A portable device for AQP4-IgG instant detection and its preparation method and application

Abstract

The present application relates to the technical field of in vitro detection, in particular to a portable device for instant detection of AQP4-IgG and a preparation method and application thereof. The portable device for instant detection of AQP4-IgG comprises a lateral flow immunochromatography test strip; a reaction pad of the lateral flow immunochromatography test strip is coated with gelated cells overexpressing AQP4 protein on a detection line. The device further comprises a blocking solution for mixing and incubating with a sample to be detected, and the blocking solution is a dispersion of the gelated cells. The back of the position where the detection line of the reaction pad is located is modified with Nafion-CNF hybrid hydrogel. The technical scheme can solve the technical problem that the prior art lacks a rapid, accurate and low-cost AQP4-IgG detection method. The present application is based on cell transfection, intracellular gelation and lateral flow immunochromatography technology, and can complete the detection within 10 minutes, with a detection limit as low as 2 ng / mL, and has an ideal application prospect.

Description

Technical Field

[0001] This invention relates to the field of in vitro detection technology, specifically to a portable device for point-of-care testing of AQP4-IgG, its preparation method, and its application. Background Technology

[0002] Rapid and accurate detection of aquaporin 4 antibodies (AQP4-IgG, anti-aquaporin 4 immunoglobulin G) plays a crucial role in the clinical diagnosis and treatment of neuromyelitis optica spectrum disorders (NMOSD).

[0003] NMOSD, an inflammatory disease affecting the central nervous system, has unique symptoms. It belongs to the category of immune-mediated inflammatory demyelinating diseases of the central nervous system, primarily damaging the optic nerve, spinal cord, and specific areas of the brain. The disease is characterized by relapsing optic neuritis and myelitis. Patients not only experience symptoms of optic neuritis such as decreased vision and abnormal visual evoked potentials, but may also exhibit symptoms of spinal cord damage, often extending to more than three spinal cord segments. It can even present with intractable hiccups, nausea, vomiting, and other symptoms of zone posterior syndrome. More seriously, its rates of blindness and disability are significantly higher than those of multiple sclerosis (MS), severely impacting patients' quality of life.

[0004] Serological studies have provided important clues for the diagnosis and pathological mechanism of NMOSD. Research shows that approximately 70%-80% of NMOSD patients are positive for AQP4-IgG. This antibody has a specific mechanism of action; it recognizes the orthogonal arrays of particles (OAP) formed by AQP4 protein tetramers at the foot processes of astrocytes on the cell membrane surface, thereby triggering complement-dependent cytotoxicity. This pathological mechanism is significant, not only explaining the high relapse rate of NMOSD but also highlighting the fundamental differences between it and MS in terms of treatment strategies and prognostic assessment. In terms of treatment, NMOSD may require more precise intervention targeting the immune response; and in terms of prognostic assessment, due to its high relapse rate, longer-term and closer follow-up observation is necessary.

[0005] It is worth noting that the OAP conformation of the native AQP4 protein is a necessary condition for its antibody binding. This conformation dependence directly leads to differences in the sensitivity of different detection methods. Currently common detection methods, such as cell-based CBA and enzyme-linked immunosorbent assay (ELISA), have different abilities to maintain the conformation of the AQP4 protein, thus affecting the detection sensitivity. The CBA method recommended by international NMOSD diagnosis and treatment guidelines has a sensitivity between 72.4% and 90% and a specificity of up to 100%, effectively maintaining the native conformation of the AQP4 protein. However, this method has many limitations. Its operation procedure is complex, requiring professional technicians and a specific experimental environment; the detection cost is high, including reagent costs and equipment usage costs; and it is dependent on imported reagents, which makes it difficult for primary healthcare institutions to obtain reagents. These factors severely restrict the clinical application of the CBA method in primary healthcare institutions, making it difficult for patients at the grassroots level to enjoy accurate testing services. Therefore, developing a rapid, accurate, and low-cost AQP4-IgG detection method is of great practical significance for improving the early diagnosis rate of NMOSD and the level of diagnosis and treatment at the grassroots level. Summary of the Invention

[0006] The present invention aims to provide a portable device for point-of-care testing of AQP4–IgG, in order to solve the technical problem that there is a lack of rapid, accurate and low-cost AQP4–IgG detection methods in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A portable device for point-of-care detection of AQP4-IgG includes a lateral flow immunochromatographic test strip; the detection line of the reaction pad of the lateral flow immunochromatographic test strip is coated with gelled cells overexpressing AQP4 protein.

[0008] Furthermore, gelled cells overexpressing AQP4 protein were prepared by the following method: engineered cells transfected with the AQP4 gene were prepared; the cells transfected with the AQP4 protein gene were resuspended in a methacrylamide hyaluronic acid solution, and after freezing and thawing, they were washed and resuspended, and then photocured to obtain gelled cells overexpressing AQP4 protein.

