibody A-2H12F8G9, fluorescent microsphere-labeled p-tau-181 antibody T1-5B9C1H7, and fluorescent microsphere-labeled p-tau-217 antibody T2-1F4A3B10; and the capture chamber is coated with Aβ42 paired antibody A-10A1 C5D11, p-tau-181 paired antibody T1-6C9E5F2, p-tau-217 paired antibody T2-8D6A7F1, and a secondary antibody, measured from the position of the reaction chamber.

[0010] Preferably, the volume ratio of the fluorescent microsphere-labeled Aβ42 antibody A-2H12F8G9, the fluorescent microsphere-labeled p-tau-181 antibody T1-5B9C1H7, and the fluorescent microsphere-labeled p-tau-217 antibody T2-1F4A3B10 in the mixture is 1:1:1.

[0011] Preferably, the amount of the mixture of fluorescent microspheres coated with antibodies in the reaction chamber is 10 μL / cm.

[0012] Preferably, the coating concentration of the Aβ42 paired antibody A-10A1C5D11 is 1.2 mg / mL, and the coating volume is 1 μL / cm; the coating concentration of the p-tau-181 paired antibody T1-6C9E5F2 is 1.5 mg / mL, and the coating volume is 1 μL / cm; the coating concentration of the p-tau-217 paired antibody T2-8D6A7F1 is 1.0 mg / mL, and the coating volume is 1 μL / cm.

[0013] Preferably, the secondary antibody is rabbit anti-mouse IgG, the coating concentration of the secondary antibody is 1.0 mg / mL, and the coating volume is 1 μL / cm.

[0014] Preferably, the distance between the Aβ42 paired antibody A-10A1C5D11 and the p-tau-181 paired antibody T1-6C9E5F2 is 1 cm, and the distance between the p-tau-181 paired antibody T1-6C9E5F2 and the p-tau-217 paired antibody T2-8D6A7F1 is 1 cm.

[0015] The present invention also provides a device for evaluating the risk of having Alzheimer's disease, comprising the above-mentioned microfluidic fluorescence immunoassay chip, a microfluidic instrument and a fluorescence immunoassay analyzer; the microfluidic instrument is equipped with an excitation light source, and the fluorescence immunoassay analyzer is equipped with a signal acquisition module and a data processing module.

[0016] Preferably, the excitation wavelength of the excitation light source is 333 nm and the emission wavelength is 613 nm; the data processing module can calculate and output the concentration values of Aβ42, p-Tau-181 and p-Tau-217 respectively according to the standard curves of Aβ42: y = 0.0075x + 0.108, p-Tau-181: y = 0.0091x + 0.608 and p-Tau-217: y = 0.0151x + 0.1513.

[0017] The present invention also provides the application of the above-mentioned microfluidic fluorescence immunoassay chip or the above-mentioned device in the preparation of products for evaluating the risk of having Alzheimer's disease or evaluating the risk of having Alzheimer's disease-related mild cognitive impairment.

[0018] Preferably, when at least one of the detection indexes Aβ42, p-Tau-181 and p-Tau-217 is high risk or medium risk, it is determined as high risk; when all three are low risk, it is determined as low risk; the risk determination of the detection index Aβ42 is: when Aβ42 ≤ 110 pg / mL, the risk is low, and when Aβ42 > 110 pg / mL, the risk is high; the risk determination of the detection index p-Tau-181 is: when p-Tau-181 ≤ 2.32 pg / mL, the risk is low, when 2.32 pg / mL < p-Tau-181 < 3.5 pg / mL, the risk is medium, and when p-Tau-181 ≥ 3.5 pg / mL, the risk is high; the risk determination of the detection index p-Tau-217 is: when p-Tau-217 < 0.40 pg / mL, the risk is low, when 0.40 ≤ p-Tau-217 ≤ 0.63 pg / mL, the risk is medium, and when p-Tau-217 > 0.63 pg / mL, the risk is high.

