Alzheimer's disease early diagnosis method based on magnetic immune micro-fluidic chip

Abstract

The invention discloses an Alzheimer's disease early diagnosis method based on a magnetic immune micro-fluidic chip, immunomagnetic beads are magnetic beads modified with Abeta1-42 and P-tau-181 fluorescent antibodies, and the detection steps are as follows: injecting a 1% BSA solution into a chip channel, i.e., each sample introduction hole, by using an injection pump, and carrying out sealing treatment; then 100 [mu] l of resuspended A [beta] 1-42 and P-tau-181 fluorescent antibody labeled magnetic beads are injected into each channel; cleaning the channel to remove unfixed magnetic beads; the magnetic bead fixing condition is observed under an inverted fluorescence microscope, and the fixing effect is confirmed; a sample liquid is injected into a sample injection hole of the chip through a micro-injection pump or a sample injection needle, and the liquid enters the fixed capture area through a channel; under a fluorescence microscope, fluorescence signals are shot and collected through a high-speed CCD, image analysis software such as ImageJ is used for carrying out qualitative and quantitative analysis on A beta 1-42 and P-tau-181, and double-index detection is achieved.

Description

Technical field:

[0001] The present invention relates to the field of biological detection technology, and in particular to a magnetic immunomicrofluidic kit for detecting early-stage Alzheimer's disease (AD)-related proteins Aβ1-42 and P-tau-181. Background technology:

[0002] AD is a neurodegenerative disease characterized by progressive cognitive decline. Patients typically experience memory loss and decreased executive function in the early stages. As the disease progresses, language skills are gradually lost, ultimately leading to the inability to take care of themselves. AD not only affects the patients themselves, but also places a heavy burden on their families and society. Because intracranial pathological changes, including widespread irreversible damage to neurons and synapses, persist for many years before clinical symptoms appear, early diagnosis is crucial for slowing disease progression and reducing the social burden.

[0003] Currently available early diagnosis technologies for AD:

[0004] 1. Cognitive function scale: Commonly used tools include the Mini-Mental State Examination, the Montreal Cognitive Assessment, the Clinical Dementia Rating Scale, and other scales.

[0005] Limitations: It is highly subjective and easily affected by educational background, language, and cultural differences, making it difficult to distinguish AD from other types of dementia (such as Lewy body dementia or frontotemporal dementia).

[0006] 2. Genetic testing: mainly detects early-onset familial AD (accounting for <5% of AD cases) and detects APP, PSEN1, and PSEN2 gene mutations.

[0007] Limitations: It has no direct diagnostic value for sporadic AD (more than 95% of cases) and is ethically controversial (e.g., APOEε4 carriers only indicate an increased risk but are not a basis for diagnosis).

[0008] 3. Cerebrospinal fluid (CSF) testing: Under the ATN testing standard, the levels of β-amyloid protein (Aβ) and phosphorylated tau protein (microtubule-associated protein tau, P-tau) in the cerebrospinal fluid are tested.

[0009] Limitations: The invasive nature of lumbar puncture results in low acceptance rates (<40%) and insufficient interlaboratory standardization (coefficient of variation 15%-20%).

[0010] 4. Blood biomarker analysis: Under the ATN detection standard, the Aβ and P-tau levels in cerebrospinal fluid and peripheral blood are highly correlated. Molecular immunoassay or mass spectrometry technology is used to analyze the Aβ and P-tau levels in the plasma of AD patients for detection.

[0011] Limitations: Sample processing requires strict standardization; testing price is too high.

[0012] 5. Exosome analysis: Because neurogenic exosomes are enriched with brain-derived Aβ and P-tau, with concentrations 10 times higher than in plasma, the Aβ and P-tau content in exosomes was tested.

[0013] Limitations: The separation process is complex, takes >8 hours, and has not yet been standardized.

[0014] 6. Imaging technology detection: positron emission tomography, single photon emission computed tomography, etc.

[0015] Limitations: The scarcity of equipment and high testing costs make this testing technology difficult to popularize.