[0009] Furthermore, engineered cells transfected with the AQP4 gene were prepared by the following method: constructing a lentiviral expression vector integrating the human AQP4-M23 gene; using the lentiviral expression vector to package the virus and prepare viral fluid; infecting HEK-293T cells with the viral fluid to obtain engineered cells transfected with the AQP4 gene. Prepare a cell hydrogel solution containing 1×10⁻⁶ cells. 6 -1×10 8Engineered cells transfected with the AQP4 gene were prepared at a concentration of 1 cell / mL, along with 2-3% (w / v) of methacrylamide hyaluronic acid and 5-10% (w / v) of dimethyl sulfoxide. The hydrogel solution was first incubated at 2-8°C for 15-30 min, then frozen at -75 to -85°C for >8 h, followed by thawing at 37-40°C for 15-30 min to obtain a freeze-thawed cell hydrogel solution. The freeze-thawed cell hydrogel solution was centrifuged and washed to obtain hydrogel-treated engineered cells. The hydrogel-treated engineered cells were resuspended and photocured with 405 nm ultraviolet light for 1-2 min to obtain gelled cells overexpressing the AQP4 protein.

[0010] Furthermore, a portable device for point-of-care detection of AQP4-IgG also includes gelled cells for incubation with the sample to be tested; The gelled cells were prepared by mixing untransfected cells, methacrylamide hyaluronic acid, and dimethyl sulfoxide to form a hydrogel solution. After freezing and thawing, the cells were washed and resuspended, and then photocured to obtain gelled cells.

[0011] Furthermore, gelled cells were added to the sample to be tested, and the final density of the gelled cells was 10. 6 -10 8 Cells / mL; the sample to be tested and the gelled cells were incubated together for 5-10 min at a temperature of 20-30℃.

[0012] Furthermore, the binding pad of the lateral flow immunochromatographic test strip is coated with labeled antibodies; The labeled antibody is composed of a marker and a molecule for specific binding to AQP4-IgG; The marker is selected from at least one of colloidal gold nanoparticles, colored latex microspheres, fluorescent microspheres, and magnetic microspheres; The molecule used for specific binding to AQP4-IgG is selected from at least one of the following: monoclonal antibody for binding to AQP4-IgG, polyclonal antibody for binding to AQP4-IgG, antibody fragment for binding to AQP4-IgG, genetically engineered antibody for binding to AQP4-IgG, and nanobody for binding to AQP4-IgG. Monoclonal antibodies, polyclonal antibodies, antibody fragments, genetically engineered antibodies, and nanobodies are all common antibody forms in existing technologies and are widely used in the binding pads of lateral flow immunochromatographic test strips. These antibodies are typically covalently coupled with markers (such as colloidal gold, fluorescent microspheres, etc.) to specifically recognize and bind to target molecules (e.g., AQP4-IgG) in the sample, thereby imparting a labeling signal to the target molecule. This labeling signal can then be visualized on the test line (T line) and control line (C line), used to determine the presence of the target molecule and verify that the detection system is functioning correctly, respectively.

[0013] Preferably, the labeled antibody is a colloidal gold-labeled antibody; the colloidal gold-labeled antibody is formed by conjugating colloidal gold nanoparticles with a monoclonal antibody for binding to AQP4-IgG.

[0014] Furthermore, the control line of the reaction pad of the lateral flow immunochromatographic test strip is coated with molecules for binding to the labeled antibody; Preferably, the labeled antibody is a colloidal gold-labeled antibody; the molecule used to bind to the colloidal gold-labeled antibody is goat anti-mouse IgG, which specifically binds to the monoclonal antibody in the colloidal gold-labeled antibody.

[0015] Furthermore, the control line of the reaction pad of the lateral flow immunochromatographic test strip is coated with goat anti-mouse IgG; the goat anti-mouse IgG is used to bind to the monoclonal antibody in the colloidal gold-labeled antibody.

[0016] Furthermore, the back side of the reaction pad at the location of the detection line is modified with Nafion-CNF hybrid hydrogel; the Nafion-CNF hybrid hydrogel contains Nafion at a final concentration of 0.1-1% by volume and CNF at a final concentration of 0.1-1% by mass. Preferably, the Nafion-CNF hybrid hydrogel contains Nafion at a final concentration of 0.2-0.3% by volume and CNF at a final concentration of 0.1-0.2% by mass.

[0017] This technical solution also provides the application of a portable device for the point-of-care detection of AQP4-IgG in the preparation of products for the diagnosis of neuromyelitis optica spectrum disorders.

[0018] This technical solution also provides a method for preparing a portable device for the point-of-care detection of AQP4-IgG, wherein a sample pad, a buffer pad, a conjugate pad, a reaction pad, and an absorbent pad are sequentially fixed on a back plate; The method for coating the conjugate pad with colloidal gold-labeled antibody is as follows: a solution of colloidal gold-labeled antibody with a concentration of 10-50 μg / mL is added dropwise to the conjugate pad, and then dried. The method for modifying the reaction pad with Nafion-CNF hybrid hydrogel is as follows: apply Nafion-CNF hybrid hydrogel at a rate of 0.1-2.0 μL / cm to the back of the detection line on the reaction pad, and dry at 20-30℃ for 8-12 hours; preferably, the amount of Nafion-CNF hybrid hydrogel used is 0.7-0.9 μL / cm. The method for drawing the detection line on the reaction pad is as follows: using a dosage of 0.1-2.0 μL / cm, the dispersion of gelled cells overexpressing AQP4 protein is drawn on the back of the modified line of the Nafion-CNF hybrid hydrogel on the reaction pad, and dried at 20-30℃ for 8-12 hours; preferably, the dosage of the dispersion of gelled cells overexpressing AQP4 protein is 0.7-0.9 μL / cm. The cell density in the dispersion of gelled cells overexpressing AQP4 protein was 10. 6 -10 8 cells / mL; The method for drawing control lines on the reaction pad is as follows: apply 0.1-2.0 μL / cm of goat anti-mouse IgG solution to the reaction pad and dry it at 20-30℃ for 8-12 hours; preferably, the amount of goat anti-mouse IgG solution used is 0.7-0.9 μL / cm. The concentration of sheep anti-mouse IgG in the sheep anti-mouse IgG solution is 1-2 mg / mL.