[0019] Advantages of the present invention:

[0020] By simultaneously coating Aβ42, p-Tau-181 and p-Tau-217 on the microfluidic fluorescence immunoassay chip, the present invention can accurately evaluate the high or low risk of an individual to be tested having Alzheimer's disease or having AD-related MCI. The microfluidic fluorescence immunoassay chip of the present invention can be used for the early screening, assisted diagnosis and evaluation of disease progression of AD-related MCI and AD, providing an effective auxiliary solution for the in vitro diagnosis of AD or AD-related MCI. Description of the Drawings

[0021] Figure 1 Schematic diagram of the structure of the microfluidic fluorescent immunoassay chip of the present invention, wherein 1 is the sample loading well, 2 is the reaction chamber, 3 is coated with Aβ42 paired antibody, 4 is coated with p-tau-181 paired antibody, 5 is the microfluidic channel, 6 is coated with p-tau-217 paired antibody, 7 is the secondary antibody, 8 is the capture chamber, and 9 is the waste liquid pool;

[0022] Figure 2 is the Aβ42 standard curve;

[0023] Figure 3 is the p-Tau-217 standard curve;

[0024] Figure 4 is the standard curve of p-Tau-181;

[0025] Figure 5 is the detection result of Aβ42;

[0026] Figure 6 This is the p-Tau-181 test result;

[0027] Figure 7 This is the p-Tau-217 test result;

[0028] Figure 8 is the detection result of Aβ42;

[0029] Figure 9 is the detection result of p-Tau-181;

[0030] Figure 10 This is the detection result of p-Tau-217. DETAILED DESCRIPTION

[0031] The present invention provides a microfluidic fluorescent immunoassay chip for assessing the risk of Alzheimer's disease, comprising a chip body, wherein the chip body is provided with a closed microchannel for sample flow, wherein the microchannel comprises a sample addition well, a reaction chamber, a capture chamber, and a waste liquid pool connected in sequence; the reaction chamber is coated with a mixture of fluorescent microsphere-labeled antibodies, wherein the mixture includes fluorescent microsphere-labeled Aβ42 antibody A-2H12F8G9, fluorescent microsphere-labeled p-tau-181 antibody T1-5B9C1H7, and fluorescent microsphere-labeled p-tau-217 antibody T2-1F4A3B10; and the capture chamber is coated with Aβ42 paired antibody A-10A1 C5D11, p-tau-181 paired antibody T1-6C9E5F2, p-tau-217 paired antibody T2-8D6A7F1, and a secondary antibody, measured from the position of the reaction chamber.

[0032] In the present invention, the antibody A-2H12F8G9 and its paired antibody A-10A1C5D11 for Aβ42, the antibody T1-5B9C1H7 and its paired antibody T1-6C9E5F2 for p-tau-181, and the antibody T2-1F4A3B10 and its paired antibody T2-8D6A7F1 for p-tau-217 are all known antibodies. For details, please see the announcement of the Ai Xiya service account.

[0033] In the present invention, the microfluidic fluorescent immunoassay chip preferably includes a top plate and a bottom plate, and the top plate and the bottom plate are preferably made of polymethyl methacrylate (PMMA). The top plate and the bottom plate are sealed by laser welding to form a channel, and the channel is a microfluidic channel, and the microfluidic channel uses capillary force to provide liquid movement power. In the present invention, the volume ratio of the fluorescent microsphere-labeled Aβ42 antibody A-2H12F8G9, the fluorescent microsphere-labeled p-tau-181 antibody T1-5B9C1H7, and the fluorescent microsphere-labeled p-tau-217 antibody T2-1F4A3B10 in the mixture is preferably 1:1:1. In the present invention, the solution used to dilute the Aβ42 antibody A-2H12F8G9, the p-tau-181 antibody T1-5B9C1H7, and the p-tau-217 antibody T2-1F4A3B10 is preferably 0.01M PBS at pH 7.4 containing 20% ​​sucrose and 2% bovine serum albumin. In the present invention, the amount of the mixture of fluorescent microspheres coated with labeled antibodies in the reaction chamber is preferably 10 μL / cm. In the present invention, the fluorescent microspheres are preferably polystyrene latex microspheres, and the mass ratio of the fluorescent microspheres to each antibody is preferably 0.2 to 0.5:1, more preferably 0.3 to 0.4:1.

