Test strip for detecting Alzheimer's disease and application thereof

Abstract

The invention discloses a test strip for detecting Alzheimer's disease and application thereof, the test strip adopts a colloidal gold-horseradish peroxidase compound labeling technology, the detection sensitivity is remarkably improved through enzyme signal amplification, and two key AD biomarkers p-Tau-181 and A beta 1-42 in blood can be synchronously detected. The test strip comprises a sample pad, a marking pad, a nitrocellulose membrane and absorbent paper, wherein the nitrocellulose membrane is provided with two detection lines (T1 and T2) for capturing p-Tau-181 and A beta1-42 respectively and a quality control line (C). The kit is high in sensitivity, rapid in detection, simple and convenient to operate, low in cost and suitable for primary medical treatment and large-scale screening scenes, and has important value in early diagnosis, popularization, prevention and control of the Alzheimer's disease.

Description

Technical Field

[0001] This application belongs to the field of biological detection technology, specifically relating to a test strip for detecting Alzheimer's disease and its application. Background Technology

[0002] Alzheimer's disease (AD), a neurodegenerative disease, has become a major public health challenge for aging societies worldwide. The core pathological features of AD are the abnormal deposition of β-amyloid protein (Aβ) in the brain, forming senile plaques, and the hyperphosphorylation of Tau protein (p-Tau) and its aggregation, forming neurofibrillary tangles. The abnormal expression of these two pathological markers is closely related to the occurrence and development of the disease and is also a key molecular basis for the clinical diagnosis of AD.

[0003] Currently, commonly used clinical methods for diagnosing Alzheimer's disease (AD) are mainly divided into three categories: imaging examinations, cerebrospinal fluid examinations, and blood biomarker examinations. However, all of these methods have significant limitations and cannot meet the needs for early, rapid, and convenient diagnosis. Imaging techniques, such as cranial magnetic resonance imaging (MRI) and positron emission tomography (PET), can aid in the diagnosis of Alzheimer's disease (AD) by observing the degree of hippocampal atrophy or using Aβ / PET imaging. However, these technologies are expensive, requiring highly specialized equipment and personnel, limiting their application to large tertiary hospitals and hindering their widespread adoption in primary healthcare institutions and community screening settings. Furthermore, imaging abnormalities typically only become significant in the middle to late stages of AD, making early disease warning difficult. Cerebrospinal fluid (CSF) testing, which involves obtaining CSF through lumbar puncture, detects Aβ levels within the fluid. 42 Changes in the concentrations of Aβ protein (the main pathogenic subtype) and p-Tau are currently considered one of the "gold standards" for early diagnosis of Alzheimer's disease (AD). However, lumbar puncture is an invasive procedure that carries risks of pain, infection, and even nerve damage, resulting in extremely low patient acceptance (especially among elderly patients). Furthermore, the procedure is complex and time-consuming, failing to meet the needs of rapid diagnosis or large-scale population screening. Traditional blood biomarker detection technologies, such as enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay, are also options. However, ELISA suffers from low sensitivity (difficulty capturing minute changes in biomarkers in the blood of early AD patients), long testing times, and cumbersome procedures. While CLIA offers improved sensitivity, it relies on large-scale instruments, preventing on-site, real-time testing, and its high cost limits its application in primary healthcare settings.

[0004] In the field of point-of-care testing, test strip technology has been widely used in areas such as blood glucose and infectious diseases due to its advantages of simple operation, rapid detection and low cost. However, its application in AD detection still faces key technical bottlenecks: insufficient sensitivity of biomarker detection. Traditional colloidal gold test strips rely on the physical color development signal of colloidal gold particles, which has weak signal intensity and is difficult to accurately capture changes in trace biomarkers, easily leading to false negative results.

[0005] Therefore, developing an AD test strip with high sensitivity and amplified detection signal for simultaneous detection of multiple biomarkers is of great clinical value and social significance for achieving early screening of AD, improving diagnostic efficiency, and reducing medical costs. It is also a current research hotspot and urgent need in the field of biodetection technology. Summary of the Invention

[0006] To address the issues of insufficient sensitivity and weak signals in the simultaneous detection of multiple biomarkers in early screening technologies for Alzheimer's disease, this application proposes a test strip for detecting Alzheimer's disease and its application. By labeling with a colloidal gold-horseradish peroxidase (HRP) complex, enzyme signal amplification technology is realized, effectively solving the problems of low accuracy and weak detection signals in the early diagnosis of AD in existing point-of-care testing methods.

[0007] This application was made based on the inventors' findings that: Tau is a normal microtubule-associated protein located inside neurons and is part of the neuronal cytoskeleton (microtubules). When excessive phosphate groups are added to Tau protein (phosphorylation), its properties change; p-Tau protein detaches from microtubules, loses its normal function, and becomes entangled with itself, forming neurofibrillary tangles. These tangles disrupt the neuronal transport system, ultimately leading to cell death. Aβ is a peptide fragment produced by enzymatic cleavage of amyloid precursor protein, and Aβ exists in different lengths. In the brains of AD patients, Aβ abnormally aggregates, forming insoluble amyloid plaques that deposit between neurons.