[0019] This invention utilizes the synergistic effect of precise cell epitope conformation maintenance technology and lateral flow immunochromatographic rapid detection technology to construct a portable device for the point-of-care detection of AQP4-IgG. The technical principle is as follows: (1) Gel cells overexpressing AQP4 protein The human AQP4-M23 gene was introduced into HEK-293T cells using lentiviral transfection technology, achieving efficient overexpression of the AQP4 protein. A composite process of embedding, freeze-thawing, and photocuring in methacryloyl hyaluronic acid hydrogel allowed the hydrogel matrix to permeate into the intercellular space and cross-link to form a three-dimensional network structure. This structure preserves the conformation of the AQP4 protein on the cell membrane surface, inhibits spontaneous distortion of its spatial conformation, and ensures that the AQP4 protein maintains its naturally formed orthogonal particle array. This conformation is a necessary prerequisite for the specific binding of AQP4-IgG. Furthermore, the gelled cells maintain structural integrity during storage and detection, further increasing the detection stability and accuracy of this portable device.

[0020] (2) Dual-effect regulation of Nafion-CNF hybrid hydrogel Nafion-CNF hybrid hydrogel was modified on the back of the test line of the lateral flow immunochromatographic test strip reaction pad. This hybrid material has two major functions: first, it utilizes the ion exchange properties of Nafion and the porous network structure of CNF to avoid the shrinkage and deformation of gelled cells caused by the loading environment of the paper-based material; second, it enhances the signal enrichment ability of the test line area through the hybrid hydrogel, reduces the non-specific diffusion of colloidal gold-labeled antibodies, and improves the intensity and clarity of the target signal.

[0021] (3) Specific detection of lateral flow immunochromatography The test strip's binding pad is coated with a colloidal gold-labeled monoclonal antibody, which specifically binds to AQP4-IgG in the sample. When the complex migrates with the sample solution to the test line, it is captured by the native conformation of AQP4 protein on the surface of gelled cells coated on the test line, forming a sandwich complex of "gelled cells-AQP4-IgG-colloidal gold antibody," which then appears as a visible colored band. The control line is coated with goat anti-mouse IgG, which captures unbound colloidal gold-labeled antibodies and is used to verify the effectiveness of the detection system. In addition, gelled cell blocking further eliminates interference and avoids false positive results.

[0022] The beneficial effects of this technical solution are as follows: (1) The detection sensitivity and specificity are significantly improved. This invention maintains the native conformation of the AQP4 protein through gelation treatment, combined with the signal enhancement effect of Nafion-CNF hybrid hydrogels, enabling the device to achieve a detection limit of as low as 1 ng / mL for AQP4-IgG, with sensitivity reaching the internationally recommended CBA level for cell-based ELISA. Simultaneously, the cell-blocking treatment through gelation effectively eliminates non-specific interference in the sample, ensuring detection specificity and solving the technical problem of insufficient sensitivity in traditional ELISA methods due to protein conformational disruption.

[0023] (2) Achieve rapid and real-time detection to adapt to grassroots application scenarios. This device requires no complex instruments or specialized operation. After sample addition, semi-qualitative interpretation can be completed within 10 minutes, significantly improving detection efficiency compared to the traditional CBA method (which takes 2-7 days) and ELISA method (which involves cumbersome steps and multiple incubation and washing processes). The device is a portable test strip structure that can operate at room temperature, requiring no special experimental environment. It overcomes the dependence of existing technologies on large instruments and specialized personnel, making it particularly suitable for rapid diagnosis in primary healthcare institutions and emergency settings.

[0024] (3) Excellent storage stability, reducing the cost of clinical application. The synergistic regulatory effect of the three-dimensional hydrogel network of gelled cells and the Nafion-CNF hybrid hydrogel enables the device to maintain a long shelf life, overcoming the drawbacks of the live cell method that requires cold chain transportation and continuous culture.

[0025] (4) The operation is simple and the results are easy to interpret. The detection process requires no complex sample pretreatment (only a brief incubation with gelled cells), and the colorimetric results can be interpreted with the naked eye. It does not require large equipment such as fluorescence microscopes or flow cytometers, reducing the technical requirements for operators and achieving the goal of instant detection with sample input and result output. This has important clinical significance for improving the early diagnosis rate of NMOSD and improving patient prognosis. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the bimodal engineered cell preparation process in Experiment Example 1 (A: engineered cells transfected with the target gene; B: engineered cells after hydrogel treatment; C: bimodal engineered cells).