[0034] In the present invention, the coating concentration of the Aβ42 paired antibody A-10A1C5D11 is preferably 1.2 mg / mL, and the coating volume is preferably 1 μL / cm; the coating concentration of the p-tau-181 paired antibody T1-6C9E5F2 is preferably 1.5 mg / mL, and the coating volume is preferably 1 μL / cm; the coating concentration of the p-tau-217 paired antibody T2-8D6A7F1 is preferably 1.0 mg / mL, and the coating volume is preferably 1 μL / cm. In the present invention, the solution for diluting the Aβ42 paired antibody A-10A1C5D11, the p-tau-181 paired antibody T1-6C9E5F2, and the p-tau-217 paired antibody T2-8D6A7F1 is preferably a 0.01 M PBS pH 7.4 buffer. In the present invention, the secondary antibody is preferably rabbit anti-mouse IgG, the coating concentration of the secondary antibody is preferably 1.0 mg / mL, and the coating amount is preferably 1 μL / cm. In the present invention, the distance between the Aβ42 paired antibody A-10A1C5D11 and the p-tau-181 paired antibody T1-6C9E5F2 is preferably 1 cm, and the distance between the p-tau-181 paired antibody T1-6C9E5F2 and the p-tau-217 paired antibody T2-8D6A7F1 is preferably 1 cm. The microfluidic fluorescent immunoassay chip provided by the present invention is preferably detected at room temperature. If the microfluidic fluorescent immunoassay chip is stored under low temperature conditions, it needs to be restored to room temperature in advance when used. The microfluidic fluorescent immunoassay chip of the present invention is preferably packaged in an aluminum foil bag.

[0035] The reaction mechanism of the microfluidic fluorescent immunoassay chip of the present invention is as follows: after the sample is added to the sample well, it fully reacts with the fluorescent microsphere-labeled Aβ42 antibody, p-Tau-181 antibody, and p-Tau-217 antibody, and then the mixed reactant flows along the microfluidic channel by capillary force, and sequentially reacts with the paired antibodies and secondary antibodies of Aβ42, p-Tau-181, and p-Tau-217, and excites fluorescence to produce a fluorescent signal. The concentration value of each detection index is converted according to the standard curve through the fluorescent signal value, and the excess liquid flows into the waste liquid pool. In the present invention, the standard curve of Aβ42 is preferably y=0.0075x+0.108, R 2 =0.9995; the standard curve of p-Tau-181 is preferably y=0.0091x+0.608, R 2 =0.9997; the standard curve of p-Tau-217 is preferably y=0.0151x+0.1513, R 2 =0.9997. In the present invention, if no fluorescent signal is collected at the secondary antibody, it indicates that the test has failed and needs to be retested.

[0036] The present invention also provides a device for assessing the risk of Alzheimer's disease, comprising the above-mentioned microfluidic fluorescence immunoassay chip, a microfluidic instrument, and a fluorescence immunoassay analyzer; the microfluidic instrument is equipped with an excitation light source, and the fluorescence immunoassay analyzer is equipped with a signal acquisition module and a data processing module.

[0037] In the present invention, the excitation wavelength of the excitation light source is 333 nm, and the emission wavelength is 613 nm; the data processing module can calculate and output the concentration values of Aβ42, p-Tau-181, and p-Tau-217 respectively according to the standard curves of Aβ42: y = 0.0075x + 0.108, p-Tau-181: y = 0.0091x + 0.608, and p-Tau-217: y = 0.0151x + 0.1513. When using the device provided by the present invention to detect a test sample, the sample is added to the sample addition hole of the microfluidic fluorescence immunoassay chip, and "start" is clicked for detection. The excitation light source emits laser light. After 15 minutes, the fluorescence immunoassay analyzer automatically displays the numerical values and units of the concentration of each detection index. In the present invention, the test sample is preferably plasma.

[0038] The present invention also provides the application of the above-mentioned microfluidic fluorescence immunoassay chip in the preparation of products for assessing the risk of Alzheimer's disease or assessing the risk of Alzheimer's disease-related mild cognitive impairment.

[0039] In the present invention, the product preferably includes a kit. In the present invention, when at least one of the detection indexes Aβ42, p-Tau-181, and p-Tau-217 is high risk or medium risk, it is determined as high risk; when all three are low risk, it is determined as low risk; the risk determination of the detection index Aβ42 is: when Aβ42 ≤ 110 pg / mL, the risk is low, and when Aβ42 > 110 pg / mL, the risk is high; the risk determination of the detection index p-Tau-181 is: when p-Tau-181 ≤ 2.32 pg / mL, the risk is low, when 2.32 pg / mL < p-Tau-181 < 3.5 pg / mL, the risk is medium, and when p-Tau-181 ≥ 3.5 pg / mL, the risk is high; the risk determination of the detection index p-Tau-217 is: when p-Tau-217 < 0.40 pg / mL, the risk is low, when 0.40 ≤ p-Tau-217 ≤ 0.63 pg / mL, the risk is medium, and when p-Tau-217 > 0.63 pg / mL, the risk is high.