[0016] Other emerging detection technologies, such as retinal imaging, saliva / tear testing, and digital biomarkers, are still in the testing phase, and their stability is difficult to guarantee. Regarding current AD detection technologies, the market lacks a new, safe, non-invasive, affordable, efficient, and sensitive detection technology. Summary of the invention:

[0017] In order to overcome the defects of the prior art, the object of the present invention is to provide a method for early diagnosis of Alzheimer's disease based on a magnetic immune microfluidic chip.

[0018] The present invention solves the technical problem by adopting the following technical solutions:

[0019] 1. An early diagnosis method for Alzheimer's disease based on a magnetic immune microfluidic chip, wherein the microfluidic chip comprises a chip body and solidified immunomagnetic beads, wherein the immunomagnetic beads are magnetic beads modified with fluorescent antibodies to Aβ1-42 and P-tau-181, and the surface of the chip body is provided with a plurality of detection channels, wherein one end of each channel is an injection hole (4) and the other end is connected to an outlet hole (5). A fixed capture area (2) is provided in the middle of each detection channel, an injection channel (1) is formed between the injection hole (4) and the fixed capture area, and an outlet channel (3) is formed between the fixed capture area (2) and the outlet hole (5). The diagnostic method comprises the following steps:

[0020] A. After washing and resuspending the streptavidin magnetic beads with PBS solution, add Aβ1-42 and P-tau-181 fluorescent antibodies and incubate. After incubation, wash again with PBS and resuspend, add BSA to block nonspecific binding sites, and finally wash with PBS to obtain the modified magnetic beads.

[0021] B. Use a syringe pump to inject 1% BSA solution into the chip channels, i.e., each injection well, for blocking. Then, inject 100 μl of resuspended Aβ1-42 and P-tau-181 fluorescent antibody-labeled magnetic beads into each channel.

[0022] C. After injecting the magnetic beads, wash the channel with PBS to remove the unfixed magnetic beads. After washing, observe the fixation of the magnetic beads under an inverted fluorescence microscope to confirm the fixation effect.

[0023] D. Pretreat the plasma sample to be tested and prepare the sample solution for detection; inject the sample solution into the chip injection hole through a microinjection pump or injection needle, and the liquid enters the fixed capture area through the channel; under a fluorescence microscope, use high-speed CCD to capture the fluorescence signal, and use image analysis software such as ImageJ to perform qualitative and quantitative analysis of Aβ1-42 and P-tau-181 to achieve dual-index detection.

[0024] There are five detection channels, and the sample outlet channels (3) of the detection channels are interconnected and connected to the sample outlet hole (5).

[0025] This invention takes the pathological changes of AD patients as the starting point to explore new detection methods. The core lesions of AD are abnormal deposition of Aβ and denaturation of tau protein. These pathological changes lead to significant changes in protein levels in the cerebrospinal fluid. Specifically, because Aβ is highly hydrophobic, it is more likely to aggregate into oligomers and deposit in brain tissue during the course of AD, thereby reducing the amount of free Aβ entering the cerebrospinal fluid; at the same time, the abnormal phosphorylation of tau protein causes it to form neurofibrillary tangles in neurons, and the level of P-tau in the cerebrospinal fluid increases accordingly.

[0026] As pathological proteins accumulate, chronic neuroinflammation and oxidative stress further damage the blood-brain barrier, allowing brain-derived Aβ and P-tau to enter the bloodstream via passive diffusion or exosomes. Simultaneously, brain Aβ clearance receptors (such as LRP1) are downregulated by 30% to 40%, exacerbating brain deposition. Aβ and P-tau clearance in peripheral blood is inefficient. Aβ is primarily taken up by hepatocytes via the low-density lipoprotein receptor (LRP1) and subsequently degraded. However, insulin-degrading enzyme activity is reduced in the liver of AD patients, resulting in a decreased rate of Aβ degradation. Furthermore, Aβ oligomers are too large to pass through glomerular filtration, and only monomeric Aβ can be excreted in the urine (accounting for 10% of total clearance). As for P-tau, oxidative stress and inflammation in the brains of AD patients impair the function of key components of the ubiquitin-proteasome system (E3 ubiquitin ligase), preventing its effective labeling and degradation. Furthermore, the human body lacks physiological neutralizing antibodies to neutralize P-tau in the blood. Therefore, the levels of Aβ and P-tau in peripheral blood are highly correlated with their levels in cerebrospinal fluid. Overall, the changes in the concentrations of the two proteins in the cerebrospinal fluid of AD patients are consistent, with the Aβ content in peripheral blood showing a downward trend and the P-tau content showing an upward trend. Related studies have found that the Aβ1-42 / P-tau-181 ratio is more scientific than a single indicator test. Fingerprint blood comes from capillaries, and capillaries and veins belong to the same systemic circulation. The blood components are basically the same after diffusion equilibrium. Several studies have confirmed that the concentrations of Aβ and P-tau in fingertip blood and venous blood are highly correlated. Relevant data show that the diagnostic sensitivity of collecting fingertip blood to detect the Aβ1-42 / P-tau-181 ratio is as high as 80% and the specificity is 85.1%.