[0027] Figure 2 The following are the Western blot (WB) results of the bimodal engineered cells in Example 1 (left side: WB image; right side: WB signal statistics, expressed as mean ± standard deviation, n=3; ordinate: ratio of target membrane protein grayscale value to internal control grayscale value; internal control is Na). + K + -ATPase; the target membrane protein is AQP4 protein; Sample A is HEK-293T cells that do not overexpress AQP4 protein; Sample B is HEK-293T cells that overexpress AQP4 protein but have not undergone intracellular gelation treatment; Sample C is bimodal engineered cells prepared according to this protocol, which overexpress AQP4 protein and have undergone intracellular gelation treatment; *** indicates P < 0.0001, ns indicates no significant difference).

[0028] Figure 3 The results show the morphological observations of the bimodal engineered cells and normal cells in Example 1.

[0029] Figure 4 The results of cell viability testing in Example 1 are as follows (the positive control is specifically normal HEK-293T cells; the negative control is specifically HEK293T cells with 10% DMSO added; the experimental results are expressed as mean ± standard deviation, n=5).

[0030] Figure 5The diagram below shows the structure of the paper-based chip layer in Example 2 (A: Paper-based chip layer structure; sample pad 1, buffer pad 2, bonding pad 3, PVC backing plate 4, reaction pad 5, absorption pad 6, detection line 7, quality control line 8, sample loading 9; B: Schematic diagram of the detection result judgment method, the appearance of bands on both detection line 7 and quality control line 8 indicates a positive result, and the appearance of a band only on quality control line 8 indicates a negative result).

[0031] Figure 6 The color intensity statistics of colloidal gold after treating NC membranes with different hydrogels in Example 3 are shown in the figure (A: control group; B: Gel-MA treatment group; C: SA-MA treatment group; D: CMCS-MA treatment group; E: Nafion-CNF hybrid hydrogel treatment group; statistical results are expressed as mean ± standard deviation, n=5).

[0032] Figure 7 The results of the experiment on the optimization of the coating concentration conditions of the colloidal gold-labeled antibody (Au-NPs@detection mAb) in Example 4 are shown (N represents negative sample; P represents positive sample).

[0033] Figure 8 The results of the experiment optimizing the gel cell coating concentration conditions in Example 5 (N represents negative sample; P represents positive sample).

[0034] Figure 9 The results of the specificity verification experiment in Example 6 are as follows (A: AQP4-IgG: aquaporin 4 antibody; B: AQP1-IgG: aquaporin 1 antibody; C: MOG-IgG: myelin oligodendrocyte glycoprotein antibody; D: RF: rheumatoid factor; E: HSA: human serum albumin).

[0035] Figure 10 The results are from the sensitivity verification experiment in Example 7.

[0036] Figure 11 The results are from the stability verification experiment of Example 8.

[0037] Figure 12 The results show the microscopic morphological observations of the dual-modal engineered cells in a comparative manner.

[0038] Figure 13 The results of confocal microscopy characterization experiments of dual-modal engineered cells are shown in the figure (A: fluorescence signal of green methacrylamide hyaluronic acid (HAMA) hydrogel, reflecting its distribution; B: orange fluorescent dye signal of target structure cells, using cell membrane dye DiI; C: bright field (BF) field, showing the morphology of the sample; D: fusion state, superposition of channels A, B, and C, observing the co-localization of HAMA and target structure).

[0039] Figure 14The fluorescence signal intensity statistics of the confocal microscopy characterization experiment of comparative dual-modal engineered cells are shown in the figure (the fluorescence integral density / area of ​​HAMA with and without DMSO is compared, i.e. the fluorescence signal intensity of HAMA per unit area; the statistical results are expressed as mean ± standard deviation, n=5; *** indicates P<0.0001). Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0041] To facilitate understanding of the technical solution, the key cells, reagents, and concepts involved in this solution are explained below: Lateral Flow Immunochromatographic Test Strip (LFICS) is a rapid detection device based on immunochromatography technology and the principle of lateral flow chromatography. It is widely used in point-of-care testing (POCT) scenarios in fields such as biomedicine, clinical diagnostics, food safety, and environmental monitoring. Using a nitrocellulose membrane as its core carrier, it consists of a sample pad, a conjugation pad, a nitrocellulose membrane containing a test line (T line) and a control line (C line), and an absorbent pad. The sample pad is responsible for adsorbing and pre-treating the sample. The conjugation pad is coated with specific probes labeled with colloidal gold, fluorescent microspheres, etc. These specific probes typically consist of labeling substances such as colloidal gold or fluorescent microspheres and substances for binding to the target analyte (e.g., monoclonal antibodies against the target analyte). The specific probes bind to and label the target analyte, forming a target-specific probe complex. The detection line on the nitrocellulose membrane immobilizes the capture probe, which is used to specifically bind the target-specific probe complex. The control line is used to verify the chromatography process and reagent effectiveness. The absorbent pad provides capillary driving force and absorbs excess sample. The working principle is that the sample flows laterally along the test strip under capillary action, first binding with the labeled probe on the conjugation pad to form a complex. When it flows through the detection line, it is enriched and colored by the capture probe. The unbound labeled probe continues to flow to the control line and is colored. Finally, the result is judged based on the color development of the T line and C line.

[0042] AQP4 protein (Aquaporin-4): Aquaporin 4 is an important member of the aquaporin (AQP) family, primarily expressed in the central nervous system (especially in the terminal foot membrane of astrocytes). AQP4 mediates the rapid transmembrane transport of water molecules and plays a crucial role in the formation and clearance of cerebral edema, neuroinflammation, and blood-brain barrier homeostasis. Its typical structure is a tetramer, with each monomer containing six transmembrane α-helices, exhibiting a highly selective ability to allow water molecules to pass through.