[0040] The technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] In the following embodiments, unless otherwise specified, all are conventional methods.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] Example 1

[0044] A microfluidic fluorescence immunoassay chip for assessing the risk of Alzheimer's disease, the structure of which is as follows Figure 1 As shown: there is a chip body, the chip body is provided with a closed microfluidic channel for sample flow, the microfluidic channel includes a sample addition hole, a reaction chamber, a capture chamber and a waste liquid pool connected in sequence; the reaction chamber is coated with a mixture of fluorescent microsphere-labeled antibodies, the coating volume is 10 μL / cm, and the mixture consists of fluorescent microsphere-labeled Aβ42 antibody A-2H12F8G9, fluorescent microsphere-labeled p-tau-181 antibody T1-5B9C1H7, and fluorescent microsphere-labeled p-tau-217 antibody T2-1F4A3B10 in a volume ratio of 1:1:1; starting from the position of the reaction chamber, the capture chamber is coated with Aβ42 paired antibody A-10A1C5D11, p-tau-181 paired antibody T1-6C9E5F2, p-tau-217 paired antibody T2-8D6A7F1 and rabbit anti-mouse IgG in sequence;

[0045] The coating concentration of the Aβ42 paired antibody A-10A1C5D11 was 1.2 mg / mL, and the coating volume was 1 μL / cm; the coating concentration of the p-tau-181 paired antibody T1-6C9E5F2 was 1.5 mg / mL, and the coating volume was 1 μL / cm; the coating concentration of the p-tau-217 paired antibody T2-8D6A7F1 was 1.0 mg / mL, and the coating volume was 1 μL / cm. cm; the coating concentration of the rabbit anti-mouse IgG (secondary antibody) was 1.0 mg / mL, and the coating volume was 1 μL / cm; the distance between the Aβ42 paired antibody A-10A1C5D11 and the p-tau-181 paired antibody T1-6C9E5F2 was 1 cm, and the distance between the p-tau-181 paired antibody T1-6C9E5F2 and the p-tau-217 paired antibody T2-8D6A7F1 was 1 cm.

[0046] The distance between the sample loading well and the reaction chamber is 22 mm, the distance between the reaction chamber and the Aβ42 paired antibody in the capture chamber is 36 mm, the distance between the reaction chamber and the p-tau-181 paired antibody in the capture chamber is 46 mm, the distance between the reaction chamber and the p-tau-217 paired antibody in the capture chamber is 56 mm, and the distance between the reaction chamber and the secondary antibody in the capture chamber is 66 mm.

[0047] The preparation method is:

[0048] (1) Reaction chamber:

[0049] Polystyrene latex microspheres were diluted with MES (0.01 M), activated by adding EDC (5 mg / mL) and NHS (5 mg / mL), centrifuged and discarded the supernatant, and then resuspended in 0.01 M MES by ultrasound to obtain activated fluorescent microspheres.

[0050] Aβ42 antibody A-2H12F8G9, p-tau-181 antibody T1-5B9C1H7, and p-tau-217 antibody T2-1F4A3B10 were diluted to 1.5 mg / mL, 1.6 mg / mL, and 1.2 mg / mL, respectively, in 0.01 M PBS (pH 7.4) containing 20% ​​sucrose and 2% bovine serum albumin. These antibodies were mixed with activated fluorescent microspheres at a 2:1 volume ratio and reacted at 37°C for 90 min. Blocking buffer (PBS buffer containing 50 mM HEPES, 1% BSA, and 0.1% ProClin 300, pH 8.0) was added and incubated for 30 min. The supernatant was discarded after centrifugation at 4°C and the cells were resuspended. Fluorescent antibody protection buffer (0.01 mol / L PBS buffer, pH 7.4) was added and sonicated for 1 min.

[0051] Mix the three fluorescent antibodies in a 1:1:1 volume ratio to obtain a mixture of fluorescent microsphere-labeled antibodies. Use a microspotter to spot the mixture of fluorescent microsphere-labeled antibodies into the reaction chamber at a coating volume of 10 μL / cm. Load a 10 μL sample and dry at 37°C.

[0052] (2) Capture chamber: Aβ42 paired antibody A-10A1C5D11, p-tau-181 paired antibody T1-6C9E5F2, p-tau-217 paired antibody T2-8D6A7F1, and rabbit anti-mouse IgG (secondary antibody) were diluted with diluent (the diluent was 0.01M PBS pH 7.4 buffer) to 1.2 mg / mL, 1.5 mg / mL, 1.0 mg / mL, and 1.0 mg / mL, respectively; the above antibodies were spotted on the corresponding positions using a micro-spotter, with a sample volume of 1 μL at each position, and dried at 37°C.