[0008] This application discovers the use of p-Tau-181 and Aβ 1-42 Combined detection, with the addition of enzyme amplification technology, can be used for early screening of Alzheimer's disease, improving sensitivity and specificity by enhancing the detection signal.

[0009] This application provides a test strip for detecting Alzheimer's disease. The test strip includes a base plate, a nitrocellulose membrane fixedly connected to the base plate, a labeling pad, and a sample pad for receiving samples. The nitrocellulose membrane is coated with test lines (T1, T2) and control lines (C), which are spaced apart. The labeling pad and absorbent paper are located on opposite sides of the nitrocellulose membrane and are both fixedly connected to it. The sample pad is fixedly connected to the labeling pad. The test strip also includes a reagent kit for detecting biomarkers of Alzheimer's disease, namely p-Tau-181 and Aβ. 1-42 The sample pad, marking pad, nitrocellulose membrane and absorbent paper are arranged from left to right, with 1-2 cm overlap between each pair to ensure good contact between the parts. The bottom of the test strip is a PVC base.

[0010] Preferably, the reagent kit uses a colloidal gold-horseradish peroxidase complex, wherein the colloidal gold surface is modified with horseradish peroxidase to achieve enzyme amplification technology; the reagent kit is coated on a labeling pad; the preparation method of colloidal gold is as follows: 1 mL of chloroauric acid solution (1%) and 99 mL of ultrapure water are added to a three-necked flask that has been soaked in aqua regia, washed and dried, and placed in an oil bath. The flask is heated and stirred until the solution boils, and then 2 mL of freshly prepared 1% trisodium citrate solution is quickly added. The solution turns black and then wine red. When the solution becomes completely transparent wine red, it is boiled and stirred for another 10 min to obtain 20 nm colloidal gold. After cooling to room temperature, it is stored at 4 °C.

[0011] Preferably, the colloidal gold surface is modified with horseradish peroxidase via covalent bonding or physical adsorption to form a colloidal gold-horseradish peroxidase complex. This complex is further labeled with anti-p-Tau-181 monoclonal antibody and anti-Aβ antibody. 1-42 Monoclonal antibodies and control antibodies were prepared. The labeling method was as follows: the pH of the colloidal gold-horseradish peroxidase complex solution was adjusted to the optimal pH using 0.1 M K₂CO₃ solution. 1 mL of the pH-adjusted colloidal gold-horseradish peroxidase complex solution was placed in each EP tube. 70 μL, 80 μL, 90 μL, and 100 μL of 100 μg / mL p-Tau-181 monoclonal antibody and Aβ were added to each tube, respectively. 1-42 Monoclonal antibodies were mixed thoroughly and reacted at room temperature for 10 min. 100 μL of 2M NaCl solution was added to each tube and allowed to stand for 1 h. The color changes in each tube were then observed.

[0012] Preferably, the method for modifying the colloidal gold surface with horseradish peroxidase includes: adjusting the colloidal gold solution to pH 7.0-8.5, adding horseradish peroxidase solution and stirring for 10-60 min, adding a stabilizer and centrifuging to purify, thereby obtaining colloidal gold particles with horseradish peroxidase surface modification.

[0013] Preferably, the stabilizer is bovine serum albumin (BSA), polyethylene glycol (PEG), or sodium caseinate, and the concentration of the stabilizer is 0.5% to 5%.

[0014] Preferably, the anti-p-Tau-181 monoclonal antibody binds to the p-Tau-181 protein to obtain an anti-p-Tau-181 protein complex; the anti-Aβ... 1-42 Monoclonal antibodies bind to β-amyloid protein to form anti-Aβ. 1-42 Complex.

[0015] Preferably, the control antibody is a rabbit IgG antibody.

[0016] Preferably, the detection lines include a first detection line (T1) and a second detection line (T2) that are independent of each other, wherein the first detection line (T1) is for capturing p-Tau-181 antibody and the second detection line (T2) is for capturing Aβ. 1-42 Antibody; the control line (C) is coated with goat anti-rabbit IgG antibody.

[0017] Preferably, the p-Tau-181-capturing antibody binds to an anti-p-Tau-181 protein complex, and the Aβ-capturing antibody... 1-42 Antibodies and anti-Aβ 1-42 Complex binding.

[0018] This application provides an application of the above-described test strip in the preparation of a reagent kit for detecting Alzheimer's disease.

[0019] The beneficial effects of the embodiments in this application are as follows: (1) The test strips of this application use markers for simultaneous combined detection: the test strips have two independent detection lines (T1 and T2) to simultaneously detect p-Tau-181 and Aβ. 1-42 Two key AD biomarkers improve the accuracy and reliability of diagnosis.

[0020] (2) Good stability and high repeatability: By optimizing the preparation process of colloidal gold-HRP complex, the stability of reagents and reaction consistency are improved, ensuring the reliability and repeatability of detection results.