[0043] AQP4-IgG (Anti-Aquaporin-4 Immunoglobulin G): An anti-AQP4 autoantibody, it is an IgG-type autoantibody that specifically recognizes and binds to the AQP4 protein. AQP4-IgG is a key pathogenic biomarker and diagnostic criterion for neuromyelitis optica spectrum disorders (NMOSD). This antibody can damage astrocytes through mechanisms such as complement activation and antibody-dependent cytotoxicity (ADCC), leading to demyelination and nerve damage.

[0044] HEK-293T cells (Human Embryonic Kidney 293T cells): A human embryonic kidney epithelial cell line widely used in molecular biology and virology research, obtained by further transfecting HEK-293 cells and stably expressing the SV40 large T antigen.

[0045] The pCDH-MSCV-MCS-EF1-puro plasmid is a commonly used lentiviral expression vector developed by System Biosciences (SB). It is widely used for the stable and efficient expression of exogenous genes in mammalian cells, and for selection using puromycin. Its target gene insertion site is located at the multiple cloning site (MCS) downstream of the MSCV promoter, driven by a potent murine stem cell virus (MSCV) promoter; simultaneously, the puromycin resistance gene (Puro...) is inserted... r It is independently driven by the human EF1α promoter (elongation factor 1-α promoter) and is used for the selection of stable transduced cells.

[0046] psPAX2, pMD2.G, and pAdvantage are plasmids commonly used in molecular biology and viral vector construction (especially lentiviral packaging). psPAX2 is a lentiviral packaging plasmid that encodes several accessory proteins required for lentiviral replication and packaging. pMD2.G is an envelope plasmid, often used in conjunction with psPAX2. pAdvantage is an auxiliary expression plasmid used to increase viral yield. All three plasmids are commercially available.

[0047] PEI transfection reagent (polyethyleneimine) is a cationic polymer widely used in in vitro cell transfection, especially in viral packaging (such as lentiviruses and adeno-associated viruses) and transient protein expression, offering advantages such as high efficiency, economy, and ease of operation. It is commercially available.

[0048] PEG-it TM This reagent, developed by System Biosciences (SBI), is a commercially available formulation for concentrating viral particles (especially lentiviruses and retroviruses). Its main component is polyethylene glycol (PEG), along with other auxiliary components. It efficiently concentrates viral supernatants via precipitation, is easy to use, requires no ultracentrifugation, and is suitable for routine viral concentration needs in laboratories.

[0049] Protease Inhibitor Mixture: The full commercial name is Protein Phosphatase Inhibitor Mixture, which contains four independent protein phosphatase inhibitors that strongly inhibit the activity of almost all important protein phosphatases, including protein serine / threonine phosphatases (PP1, PP2A, PP2B, PP2C), protein tyrosine phosphatases (PTPs), alkaline phosphatases, acid phosphatases, etc.

[0050] HAMA (Hyaluronic Acid Methacryloyl): A photocrosslinked derivative obtained by introducing methacryloyl groups onto the hydroxyl or carboxyl groups of the hyaluronic acid (HA) molecule. HAMA can form hydrogels under ultraviolet or visible light initiation.

[0051] Gel-MA (Gelatin Methacryloyl): A derivative obtained by reacting the amino or hydroxyl groups in gelatin with reagents such as methacrylic anhydride to introduce photocrosslinkable methacryloyl groups.

[0052] SA-MA (Methacrylated Sodium Alginate): By modifying the hydroxyl groups in the sodium alginate molecule with methacrylamide, it is endowed with photocrosslinking ability.

[0053] CMCS-MA (Carboxymethyl Chitosan Methacryloyl): A photocrosslinkable derivative obtained by introducing methacryloyl groups into water-soluble carboxymethyl chitosan (CMCS).

[0054] Monoclonal antibodies are homologous antibodies secreted by a single B lymphocyte clone, targeting a single antigenic epitope. Their preparation relies heavily on hybridoma technology, which involves fusing B cells from immunized animals with myeloma cells, screening for hybridoma cell lines that stably secrete the target antibody, and then mass-producing them through in vitro culture or in vivo induction. The core advantages of these antibodies are extremely high specificity, high molecular homogeneity, and minimal batch-to-batch variation. They can accurately recognize specific epitopes of AQP4-IgG and are less prone to cross-reactivity with other proteins in the sample. They are the traditional preferred type of labeled antibody for AQP4-IgG point-of-care testing devices. The disadvantages are a longer preparation cycle and relatively higher production costs.

[0055] Polyclonal antibodies are heterogeneous mixtures of antibodies secreted by multiple B lymphocyte clones, targeting multiple different epitopes of the same antigen. Their preparation is relatively simple; experimental animals such as rabbits and sheep are directly immunized with AQP4-IgG. Once the antibody titer in the animal serum reaches a preset standard, blood is collected and the antibodies are separated from the serum through purification. These antibodies are characterized by low preparation cost, high antigen-binding affinity, and the ability to simultaneously bind to multiple epitopes of AQP4-IgG, improving the capture efficiency of the target analyte and meeting the needs of rapid detection. However, they suffer from poor specificity and significant batch-to-batch variability, and in complex sample matrices, non-specific binding may lead to false positive results.