[0053] Example 2

[0054] Preparation of standard curve:

[0055] Linear dilution of standard antigens: Aβ42 standard antigen was linearly diluted using negative plasma at the following concentrations: 0.1, 5, 35, 70, 110, 220, and 380 pg / mL. p-Tau-181 standard antigen was linearly diluted using negative plasma at the following concentrations: 0.1, 5, 20, 30, 50, 80, and 100 pg / mL. p-Tau-217 standard antigen was linearly diluted using negative plasma at the following concentrations: 0.1, 5, 20, 30, 50, 80, and 100 pg / mL.

[0056] Add the standard sample to the sample well of the microfluidic fluorescent immunoassay chip obtained in Example 1, use the same batch of reagents, and repeat the measurement for more than 3 times at each concentration. Use the immunofluorescence analyzer to obtain the fluorescence intensity data of the test line and the quality control line for analysis. The concentration of the standard is used as the horizontal axis, and the fluorescence intensity ratio (T / C) of the test line and the quality control line is used as the vertical axis to fit the standard curve. The results are as follows: Figures 2 to 4 shown.

[0057] Depend on Figure 2 It can be seen that the detection sensitivity of Aβ42 is 0.1pg / mL, and the maximum detection range is 380pg / mL; Figure 4 It can be seen that the detection sensitivity of p-Tau-181 is 0.1pg / mL, and the maximum detection range is 100pg / mL; Figure 3 It can be seen that the detection sensitivity of p-Tau-217 is 0.1pg / mL, and the maximum detection range is 100pg / mL. The linear correlation coefficients R 2 All are greater than 0.999.

[0058] Comparative Example 1

[0059] The difference from Example 1 is that the fluorescent microsphere-labeled Aβ42 antibody is changed from A-2H12F8G9 to A-10A1C5D11, and the Aβ42 paired antibody is changed from A-10A1C5D11 to A-2H12F8G9; the fluorescent microsphere-labeled p-tau-181 antibody is changed from T1-5B9C1H7 to T1-6C9E5F2, and the p-tau-181 paired antibody is changed from T1-6C9E5F2 to T1-5B9C1H7; the fluorescent microsphere-labeled p-tau-217 antibody is changed from T2-1F4A3B10 to T2-8D6A7F1, and the p-tau-217 paired antibody is changed from T2-8D6A7F1 to T2-1F4A3B10. The rest is the same as Example 1.

[0060] Example 1 is recorded as system A, and Comparative Example 1 is recorded as system B. The following tests are performed:

[0061] Different concentrations of Aβ42 standard antigen (0.5, 5, 30, 70, 110, 230, 380 pg / mL), p-Tau-181 standard antigen (0.1, 5, 20, 30, 50, 80, 100 pg / mL) obtained in Example 2, and p-Tau-217 standard antigen (0.1, 5, 20, 30, 50, 80, 100 pg / mL) were respectively added dropwise to the sample wells of the microfluidic fluorescent immunoassay chip obtained in Example 1 and the sample wells of the microfluidic fluorescent immunoassay chip obtained in Comparative Example 1. Irradiation was performed using a light source with an excitation wavelength of 333 nm and an emission wavelength of 613 nm. Capillary force was used to drive the liquid to flow. After the antigen and antibody fully reacted, the fluorescence signal was scanned, and the concentration of each detection indicator was calculated using the standard curve obtained in Example 2.

[0062] The test results of each test index of the two systems (A and B) are as follows: Figures 5 to 7 As shown, it is shown that the signal value of system A (Example 1) is stronger.

[0063] Comparative Example 2

[0064] The difference from Example 1 is that the coating concentration of Aβ42 paired antibody A-10A1C5D11 is changed to 1.0 mg / mL, the coating concentration of p-tau-181 paired antibody T1-6C9E5F2 is changed to 1.2 mg / mL, the coating concentration of p-tau-217 paired antibody T2-8D6A7F1 is changed to 0.8 mg / mL, and the coating concentration of the secondary antibody is changed to 0.8 mg / mL. The rest is the same as Example 1.