[0027] The team focused on microfluidics. Microfluidics is a miniaturized, integrated, high-throughput, and highly sensitive detection method that has emerged worldwide in recent years. Microfluidics encompasses multiple disciplines, including physics, chemistry, biology, and engineering. Often combined with magnetic immunoassays, it rapidly detects specific antigens by manipulating microfluidics at the micrometer scale. This technology overcomes the time-consuming, material-consumable, and instrument-dependent shortcomings of traditional detection methods, offering significant potential for disease diagnosis, chemical and biological analysis, and the development of innovative medical devices.

[0028] Based on the above principle, the present invention intends to develop a magnetic immune microfluidic chip to detect the fingertip blood of AD patients, which is used to distinguish AD patients from normal people, and provide a new safe, non-invasive, low-cost, efficient and sensitive way for the early diagnosis of AD.

[0029] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0030] 1. The first joint development based on Aβ1-42 and P-tau-181 to achieve efficient detection of AD markers;

[0031] 2. It is designed with multiple independent detection channels, which can be used to draw standard curves and realize simultaneous detection of multiple samples;

[0032] 3. Combined with the immunofluorescence detection system, the detection can be completed within 30 minutes, significantly improving the detection efficiency, specificity and sensitivity without increasing the detection cost, and has good economic applicability and promotion value. Description of the drawings:

[0033] Figure 1 It is a structural schematic diagram of the magnetic immune microfluidic chip device of the present invention.

[0034] Numbers in the figure: 1 injection channel, 2 fixed capture area, 3 sample outlet channel, 4 injection hole, 5 sample outlet hole.

[0035] The present invention will be further described below through specific implementation methods in conjunction with the accompanying drawings. Specific implementation method:

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0037] The materials and reagents used in the following examples include:

[0038] Unless otherwise specified, other materials are conventional materials and can be obtained from commercial sources.

[0039]

[0040] Example 1:

[0041] Chip Fabrication: The chip body can be made of materials such as quartz glass, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC), or cycloolefin copolymer (COC). Its detection channel, injection hole, output hole, and fixed capture area can all be fabricated using conventional micromachining methods such as laser engraving.

[0042] In a Class 100,000 cleanroom environment, an engraving machine was used to carve five parallel detection channels into the center of a polymethyl methacrylate (PMMA) test sheet. Each channel has a sample inlet 4 at one end, which connects to the sample inlet channel 1, the fixed capture area 2, and the sample outlet channel 3 in sequence. The five sample outlet channels converge into a connecting channel and ultimately lead to a sample outlet 5.

[0043] The specific structural parameters are as follows:

[0044] There are five detection channels, arranged in parallel, with a width of 0.2mm and a depth of 20μm;

[0045] The injection and discharge holes are circular holes with a diameter of 2 mm and a depth of 25 μm.

[0046] The fixed capture zone is an elliptical structure with a length of 6 mm, a width of 3 mm, and a depth of 25 μm.

[0047] The length of the sample inlet channel is 10mm, the length of the sample outlet channel is 15mm, and the length of the horizontal connecting channel is 20mm;

[0048] The distance between injection holes of adjacent detection channels is 5mm, and the distance between capture areas is 2mm.

[0049] Example 2:

[0050] Detection method:

[0051] 1) Add 20 μl of streptavidin magnetic beads to a centrifuge tube containing 1 ml of PBS. Shake well and place on a magnetic rack. Allow the beads to settle and the liquid to clear before discarding the supernatant. Repeat this washing process three times to complete the bead wash.