[0021] (3) High sensitivity: By employing colloidal gold-horseradish peroxidase (HRP) complex labeling technology, enzyme signal amplification was achieved, significantly enhancing the intensity of the detection signal and enabling accurate capture of p-Tau-181 and Aβ in the blood. 1-42 Biomarkers effectively reduce the risk of false negatives and are suitable for early screening of Alzheimer's disease (AD). Attached Figure Description

[0022] Figure 1 Example 9 shows the color pattern in the antibody-labeled tube prepared for this application; Figure 2 This is a schematic diagram of the test strip assembly in Embodiment 1 of this application; Figure 3 This is a graph showing a good linear relationship between color signal intensity and concentration when the concentration of p-Tau-181 in Example 4 of this application is 40-200 pg / mL; Figure 4 Aβ in Embodiment 4 of this application 1-42 The color signal intensity showed a good linear relationship with the concentration when the concentration was between 120 and 600 pg / mL. Detailed Implementation

[0023] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0024] It should be noted that: in this document, the terms "comprising" or "including" are open-ended, meaning they include the content specified in this application but do not exclude other aspects; in this document, the terms "marker" or "biomarker" should be interpreted broadly, including any detectable biological indicator that can reflect an abnormal state, which may include genetic markers, species markers (genus markers), and functional markers. The meaning of genetic markers is not limited to existing genes that can be expressed as biologically active proteins, but also includes any nucleic acid fragment, which can be DNA or RNA, modified DNA or RNA, or unmodified DNA or RNA. Specifically, the biomarker proteins (e.g., p-Tau, Aβ) in this application are protein or nucleic acid fragments; the detection principle is as follows: A rapid detection strip for p-Tau and Aβ is prepared using a double-antibody sandwich combined with enzyme signal amplification technology. Antibodies and horseradish peroxidase are conjugated onto colloidal gold. The colloidal gold is immobilized on the test strip through the bridging effect of the target analytes. Then, HRP catalyzes the reaction of the substrate 3-amino-9-ethylcarbazole (AEC) and H2O2 to amplify the color signal. After the sample is added to the sample pad, it travels forward from the sample pad to the absorbent pad under capillary action, passing through the labeling pad and nitrocellulose membrane. In the positive sample, p-Tau-181 binds to the p-Tau-181 antibody in the labeling pad, forming an anti-p-Tau-181 protein complex; Aβ… 1-42 It binds to the Aβ monoclonal antibody in the labeling pad to form anti-Aβ. 1-42Complex; anti-p-Tau-181 protein complex, anti-Aβ 1-42 The complex and rabbit IgG antibody were chromatographically deposited onto a nitrocellulose membrane. The p-Tau-181 antibody on detection line T1 captured the anti-p-Tau-181 protein complex, forming p-Tau-181-anti-p-Tau-181 protein complex-colloidal gold-horseradish peroxidase; the Aβ antibody on detection line T2... 1-42 Antibody captures anti-Aβ 1-42 Complex, forming Aβ 1-42 -Anti-Aβ 1-42 The complex – colloidal gold-horseradish peroxidase; rabbit IgG-colloidal gold-horseradish peroxidase reaches the control line and is captured by goat anti-rabbit IgG on the control line, forming goat anti-rabbit IgG-rabbit IgG-colloidal gold-horseradish peroxidase, resulting in specific signal peaks on both the test line and the control line. Negative samples, however, do not contain the target antigen or contain trace amounts of it, and therefore cannot form a complex with the colloidal gold-horseradish peroxidase-labeled antibody on the labeling pad; thus, only the control line shows a specific signal peak.

[0025] Preparation Example 1 Solution preparation (1) Reagent preparation: H2O2, sucrose, Tween-20 polyethylene glycol (PEG), and sodium caseinate were purchased from Sinopharm Chemical Reagent Co., Ltd.; horseradish peroxidase (HRP) was purchased from Aladdin Reagent (Shanghai) Co., Ltd.; 3-amino-9-ethylcarbazole (AEC) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; Antibodies bound to colloidal gold: anti-β-amyloid antibody (6E10) was purchased from BioLegend (catalog number 824201), and anti-phosphorylated Tau (Thr231) antibody was purchased from Cell Signaling Technology (catalog number 4539S); membrane scratching antibodies: anti-β-amyloid 1-42 antibody (EPR18841) was purchased from Abcam (catalog number ab227378), anti-phosphorylated Tau (Ser396) antibody was purchased from Thermo Fisher Scientific (catalog number: PA5-27414), and goat anti-rabbit IgG was purchased from Jackson. ImmunoResearch (catalog number 111-035-003); all water used in this experiment was ultrapure water, and all chemical reagents were analytical grade.

[0026] (2) Solution preparation 1) Sample pad treatment solution: Weigh 0.5g BSA and 2.5g sucrose using an electronic analytical balance, add 40mL of PB (pH 7.4, 10 mM), transfer 227.5μL of Tween-20 with a pipette, add 10mL of 20x SSC, dissolve thoroughly, and store at 4℃.

[0027] 2) Labeling pad treatment solution: Weigh 0.5 g BSA and 2.5 g sucrose using an electronic analytical balance, add 50 mL PB (pH 7.4, 10 mM), transfer 227.5 μL Tween-20 with a pipette, dissolve completely, and store at 4°C.