[0065] Comparative Example 3

[0066] The difference from Example 1 is that the coating concentration of Aβ42 paired antibody A-10A1C5D11 is changed to 1.5 mg / mL, the coating concentration of p-tau-181 paired antibody T1-6C9E5F2 is changed to 1.8 mg / mL, the coating concentration of p-tau-217 paired antibody T2-8D6A7F1 is changed to 1.2 mg / mL, and the coating concentration of the secondary antibody is changed to 1.2 mg / mL. The rest is the same as Example 1.

[0067] Example 1 is recorded as System 2, Comparative Example 2 is recorded as System 1, and Comparative Example 3 is recorded as System 3. The following tests are performed:

[0068] Different concentrations of Aβ42 standard antigen (0.1, 5, 35, 70, 110, 220, 380 pg / mL), p-Tau-181 standard antigen (0.1, 5, 20, 30, 50, 80, 100 pg / mL), and p-Tau-217 standard antigen (0.1, 5, 20, 30, 50, 80, 100 pg / mL) obtained in Example 2 were respectively added dropwise to the sample wells of the microfluidic fluorescent immunoassay chip obtained in Example 1, the sample wells of the microfluidic fluorescent immunoassay chip obtained in Comparative Example 2, and the sample wells of the microfluidic fluorescent immunoassay chip obtained in Comparative Example 3. Irradiation was performed using a light source with an excitation wavelength of 333 nm and an emission wavelength of 613 nm. Capillary force was used to drive the liquid to flow. After the antigen and antibody fully reacted, the fluorescence signal was scanned, and the concentration of each detection index was calculated using the standard curve obtained in Example 2.

[0069] The test results of each test index of the three systems (1, 2 and 3) are as follows: Figures 8 to 10 As shown, it is shown that the signal value of system 2 (Example 1) is stronger and the amount of antibody used is more economical.

[0070] Example 3

[0071] A device for assessing the risk of Alzheimer's disease comprises the microfluidic fluorescent immunoassay chip obtained in Example 1, a microfluidic instrument, and a fluorescent immunoassay analyzer; the microfluidic instrument is equipped with an excitation light source with an excitation wavelength of 333 nm and an emission wavelength of 613 nm, and the fluorescent immunoassay analyzer is equipped with a signal acquisition module and a data processing module. The data processing module can calculate and output the concentration values ​​of Aβ42, p-Tau-181, and p-Tau-217 based on the standard curve y=0.0075x+0.108 of Aβ42, the standard curve y=0.0091x+0.608 of p-Tau-181, and the standard curve y=0.0151x+0.1513 of p-Tau-217, respectively.

[0072] Example 4

[0073] A method for assessing the risk of Alzheimer's disease in a test sample using the microfluidic fluorescent immunoassay chip obtained in Example 1, comprising the following steps: dripping the test sample (plasma) into the sample well of the microfluidic fluorescent immunoassay chip obtained in Example 1, irradiating the sample with an excitation wavelength of 333 nm and an emission wavelength of 613 nm, driving the liquid to flow using capillary force, scanning the fluorescence signal after the antigen and antibody fully react, and calculating the concentration of each detection indicator using the standard curve obtained in Example 2.

[0074] When at least one of the detection indicators Aβ42, p-Tau-181, and p-Tau-217 is high-risk or medium-risk, it is determined to be high-risk; when all three are low-risk, it is determined to be low-risk. The risk determination for the detection indicator Aβ42 is: when Aβ42 ≤ 110 pg / mL, the risk is low; when Aβ42 > 110 pg / mL, the risk is high. The risk determination for the detection indicator p-Tau-181 is: when p-Tau-181 ≤ 2.32 pg / mL, the risk is low; when 2.32 pg / mL < p-Tau-181 < 3.5 pg / mL, the risk is medium; when p-Tau-181 ≥ 3.5 pg / mL, the risk is high. The risk determination for the detection indicator p-Tau-217 is: when p-Tau-217 < 0.40 pg / mL, the risk is low; when 0.40 ≤ p-Tau-217 ≤ 0.63 pg / mL, the risk is medium; when p-Tau-217 > 0.63 pg / mL, the risk is high.

[0075] Example 5

[0076] A method for evaluating the risk of Alzheimer's disease in a test sample using the device obtained in Example 3 is as follows:

[0077] Drop the test sample (plasma) into the sample addition hole of the microfluidic fluorescence immunoassay chip, and click the "Start" button on the microfluidic instrument to perform the detection. After 15 minutes, the fluorescence immunoassay analyzer automatically displays the concentration values of each detection indicator (Aβ42, p-Tau-181, and p-Tau-217).