[0056] Antibody fragments are molecular fragments obtained by cleaving and expressing intact antibody molecules using enzymatic digestion or gene recombination techniques. They retain antigen-binding function but are not complete antibody structures. Common types include Fab fragments (containing one antigen-binding site), F(ab')2 fragments (containing two antigen-binding sites), and scFv single-chain variable region fragments (composed of light chain variable regions and heavy chain variable regions linked by short peptides). The core advantage of these fragments is that their molecular weight is much smaller than that of intact antibodies, resulting in faster migration in lateral flow immunochromatographic test strips and significantly shortening detection time. Simultaneously, they lack the constant region (Fc segment), effectively reducing non-specific adsorption of Fc receptor proteins in the sample and improving detection accuracy. They are highly suitable for portable point-of-care testing devices. The disadvantage is that their structural stability is slightly lower than that of intact antibody molecules.

[0057] Genetically engineered antibodies are a general term for antibody molecules produced by artificially modifying and recombining the gene sequences of natural antibodies using molecular biology techniques such as gene recombination, gene editing, and phage display, and then expressing them in engineered cells (such as E. coli, CHO cells, and yeast cells). They encompass various types, including humanized antibodies, fully human antibodies, bispecific antibodies, and single-chain antibodies. These antibodies overcome the limitations of traditional monoclonal and polyclonal antibody production, which relies on animal immunization. They allow for targeted modification of antigen-binding sites to enhance affinity for AQP4-IgG, or to reduce the immunogenicity of murine proteins through humanization, thus minimizing detection interference. Furthermore, they can be produced on a large scale through fermentation in engineered cells, meeting industrialization needs. Including them within the scope of specific binding reagents in patent claims effectively expands the protection boundary of the technical solution.

[0058] Nanobodies are a special subtype of genetically engineered antibodies. Essentially, they are variable region fragments (VHH) of naturally occurring heavy chain antibodies found in camel (camel, alpaca) or shark animals. Their molecular weight is only about 1 / 10 that of traditional antibodies, making them the smallest known functional antigen-binding fragment. The preparation process involves immunizing alpacas or other animals with AQP4-IgG, extracting RNA from peripheral blood lymphocytes, amplifying the VHH gene using RT-PCR, screening with phage display technology, and expressing the target nanobody in engineered cells. The core advantages of these antibodies are their extremely small molecular weight, extremely high structural stability, resistance to acids, alkalis, high temperatures, and protease degradation, enabling them to withstand harsh testing environments such as outdoor activities and primary healthcare settings. Furthermore, they can bind to occult epitopes that are difficult for traditional antibodies to recognize, further enhancing detection specificity, making them a preferred option for next-generation AQP4-IgG point-of-care testing reagents.

[0059] Example 1: Preparation of gelled HEK-293T cells (G-AQP4-293T) overexpressing AQP4 protein (1) Construction of cell lines overexpressing AQP4 protein The cell line used for gelling in this study was HEK-293T (human embryonic kidney epithelial cells). HEK-293T is a highly efficient tool cell for transfection. After gelling, the transfected cells were integrated into the test strip detection line (T-line) to capture the target. The untransfected cells were gelled and used as control cells for blocking treatment to eliminate non-specific interference in the sample.

[0060] The mRNA sequence of the human AQP4-M23 gene (NCBI database ID: NM_001317384.3) was obtained from the NCBI database as a template to design and synthesize the target gene sequence for expressing the target protein (the specific sequence of the human AQP4 gene CDS fragment is shown in SEQ ID NO.1). A lentiviral vector carrying the puromycin N-acetyltransferase (Puro) gene was then constructed using this template. Specifically, the pCDH-MSCV-MCS-EF1-puro plasmid (System Biosciences, SBI) was used as the vector backbone, and the synthesized human AQP4 gene CDS fragment was inserted into the plasmid's multiple cloning site using conventional molecular cloning techniques to form the target gene expression plasmid. The human AQP4 gene CDS fragment (SEQ ID NO.1) is as follows:

[0061] Then, HEK-293T cells were transfected using a four-plasmid co-transfection system and PEI transfection reagent. The specific transfection procedure was as follows: 24 hours before transfection, HEK-293T cells in the logarithmic growth phase were transfected at a concentration of 2 × 10⁻⁶ cells / mL. 6 Seeds were placed at a density of cells / well into large culture dishes (10 cm), and 7 mL of DMEM high-glucose medium containing 10% fetal bovine serum (FBS) was added. The dishes were then incubated at 37°C in a 5% CO2 incubator. At transfection, cell confluence should reach 70%-90%, and cells should be morphologically uniform, firmly adherent, and free from significant contamination (microscopic observation showed no suspended impurities, abnormal cell aggregation, or lysis). The constructed recombinant plasmid (i.e., the aforementioned target gene expression plasmid, synthesized by General Biotechnology Co., Ltd.), psPAX2, pMD2.G, and pAdvantage plasmids were added to 500 µL of antibiotic- and serum-free DMEM medium at amounts of 10 µg, 7.5 µg, 2.5 µg, and 5 µg, respectively, and gently mixed. Simultaneously, 75 µL of PEI transfection reagent (1 mg / mL) was added to 500 µL of antibiotic- and serum-free DMEM medium, and the mixture was allowed to stand at room temperature for 5-10 min to allow the PEI to fully dissolve, forming a PEI solution. Then, the PEI solution is slowly added to the culture medium containing the plasmid, while gently blowing on the side wall of the centrifuge tube to ensure that the two solutions are fully mixed. After the addition is complete, continue blowing 3-4 times and let it stand at room temperature for 20-25 minutes to allow the plasmid and PEI to fully combine and form a complex, thus obtaining the plasmid-PEI complex.