[0028] Preparation Example 2 Preparation of colloidal gold Take 1 mL of chloroauric acid solution (1%) and 99 mL of ultrapure water and add them to a three-necked flask that has been soaked in aqua regia, washed and dried. Place the flask in an oil bath and heat and stir until the solution boils. Then quickly add 2 mL of freshly prepared 1% trisodium citrate solution. The solution will turn black and then wine red. When the solution is completely transparent wine red, continue to boil and stir for 10 minutes to obtain 20 nm colloidal gold. After cooling to room temperature, store at 4°C.

[0029] Preparation Example 3 Preparation of labeling pads (low concentration of BSA) (1) Colloidal gold-horseradish peroxidase complex: The colloidal gold solution was adjusted to pH 7.0, horseradish peroxidase solution was added and stirred for 10 min, and stabilizer BSA was added to make the concentration of BSA in the solution 0.5% to obtain colloidal gold-horseradish peroxidase complex solution. (2) Antibody labeling: The pH of the colloidal gold-horseradish peroxidase complex solution was adjusted to 8.5 with 0.1M K2CO3 solution. 1 mL of the pH-adjusted colloidal gold-horseradish peroxidase complex was placed in each EP tube. 70 μL, 80 μL, 90 μL, and 100 μL of 100 μg / mL p-Tau-181 monoclonal antibody and Aβ monoclonal antibody were added to each tube, respectively. The mixture was stirred evenly and reacted at room temperature for 10 min. 100 μL of 2M NaCl solution was added to each tube and allowed to stand for 1 h. (3) Antibody labeling results: The color change in each tube was red. TEM characterization showed that the colloidal gold particles were spherical, well-dispersed, uniform in size, and about 21 nm in diameter, indicating that antibody labeling was successful. (4) Preparation of labeling pads: Immerse the labeling pads in the labeling pad treatment solution for 10 min, resuspend the antibody markers obtained in the previous step in the labeling pad treatment solution, spray them onto the immersed labeling pads, freeze dry them in a freeze dryer for 3 h to prepare the labeling pads, and store them in a dry environment.

[0030] Preparation Example 4 Preparation of labeling pads (high concentration of BSA) (1) Colloidal gold-horseradish peroxidase complex: The colloidal gold solution was adjusted to pH 8.5, horseradish peroxidase solution was added and stirred for 10 min, and stabilizer BSA was added to make the concentration of BSA in the solution 5% to obtain colloidal gold-horseradish peroxidase complex solution. (2) Antibody labeling: The pH of the colloidal gold-horseradish peroxidase complex solution was adjusted to 8.5 with 0.1M K2CO3 solution. 1 mL of the pH-adjusted colloidal gold-horseradish peroxidase complex was placed in each EP tube. 70 μL, 80 μL, 90 μL, and 100 μL of 100 μg / mL p-Tau-181 monoclonal antibody and Aβ monoclonal antibody were added to each tube, respectively. The mixture was stirred evenly and reacted at room temperature for 10 min. 100 μL of 2M NaCl solution was added to each tube and allowed to stand for 1 h. (3) Antibody labeling results: The color change in each tube was red. TEM characterization showed that the colloidal gold particles were spherical, well-dispersed, uniform in size, and about 20 nm in diameter, indicating that antibody labeling was successful. (4) Preparation of labeling pads: Immerse the labeling pads in the labeling pad treatment solution for 10 min, resuspend the antibody markers obtained in the previous step in the labeling pad treatment solution, spray them onto the immersed labeling pads, freeze dry them in a freeze dryer for 3 h to prepare the labeling pads, and store them in a dry environment.

[0031] Preparation Example 5 Preparation of labeled pads (concentration in BSA) (1) Colloidal gold-horseradish peroxidase complex: The colloidal gold solution was adjusted to pH 7.8, horseradish peroxidase solution was added and stirred for 10 min, and stabilizer BSA was added to make the concentration of BSA in the solution 3% to obtain colloidal gold-horseradish peroxidase complex solution. (2) Antibody labeling: The pH of the colloidal gold-horseradish peroxidase complex solution was adjusted to 8.5 with 0.1M K2CO3 solution. 1 mL of the pH-adjusted colloidal gold-horseradish peroxidase complex was placed in each EP tube. 70 μL, 80 μL, 90 μL, and 100 μL of 100 μg / mL p-Tau-181 monoclonal antibody and Aβ monoclonal antibody were added to each tube, respectively. The mixture was stirred evenly and reacted at room temperature for 10 min. 100 μL of 2M NaCl solution was added to each tube and allowed to stand for 1 h. (3) Antibody labeling results: The color change in each tube was red. TEM characterization showed that the colloidal gold particles were spherical, well-dispersed, uniform in size, and about 19 nm in diameter, indicating that antibody labeling was successful. (4) Preparation of labeling pads: Immerse the labeling pads in the labeling pad treatment solution for 10 min, resuspend the antibody markers obtained in the previous step in the labeling pad treatment solution, spray them onto the immersed labeling pads, freeze dry them in a freeze dryer for 3 h to prepare the labeling pads, and store them in a dry environment.