[0078] When at least one of the detection indicators Aβ42, p-Tau-181, and p-Tau-217 is high-risk or medium-risk, it is determined to be high-risk; when all three are low-risk, it is determined to be low-risk. The risk determination for the detection indicator Aβ42 is: when Aβ42 ≤ 110 pg / mL, the risk is low; when Aβ42 > 110 pg / mL, the risk is high. The risk determination for the detection indicator p-Tau-181 is: when p-Tau-181 ≤ 2.32 pg / mL, the risk is low; when 2.32 pg / mL < p-Tau-181 < 3.5 pg / mL, the risk is medium; when p-Tau-181 ≥ 3.5 pg / mL, the risk is high. The risk determination for the detection indicator p-Tau-217 is: when p-Tau-217 < 0.40 pg / mL, the risk is low; when 0.40 ≤ p-Tau-217 ≤ 0.63 pg / mL, the risk is medium; when p-Tau-217 > 0.63 pg / mL, the risk is high.

[0079] Example 6

[0080] Plasma was collected from 36 volunteers aged 60 to 85 years old randomly selected from three provincial hospitals in Hebei Province, and the test was performed using the method of Example 4. At the same time, a comparative test was performed using the hospital's clinical test methods. After testing using the method of Example 4, it was suggested that 11 of them were at high risk and the other 25 were at low risk. Among the 11 people who were determined to be at high risk by clinical testing, 3 were diagnosed with preclinical Alzheimer's disease (AD), 6 were diagnosed with mild cognitive impairment (MCI), and 2 were diagnosed with Alzheimer's disease (AD) dementia; the 25 people at low risk were determined to have no Alzheimer's disease or AD-induced MCI by clinical testing.

[0081] This indicates that the microfluidic fluorescent immunoassay chip provided by the present invention can accurately assess the risk of an individual suffering from Alzheimer's disease and can assist doctors in diagnosis.

[0082] Example 7

[0083] The preparation process of the Aβ42 antibody A-2H12F8G9, the p-tau-181 antibody T1-5B9C1H7 and the p-tau-217 antibody T2-1F4A3B10 of the present invention, as well as the Aβ42 paired antibody A-10A1C5D11, the p-tau-181 paired antibody T1-6C9E5F2 and the p-tau-217 paired antibody T2-8D6A7F1 are as follows:

[0084] Preparation and purification of monoclonal antibodies: Aβ42, p-Tau-181, and p-Tau-217 proteins were purchased as immune antigens and immunized into 6-week-old healthy BALB / c female mice. Hybridoma technology was used to prepare and screen monoclonal antibody cell lines. The screened specific antibody cell lines were injected into mice to prepare ascites, which was then purified by octanoic acid-ammonium sulfate precipitation to obtain Aβ42 antibody A-2H12F8G9, p-tau-181 antibody T1-5B9C1H7, and p-tau-217 antibody T2-1F4A3B10.

[0085] Screening of three paired monoclonal antibodies: The monoclonal cell lines obtained in the above steps were tested by ELISA and the titers were required to be within 9×10 6 In the above, monoclonal antibodies of IgG1, IgG1, and IgG2a subtypes were selected; after protein purification, the purified protein content was 6.22 to 12.8 mg / mL; each paired antibody was screened by ELISA addition test, and the addition index AI was calculated according to the following formula: AI>50% indicates that they are directed against different antigenic determinants, and AI<50% indicates that they are directed against the same antigenic determinant. The present invention requires that the addition index AI of each paired antibody be above 95% to ensure that the two paired monoclonal antibodies recognize different antigenic epitopes.

[0086]

[0087] A1 and A2 are the OD values ​​measured using a single monoclonal cell line, 1+2 The OD values ​​of the two monoclonal cell lines were obtained. Finally, the Aβ42 paired antibody A-10A1C5D11, the p-tau-181 paired antibody T1-6C9E5F2, and the p-tau-217 paired antibody T2-8D6A7F1 were obtained.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A microfluidic fluorescence immunoassay chip for assessing the risk of Alzheimer's disease, characterized in that: The chip comprises a chip body, which is provided with a closed microfluidic channel for sample flow, wherein the microfluidic channel comprises a sample addition hole, a reaction chamber, a capture chamber and a waste liquid pool connected in sequence; the reaction chamber is coated with a mixture of fluorescent microsphere-labeled antibodies, wherein the mixture includes fluorescent microsphere-labeled Aβ42 antibody A-2H12F8G9, fluorescent microsphere-labeled p-tau-181 antibody T1-5B9C1H7 and fluorescent microsphere-labeled p-tau-217 antibody T2-1F4A3B10; and the capture chamber is coated with Aβ42 paired antibody A-10A1 C5D11, p-tau-181 paired antibody T1-6C9E5F2, p-tau-217 paired antibody T2-8D6A7F1 and a secondary antibody in sequence, measured from the position of the reaction chamber.