[0052] 2) After washing, resuspend the magnetic beads in PBS, add 2 μl of Aβ1-42 fluorescent antibody and 2 μl of P-tau-181 fluorescent antibody, mix well, place on a shaker, and incubate overnight at 4°C.

[0053] 3) After incubation, wash the beads three times with PBS, resuspend in 1 ml of PBS, block with an appropriate amount of BSA, and incubate on a shaker at 25°C and 100 rpm for 30 minutes.

[0054] 4) After incubation, wash again three times, resuspend the magnetic beads in PBS, and store at 4°C until use.

[0055] 5) Immobilization of the immunomagnetic beads: Use a syringe pump to inject 1% BSA solution into the chip channels for blocking. Subsequently, inject 100 μl of resuspended Aβ1-42 and P-tau-181 fluorescent antibody-labeled magnetic beads into each channel. After injection, wash the channels with PBS to remove unfixed magnetic beads. After washing, observe the magnetic bead fixation under an inverted fluorescence microscope to confirm the fixation effect.

[0056] 6) Chip sealing test: randomly select the prepared chips, inject the colored test solution from the injection hole, and observe whether there is liquid seepage or leakage to evaluate the chip sealing performance.

[0057] 7) Chip detection function test: The human plasma sample to be tested is pretreated to prepare the sample liquid for detection; the sample liquid is injected into the chip sampling hole via a microinjection pump or injection needle, and the liquid enters the fixed capture area through the channel; under a fluorescence microscope, the fluorescence signal is captured by high-speed CCD imaging, and image analysis software such as ImageJ is used to perform qualitative and quantitative analysis of Aβ1-42 and P-tau-181 to achieve dual-index detection.

[0058] It should be noted that some parts not described in detail in the present invention belong to the common knowledge in the art, or can be directly purchased from the market, and those skilled in the art can obtain them without creative work. The specific connection method has extremely wide applications in this art or daily life and will not be described in detail here.

[0059] In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are mutually inconsistent. Although the embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art may make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An early diagnosis method for Alzheimer's disease based on a magnetic immune microfluidic chip, wherein the microfluidic chip comprises a chip body and solidified immunomagnetic beads, wherein the immunomagnetic beads are magnetic beads modified with Aβ1-42 and P-tau-181 fluorescent antibodies, and the surface of the chip body is provided with a plurality of detection channels, wherein one end of each channel is an injection hole (4) and the other end is connected to a sample outlet (5). A fixed capture area (2) is provided in the middle of each detection channel, an injection channel (1) is formed between the injection hole (4) and the fixed capture area, and a sample outlet channel (3) is formed between the fixed capture area (2) and the sample outlet (5), characterized in that: The diagnostic method comprises the following steps: A. After washing and resuspending the streptavidin magnetic beads with PBS solution, add Aβ1-42 and P-tau-181 fluorescent antibodies and incubate. After incubation, wash again with PBS and resuspend, add BSA to block nonspecific binding sites, and finally wash with PBS to obtain the modified magnetic beads. B. Use a syringe pump to inject 1% BSA solution into the chip channels, i.e., each injection well, for blocking. Then, inject 100 μl of resuspended Aβ1-42 and P-tau-181 fluorescent antibody-labeled magnetic beads into each channel. C. After injecting the magnetic beads, wash the channel with PBS to remove the unfixed magnetic beads. After washing, observe the fixation of the magnetic beads under an inverted fluorescence microscope to confirm the fixation effect. D. Pretreat the plasma sample to be tested and prepare the sample solution for detection; inject the sample solution into the chip injection hole through a microinjection pump or injection needle, and the liquid enters the fixed capture area through the channel; under a fluorescence microscope, use high-speed CCD to capture the fluorescence signal, and use image analysis software such as ImageJ to perform qualitative and quantitative analysis of Aβ1-42 and P-tau-181 to achieve dual-index detection.

2. The method for early diagnosis of Alzheimer's disease based on a magnetic immune microfluidic chip according to claim 1, characterized in that: There are five detection channels, and the sample outlet channels (3) of the detection channels are interconnected and connected to the sample outlet hole (5).

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