[0032] Preparation Example 6 Preparation of labeling pads (low concentration of PEG) (1) Colloidal gold-horseradish peroxidase complex: The colloidal gold solution was adjusted to pH 7.0, horseradish peroxidase solution was added and stirred for 10 min, and stabilizer PEG was added to make the concentration of PEG in the solution 0.5% to obtain colloidal gold-horseradish peroxidase complex solution. (2) Antibody labeling: The pH of the colloidal gold-horseradish peroxidase complex solution was adjusted to 8.5 with 0.1M K2CO3 solution. 1 mL of the pH-adjusted colloidal gold-horseradish peroxidase complex was placed in each EP tube. 70 μL, 80 μL, 90 μL, and 100 μL of 100 μg / mL p-Tau-181 monoclonal antibody and Aβ monoclonal antibody were added to each tube, respectively. The mixture was stirred evenly and reacted at room temperature for 10 min. 100 μL of 2M NaCl solution was added to each tube and allowed to stand for 1 h. (3) Antibody labeling results: The color change in each tube was red. TEM characterization showed that the colloidal gold particles were spherical, well-dispersed, uniform in size, and about 20 nm in diameter, indicating that antibody labeling was successful. (4) Preparation of labeling pads: Immerse the labeling pads in the labeling pad treatment solution for 10 min, resuspend the antibody markers obtained in the previous step in the labeling pad treatment solution, spray them onto the immersed labeling pads, freeze dry them in a freeze dryer for 3 h to prepare the labeling pads, and store them in a dry environment.

[0033] Preparation Example 7 Preparation of labeling pads (high concentration of PEG) (1) Colloidal gold-horseradish peroxidase complex: Adjust the colloidal gold solution to pH 8.5, add horseradish peroxidase solution and stir for 10 min, add stabilizer PEG to make the concentration of PEG in the solution 5% to obtain colloidal gold-horseradish peroxidase complex solution. (2) Antibody labeling: The pH of the colloidal gold-horseradish peroxidase complex solution was adjusted to 8.5 with 0.1M K2CO3 solution. 1 mL of the pH-adjusted colloidal gold-horseradish peroxidase complex was placed in each EP tube. 70 μL, 80 μL, 90 μL, and 100 μL of 100 μg / mL p-Tau-181 monoclonal antibody and Aβ monoclonal antibody were added to each tube, respectively. The mixture was stirred evenly and reacted at room temperature for 10 min. 100 μL of 2M NaCl solution was added to each tube and allowed to stand for 1 h. (3) Antibody labeling results: The color change in each tube was red. TEM characterization showed that the colloidal gold particles were spherical, well-dispersed, uniform in size, and about 19 nm in diameter, indicating that antibody labeling was successful. (4) Preparation of labeling pads: Immerse the labeling pads in the labeling pad treatment solution for 10 min, resuspend the antibody markers obtained in the previous step in the labeling pad treatment solution, spray them onto the immersed labeling pads, freeze dry them in a freeze dryer for 3 h to prepare the labeling pads, and store them in a dry environment.

[0034] Preparation Example 8 Preparation of labeling pads (concentration in PEG) (1) Colloidal gold-horseradish peroxidase complex: The colloidal gold solution was adjusted to pH 7.8, horseradish peroxidase solution was added and stirred for 10 min, and stabilizer PEG was added to make the concentration of PEG in the solution 3% to obtain colloidal gold-horseradish peroxidase complex solution. (2) Antibody labeling: The pH of the colloidal gold-horseradish peroxidase complex solution was adjusted to 8.5 with 0.1M K2CO3 solution. 1 mL of the pH-adjusted colloidal gold-horseradish peroxidase complex was placed in each EP tube. 70 μL, 80 μL, 90 μL, and 100 μL of 100 μg / mL p-Tau-181 monoclonal antibody and Aβ monoclonal antibody were added to each tube, respectively. The mixture was stirred evenly and reacted at room temperature for 10 min. 100 μL of 2M NaCl solution was added to each tube and allowed to stand for 1 h. (3) Antibody labeling results: The color change in each tube was red. TEM characterization showed that the colloidal gold particles were spherical, well-dispersed, uniform in size, and about 20 nm in diameter, indicating that antibody labeling was successful. (4) Preparation of labeling pads: Immerse the labeling pads in the labeling pad treatment solution for 10 min, resuspend the antibody markers obtained in the previous step in the labeling pad treatment solution, spray them onto the immersed labeling pads, freeze dry them in a freeze dryer for 3 h to prepare the labeling pads, and store them in a dry environment.