[0062] Ten minutes before transfection, aspirate the cell culture medium from the culture dish and gently rinse the cell surface with serum-free DMEM medium. Then, add 6 mL of serum-free DMEM medium to the culture dish. Slowly add the plasmid-PEI complex, after allowing it to stand, to the corresponding cell culture medium. After adding, gently pipette the medium 1-2 times to ensure uniform dispersion of the complex. Return the culture dishes to a 37°C, 5% CO2 incubator and incubate statically. After 6 hours, replace with fresh medium (DMEM high-glucose medium containing 10% fetal bovine serum). Collect the culture supernatant containing virus particles at 48 and 72 hours after transfection, combine them, and centrifuge at 4°C, 3000×g for 10 minutes to remove cell debris. Add 1 / 4 volume of PEG-it to the supernatant. TM Reagent (lentivirus concentrate, System Biosciences), mix and incubate overnight (≥ 8 h) at 4°C. Centrifuge for 30 min at 4°C and 1500×g, discard the supernatant, and resuspend the virus pellet in PBS to a final concentration of 1×10⁻⁶. 8 Infectivity units / mL. Filter through a 0.45 μm filter, aliquot, and store at -80°C.

[0063] Next, cell transfection is performed. HEK-293T cells are seeded in 6-well (or 12-well, 24-well) plates until confluence reaches 30-60%. The original culture medium in the wells is discarded, and a virus titer of 1-5 × 10⁻⁶ is added. 5 Transfection began with complete culture medium (49500 μL DMEM high-glucose medium + 500 μL fetal bovine serum) at an infection unit / mL level. The medium was changed every 24 hours (no virus was added to the medium after the medium change, meaning the virus transfection time to cells was 24 hours; subsequent cell culture was performed using virus-free medium). After observing confluence at 48 and 72 hours, the cells were re-digested and plated. When the confluence reached 70-80%, puromycin was added at a final concentration of 1.5 μg / mL for selection. Stable cell lines expressing the target gene were obtained through continuous passage culture for subsequent experiments, resulting in engineered cells transfected with the target gene. More specifically, engineered cells stained with the AQP4 gene were obtained, i.e., engineered cells overexpressing the AQP4 protein on their surface.

[0064] (2) Intracellular gelation treatment Prepare a cell hydrogel solution containing engineered cells transfected with the AQP4 gene, 2% (w / v, optional 2-3%) methacrylamide hyaluronic acid (HAMA; EFL Tech, model EFL-HAMA-150K), and 5-10% (v / v, preferably 5%) dimethyl sulfoxide (DMSO) for co-incubation. The cell density is controlled at 1×10⁻⁶ cells / year. 7 Approximately 10 cells / mL (selectable range 1×10⁻⁶) 6 -1×10 8The cell hydrogel solution, consisting of 2-5 mL of cells / mL, is prepared by incubating at 4°C (optional range 2-8°C) for 30 min (optional range 15-30 min), then at -80°C (optional range -75 to -85°C) overnight for approximately 10 h (generally >8 h, preferably 8 h-30 days, further preferably 8 h-12 h; if inconvenient to operate, storage under these conditions can be up to 30 days). The cells are then thawed in a water bath at 37°C (optional range 37-40°C) for 30 min (optional range 15-30 min) to obtain the hydrogel-treated engineered cells in the freeze-thawed cell hydrogel solution. Next, the freeze-thawed cell hydrogel solution is centrifuged at 1200 rpm for 5 min (optional range 1200-1500 rpm for 3-5 min). Add 5 mL (optional range 5-8 mL) of PBS to resuspend the lower cell pellet, centrifuge at 800 rpm for 5 min (optional range 800-1200 rpm, 3-5 min), and repeat the washing process 3 times (can be repeated 2-3 times). After washing, add an equal volume of PBS to resuspend the cells. Finally, light-cur at 405 nm for 2 min (optional range 1-2 min). Finally, add a protease inhibitor mixture at a volume ratio of 1:200 (optional range 1:100-200), and store at 4°C for subsequent use. After the above steps, the bimodal engineered cells of this scheme are obtained, more specifically, bimodal engineered cells overexpressing AQP4 protein on the cell surface, that is, gelled HEK-293T cells (G-AQP4-293T) overexpressing AQP4 protein in this embodiment are obtained.

[0065] This technical solution also prepares gelled HEK-293T cells (G-293T), which are prepared in the same way as gelled HEK-293T cells overexpressing AQP4 protein (G-AQP4-293T), except that the starting cells are changed from engineered cells transfected with the AQP4 gene to HEK-293T cells.

[0066] A dual-modal engineered cell was constructed using a synergistic strategy of cell transfection and intracellular gelation. The schematic diagram of its preparation process is shown below. Figure 1 As shown, the preparation process of bimodal engineered cells includes: firstly, cell membrane protein overexpression is performed. Ordinary cells are taken and transfected to carry the target gene, forming engineered cells capable of overexpressing specific membrane proteins. Then, the engineered cells undergo freeze-thaw treatment, with DMSO and hydrogel added simultaneously, allowing the hydrogel to penetrate the cells, forming hydrogel-treated engineered cells. Finally, photocuring is performed to form the finished bimodal engineered cell product.