2. The microfluidic fluorescent immunoassay chip according to claim 1, characterized in that: The volume ratio of the fluorescent microsphere-labeled Aβ42 antibody A-2H12F8G9, the fluorescent microsphere-labeled p-tau-181 antibody T1-5B9C1H7, and the fluorescent microsphere-labeled p-tau-217 antibody T2-1F4A3B10 in the mixture is 1:1:

1.

3. The microfluidic fluorescent immunoassay chip according to claim 1, characterized in that: The amount of the mixture of fluorescent microspheres coated with antibodies in the reaction chamber is 10 μL / cm.

4. The microfluidic fluorescent immunoassay chip according to claim 1, characterized in that: The coating concentration of the Aβ42 paired antibody A-10A1C5D11 was 1.2 mg / mL, and the coating volume was 1 μL / cm; the coating concentration of the p-tau-181 paired antibody T1-6C9E5F2 was 1.5 mg / mL, and the coating volume was 1 μL / cm; the coating concentration of the p-tau-217 paired antibody T2-8D6A7F1 was 1.0 mg / mL, and the coating volume was 1 μL / cm.

5. The microfluidic fluorescent immunoassay chip according to claim 1, characterized in that: The secondary antibody was rabbit anti-mouse IgG, the coating concentration of the secondary antibody was 1.0 mg / mL, and the coating volume was 1 μL / cm.

6. The microfluidic fluorescent immunoassay chip according to claim 1, characterized in that: The distance between the Aβ42 paired antibody A-10A1C5D11 and the p-tau-181 paired antibody T1-6C9E5F2 was 1 cm, and the distance between the p-tau-181 paired antibody T1-6C9E5F2 and the p-tau-217 paired antibody T2-8D6A7F1 was 1 cm.

7. A device for assessing the risk of developing Alzheimer's disease, characterized in that: The invention comprises the microfluidic fluorescent immunoassay chip according to any one of claims 1 to 6, a microfluidic instrument and a fluorescent immunoassay analyzer; the microfluidic instrument is equipped with an excitation light source, and the fluorescent immunoassay analyzer is equipped with a signal acquisition module and a data processing module.

8. The device according to claim 7, characterized in that The excitation wavelength of the excitation light source is 333 nm, and the emission wavelength is 613 nm; the data processing module can calculate and output the concentration values ​​of Aβ42, p-Tau-181 and p-Tau-217 based on the standard curve y=0.0075x+0.108 of Aβ42, the standard curve y=0.0091x+0.608 of p-Tau-181 and the standard curve y=0.0151x+0.1513 of p-Tau-217, respectively.

9. Use of the microfluidic fluorescence immunoassay chip according to any one of claims 1 to 6 or the device according to any one of claims 7 to 8 in the preparation of a product for assessing the risk of Alzheimer's disease or the risk of Alzheimer's disease-related mild cognitive impairment.

10. The use according to claim 9, characterized in that When at least one of the detection indicators Aβ42, p-Tau-181, and p-Tau-217 is high risk or medium risk, it is determined to be high risk; when all three are low risk, it is determined to be low risk. The risk determination of the detection indicator Aβ42 is as follows: when Aβ42 ≤ 110 pg / mL, the risk is low; when Aβ42 > 110 pg / mL, the risk is high. The risk determination of the detection indicator p-Tau-181 is as follows: when p-Tau-181 ≤ 2.32 pg / mL, the risk is low; when 2.32 pg / mL < p-Tau-181 < 3.5 pg / mL, the risk is medium; when p-Tau-181 ≥ 3.5 pg / mL, the risk is high. The risk determination of the detection indicator p-Tau-217 is as follows: when p-Tau-217 < 0.40 pg / mL, the risk is low; when 0.40 ≤ p-Tau-217 ≤ 0.63 pg / mL, the risk is medium; when p-Tau-217 > 0.63 pg / mL, the risk is high.

Images