[0035] Preparation Example 9 Preparation of labeling pads (low concentration of sodium caseinate) (1) Colloidal gold-horseradish peroxidase complex: The colloidal gold solution was adjusted to pH 7.0, horseradish peroxidase solution was added and stirred for 10 min, and sodium caseinate was added as a stabilizer to make the concentration of sodium caseinate in the solution 0.5% to obtain colloidal gold-horseradish peroxidase complex solution. (2) Antibody labeling: The pH of the colloidal gold-horseradish peroxidase complex solution was adjusted to 8.5 with 0.1M K2CO3 solution. 1 mL of the pH-adjusted colloidal gold-horseradish peroxidase complex was placed in each EP tube. 70 μL, 80 μL, 90 μL, and 100 μL of 100 μg / mL p-Tau-181 monoclonal antibody and Aβ monoclonal antibody were added to each tube, respectively. The mixture was stirred evenly and reacted at room temperature for 10 min. 100 μL of 2M NaCl solution was added to each tube and allowed to stand for 1 h. (3) Antibody marker results: The color change in each tube was red, as shown in the following figures. Figure 1 As shown, TEM characterization revealed that the colloidal gold particles were spherical, well-dispersed, uniform in size, and approximately 21 nm in diameter, indicating successful antibody labeling. (4) Preparation of labeling pads: Immerse the labeling pads in the labeling pad treatment solution for 10 min, resuspend the antibody markers obtained in the previous step in the labeling pad treatment solution, spray them onto the immersed labeling pads, freeze dry them in a freeze dryer for 3 h to prepare the labeling pads, and store them in a dry environment.

[0036] Preparation Example 10 Preparation of labeling pads (high concentration of sodium caseinate) (1) Colloidal gold-horseradish peroxidase complex: The colloidal gold solution was adjusted to pH 8.5, horseradish peroxidase solution was added and stirred for 10 min, and sodium caseinate was added as a stabilizer to make the concentration of sodium caseinate in the solution 5% to obtain colloidal gold-horseradish peroxidase complex solution. (2) Antibody labeling: The pH of the colloidal gold-horseradish peroxidase complex solution was adjusted to 8.5 with 0.1M K2CO3 solution. 1 mL of the pH-adjusted colloidal gold-horseradish peroxidase complex was placed in each EP tube. 70 μL, 80 μL, 90 μL, and 100 μL of 100 μg / mL p-Tau-181 monoclonal antibody and Aβ monoclonal antibody were added to each tube, respectively. The mixture was stirred evenly and reacted at room temperature for 10 min. 100 μL of 2M NaCl solution was added to each tube and allowed to stand for 1 h. (3) Antibody labeling results: The color change in each tube was red. TEM characterization showed that the colloidal gold particles were spherical, well-dispersed, uniform in size, and about 21 nm in diameter, indicating that antibody labeling was successful. (4) Preparation of labeling pads: Immerse the labeling pads in the labeling pad treatment solution for 10 min, resuspend the antibody markers obtained in the previous step in the labeling pad treatment solution, spray them onto the immersed labeling pads, freeze dry them in a freeze dryer for 3 h to prepare the labeling pads, and store them in a dry environment.

[0037] Preparation Example 11 Preparation of labeling pads (concentration in sodium caseinate) (1) Colloidal gold-horseradish peroxidase complex: The colloidal gold solution was adjusted to pH 7.8, horseradish peroxidase solution was added and stirred for 10 min, and sodium caseinate was added as a stabilizer to make the concentration of sodium caseinate in the solution 3% to obtain colloidal gold-horseradish peroxidase complex solution. (2) Antibody labeling: The pH of the colloidal gold-horseradish peroxidase complex solution was adjusted to 8.5 with 0.1M K2CO3 solution. 1 mL of the pH-adjusted colloidal gold-horseradish peroxidase complex was placed in each EP tube. 70 μL, 80 μL, 90 μL, and 100 μL of 100 μg / mL p-Tau-181 monoclonal antibody and Aβ monoclonal antibody were added to each tube, respectively. The mixture was stirred evenly and reacted at room temperature for 10 min. 100 μL of 2M NaCl solution was added to each tube and allowed to stand for 1 h. (3) Antibody labeling results: The color change in each tube was red. TEM characterization showed that the colloidal gold particles were spherical, well-dispersed, uniform in size, and about 20 nm in diameter, indicating that antibody labeling was successful. (4) Preparation of labeling pads: Immerse the labeling pads in the labeling pad treatment solution for 10 min, resuspend the antibody markers obtained in the previous step in the labeling pad treatment solution, spray them onto the immersed labeling pads, freeze dry them in a freeze dryer for 3 h to prepare the labeling pads, and store them in a dry environment.

[0038] Comparative Example Label pad preparation (enzyme-free signal amplification technique) (1) Antibody labeling: The pH of the colloidal gold solution was adjusted to 8.5 with 0.1M K2CO3 solution. 1 mL of the pH-adjusted colloidal gold-horseradish peroxidase complex was placed in each EP tube. 70 μL, 80 μL, 90 μL, and 100 μL of 100 μg / mL p-Tau-181 monoclonal antibody and Aβ monoclonal antibody were added to each tube, respectively. The mixture was stirred evenly and reacted at room temperature for 10 min. 100 μL of 2M NaCl solution was added to each tube and allowed to stand for 1 h. (2) Labeling results: Observe the color changes in each tube and perform TEM characterization. The colloidal gold particles are spherical, well dispersed, uniform in size, and have a particle size of about 16 nm, indicating that antibody labeling was successful. (3) Preparation of labeling pads: Immerse the labeling pads in the labeling pad treatment solution for 10 min, resuspend the antibody markers obtained in the previous step in the labeling pad treatment solution, spray them onto the immersed labeling pads, freeze dry them in a freeze dryer for 3 h to prepare the labeling pads, and store them in a dry environment. Example

[0039] Paper strip preparation and assembly (1) The chromatography strip used in this experiment consists of four parts, from left to right: sample pad, label pad, nitrocellulose membrane, and absorbent pad. Each pair overlaps by 1-2 cm to ensure good contact between the parts. The bottom of the strip is a PVC base plate. (2) Pretreatment of sample pads: The glass fiber membrane was immersed in the sample pad treatment solution (containing PB (pH 7.4, 10mM), 4 xSSC, 1% BSA, 5% sucrose, 0.5% Tween-20) for 30 min, dried in an oven at 37°C, and stored at room temperature. (3) Preparation of nitrocellulose membrane reaction membrane: Draw detection lines T1, T2, and control lines on the nitrocellulose membrane. Dilute the required detection line antibodies Aβ and p-Tau-181 to 2 mg / mL with antibody diluent. Adjust the membrane drawing and spraying device and spray onto the detection lines at a rate of 2.0 μL / cm. Dilute the required control line antibody goat anti-rabbit to 0.5 mg / mL with antibody diluent. Adjust the membrane drawing and spraying device and spray onto the control lines at a rate of 2.0 μL / cm. The antibody diluent formulation is: 1% BSA, 2% sucrose, and 0.1 Tween-100. Dry the sprayed nitrocellulose membrane at 37℃ for later use. (4) Then, attach the sample pad, marking pad, nitrocellulose membrane, and absorbent pad to the corresponding positions on the PVC base plate in sequence. Use a microcomputer-controlled automatic cutting machine ZQ2000 to cut the attached base plate into 4mm wide test strips, as shown below. Figure 2 As shown. Example

[0040] Sample detection enzyme reaction test Experimental Method: The Aβ and p-Tau-181 antigens to be tested were added to 25 mM HEPES buffer (pH 7.2). After thorough mixing and reaction for 10 min, 120 μL of the reaction solution was added to the sample pad of the test strip. Chromatography was performed for 20 min, followed by adding HEPES buffer to the sample pad to rinse the test strip. Results were read after 10 min. Due to the accumulation of colloidal gold complexes in the detection and control areas, a visible red band was formed. 80 μL of HRP substrate containing 0.05% AEC and 0.03% H2O2 in 50 mM HAC-NaAC buffer (pH 5.4) was added. The enzymatic reaction was carried out for 8 min. The experimental results were recorded by photographing with a digital camera and analyzed using ImageJ software.

[0041] Experimental results: After 8 minutes of enzymatic reaction, the red bands of the labeled pads prepared in Preparation Examples 3 to 11 all deepened in color, while the comparative examples showed no color change. This indicates that the horseradish peroxidase-modified colloidal gold amplified the signal and made the color more obvious. Example

[0042] Specificity comparison test (1) Forty Alzheimer's disease (AD) patients admitted between October 2023 and June 2024 were selected as the AD group, and another 40 healthy elderly individuals who underwent physical examinations during the same period were selected as the healthy group. The AD group consisted of 21 females and 19 males, aged 60–88 years (mean 75.53 ± 9.83 years). The healthy group consisted of 23 females and 17 males, aged 61–87 years (mean 76.50 ± 9.92 years). There were no statistically significant differences in general characteristics between the two groups (P>0.05), indicating comparability. This study complied with the requirements of the Declaration of Helsinki, and all participants provided informed consent.

[0043] (2) In both groups, 5 ml of venous blood was drawn in the morning on an empty stomach. The blood was allowed to coagulate for 15 min, then centrifuged at a radius of 16 cm and a speed of 4200 r / min. After 10 min, the serum was separated and stored at -80°C for later testing. The following methods were used: Group A: Detection of horseradish peroxidase modification by Aβ and p-Tau-181 combined; Group B: Detection of Aβ and p-Tau-181 without horseradish peroxidase modification; Group C: Detection of Aβ horseradish peroxidase modification; Group D: Detection of p-Tau-181 horseradish peroxidase modification; Two drops of serum samples were added to the sample pads of groups A, B, C, and D respectively, and the test results were observed. It should be noted that: if C shows color development, at least one T1 or T2 test result is positive; if C shows color development, neither T1 nor T2 test result is negative; if C does not show color development, the result is invalid.

[0044] (3) The test results of different groups are shown in Table 1. Statistical analysis was performed using SPSS 25.0 software. Measurement data are presented as follows: The data are expressed as t-tests. Count data are expressed as rates. The positive detection rate was calculated as follows: P < 0.05 indicates statistical significance. The positive detection rate was calculated as: (Number of positive cases / Total number of cases) × 100%. Table 1 Test Results

[0045] (4) Experimental results: Based on the above data, the positive detection rate of group A was higher than that of the other three groups, with a positive detection rate of 97.5%. The specificity was strong, and the test strip modified with Aβ and p-Tau-181 combined with horseradish peroxidase had the best effect.

[0046] (5) Selectivity: Using several common tumor markers and proteins such as CEA, HCG, IgG, GSH, and BSA as interfering agents, the results showed that they had virtually no interference with the signals of Aβ and p-Tau-181. Even when the concentration of these proteins was 100 times that of Aβ and p-Tau-181, the response of Aβ and p-Tau-181 to the detection line was far superior to that of these interfering substances. Therefore, this method has good selectivity for Aβ and p-Tau-181. Example

[0047] Sensitivity test (1) To verify the sensitivity of this detection method, different concentrations of Aβ and p-Tau-181 standard samples (Aβ: 30, 60, 90, 120, 150, 300, 450, 600, 800 and 1000 pg / mL; p-Tau-181: 10, 20, 30, 40, 50, 100, 200, 300, 600 and 1000 pg / mL) were detected using this sensor. The results showed that as the concentrations of Aβ and p-Tau-181 increased, the color of the detection line gradually deepened. The p-Tau-181 and Aβ data are shown in Table 2. When the p-Tau-181 concentration was between 40-200 pg / mL, the color signal intensity showed a good linear relationship with the concentration (R² = 0.997). Figure 3 As shown, the color signal intensity exhibits a good linear relationship with concentration (R² = 0.996) when Aβ concentration is between 120-600 pg / mL. Figure 4As shown, when the concentration of p-Tau-181 is higher than 200 pg / mL and the concentration of Aβ is higher than 600 pg / mL, the color deepening of the detection line becomes slower. This is because there is too much Aβ and p-Tau-181, and the binding eventually approaches saturation.

[0048] Table 2 p-Tau-181, Aβ data

[0049] (2) Experimental results: Sensitivity test results showed that the detection limit of p-Tau-181 was 40 pg / mL and the detection limit of Aβ was 120 pg / mL.

[0050] In summary, the Alzheimer's disease test strip developed in this application combines high sensitivity, high specificity, convenience, and low cost through colloidal gold-horseradish peroxidase enzyme signal amplification and simultaneous detection of dual biomarkers. It can effectively meet the needs of early screening for AD and application in primary healthcare, and is of great significance for promoting the popularization of AD diagnostic technology and improving the level of public health prevention and control.

[0051] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. A test strip for detecting Alzheimer's disease, characterized in that, The test strip includes a base plate, a nitrocellulose membrane fixedly connected to the base plate, a labeling pad, and a sample pad for receiving the sample. The nitrocellulose membrane is coated with detection lines (T1, T2) and control lines (C), which are spaced apart. The labeling pad and absorbent paper are located on opposite sides of the nitrocellulose membrane and are both fixedly connected to it. The sample pad is fixedly connected to the labeling pad. The test strip also includes a reagent kit for detecting biomarkers of Alzheimer's disease, namely p-Tau-181 and Aβ. 1-42 .

2. The test strip for detecting Alzheimer's disease as described in claim 1, characterized in that, The reagent kit uses a colloidal gold-horseradish peroxidase complex, wherein the colloidal gold surface is modified with horseradish peroxidase to achieve enzyme amplification technology; the reagent kit is coated on a labeling pad.

3. The test strip for detecting Alzheimer's disease as described in claim 1, characterized in that, The colloidal gold surface is modified with horseradish peroxidase via covalent bonding or physical adsorption to form a colloidal gold-horseradish peroxidase complex. This complex is further labeled with anti-p-Tau-181 monoclonal antibody and anti-Aβ antibody. 1-42 Monoclonal antibodies and control antibodies.

4. The test strip for detecting Alzheimer's disease as described in claim 3, characterized in that, The method for modifying the colloidal gold surface with horseradish peroxidase includes: adjusting the colloidal gold solution to pH 7.0-8.5, adding horseradish peroxidase solution and stirring for 10-60 min, adding a stabilizer and centrifuging to purify, thereby obtaining colloidal gold particles with horseradish peroxidase surface modification.

5. The test strip for detecting Alzheimer's disease as described in claim 4, characterized in that, The stabilizer is bovine serum albumin (BSA), polyethylene glycol (PEG), or sodium caseinate, and the concentration of the stabilizer is 0.5% to 5%.

6. The test strip for detecting Alzheimer's disease as described in claim 3, characterized in that, The anti-p-Tau-181 monoclonal antibody binds to the p-Tau-181 protein to obtain an anti-p-Tau-181 protein complex; the anti-Aβ... 1-42 Monoclonal antibodies bind to β-amyloid protein to form anti-Aβ. 1-42 Complex.

7. The test strip for detecting Alzheimer's disease as described in claim 3, characterized in that, The control antibody was a rabbit IgG antibody.

8. The test strip for detecting Alzheimer's disease as described in claim 1, characterized in that, The detection lines include a first detection line (T1) and a second detection line (T2) that are independent of each other. The first detection line (T1) is for capturing p-Tau-181 antibody, and the second detection line (T2) is for capturing Aβ. 1-42 Antibody; the control line (C) is coated with goat anti-rabbit IgG antibody.

9. The test strip for detecting Alzheimer's disease as described in claim 8, characterized in that, The captured p-Tau-181 antibody binds to the anti-p-Tau-181 protein complex, and the captured Aβ... 1-42 Antibodies and anti-Aβ 1-42 Complex binding.

10. The use of a test strip as described in any one of claims 1-9 in the preparation of an Alzheimer's disease diagnostic kit.

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