[0067] Engineered cells transfected with the target gene (HEK-293T cells overexpressing AQP4 protein but not subjected to intracellular gelation) and dual-modal engineered cells (cells expressing AQP4 protein and subjected to gelation) were prepared according to the preferred method of this embodiment, and then subsequent analyses were performed. Western blot analysis showed that transfection treatment induced overexpression of the target membrane protein on the cell surface (p < 0.01), and after intracellular gelation treatment, the overexpressed membrane protein was not significantly different from that before treatment. For detailed experimental results, please refer to [link to relevant documentation]. Figure 2 .

[0068] Continuous microscopic observation revealed that the bimodal engineered cells, after cell transfection and intracellular gelation, exhibited significantly better morphological maintenance than normal cells: normal cells showed obvious morphological damage after 12 hours, while the bimodal engineered cells retained intact cell morphology even after 72 hours. See detailed experimental results below. Figure 3 In this case, normal cells or bimodal engineered cells are preserved in a mixture of protease inhibitors, as described in “(2) Intracellular gelation treatment”.

[0069] CCK8 cell viability assays revealed that bimodal engineered cells, after transfection and intracellular gelation, lacked the metabolic activity of normal cells. This indicates that when used for drug delivery or selective bioelectronic affinity sensing, they will not induce endocytosis, generate metabolites, undergo apoptosis, structural disintegration, or interfere with the in vivo environment or detection. For detailed experimental results, please refer to [link to relevant documentation]. Figure 4 .

[0070] Example 2: Test strip assembly and usage method The portable device for AQP4-IgG point-of-care testing consists of a shell formed by the snap-fit ​​of a top and bottom layer, and a middle paper-based chip layer. The shell is made of modified plastic or polyvinyl chloride. See the paper-based chip layer design for details. Figure 5 A. The paper-based chip layer, from left to right in the order of liquid flow, includes sample pad 1, buffer pad 2, binding pad 3, reaction pad 5, and absorbent pad 6. Sample pad 1, buffer pad 2, binding pad 3, reaction pad 5, and absorbent pad 6 are all adhered to a PVC backing plate 4 (PVC is polyvinyl chloride, a commonly used thermoplastic polymer material, used as the support backing for this device). The right end of sample pad 1 overlaps with the left end of buffer pad 2, and the right end of sample pad 1 is on top. The right end of buffer pad 2 overlaps with the left end of binding pad 3, and the right end of buffer pad 2 is on top. The left end of reaction pad 5 overlaps with the right end of binding pad 3, and the right end of reaction pad 5 overlaps with the left end of absorbent pad 6, and reaction pad 5 is located below binding pad 3 and absorbent pad 6. The following provides a detailed description of each part: (1) Sample pad 1, buffer pad 2 and absorbent pad 6 Sample pad 1 is made using a separation membrane (plasma separation membrane, purchased from Jieyi Biotechnology) to retain control cells (e.g., gelled HEK-293T cells, G-293T) in the mixture formed by the sample and blocking solution. Buffer pad 2 uses a separation membrane (plasma separation membrane, purchased from Jieyi Biotechnology) to prevent control cells (e.g., gelled HEK-293T cells, G-293T) from entering subsequent areas. Absorbent pad 6 uses cotton pulp paper to absorb residual liquid after the reaction.

[0071] (2) Combination pad 3 Binding pad 3 uses a glass fiber membrane (Shanghai Jiening Biotechnology, GF06) pre-coated with colloidal gold-labeled antibody (Au-NPs@detection mAb). The colloidal gold-labeled antibody is composed of colloidal gold nanoparticles (Au-NPs) conjugated to a monoclonal antibody for binding to AQP4-IgG. The monoclonal antibody, acting as the primary antibody (detection mAb), specifically recognizes and binds to the target molecule AQP4-IgG, forming an AQP4-IgG-colloidal gold-labeled antibody complex. Specifically, this technique uses a murine monoclonal antibody capable of binding to AQP4-IgG; both the aforementioned monoclonal antibody and its colloidal gold-labeled form are commercially available. The method for pre-coating the colloidal gold-labeled antibody with pad 3 is as follows: cut the above glass fiber into strips of 3mm × 8cm, take a 30μg / mL (optional range 10μg / mL-50μg / mL, more preferably 20μg / mL-40μg / mL) colloidal gold-labeled antibody (Au-NPs@detection mAb) solution, and uniformly drop it onto the cut glass fiber (120μL / strip; optional range 100-120μL / strip), and dry it to obtain the final product. The diluent for diluting the colloidal gold-labeled antibody was prepared as follows: 50 mL of ultrapure water was taken, and 0.0406 g of disodium hydrogen phosphate (Na2HPO4), 0.0335 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), 0.05 g of sodium chloride (NaCl), 5 g of sucrose, 0.25 g of bovine serum albumin (BSA), 2.5 g of trehalose, and 25 μL of Tween-20 were added. The mixture was vortexed until completely dissolved, filtered, and dispensed into 1.5 mL vials. The vials were stored at 4 °C.

[0072] For the coating material in the conjugation pad 3, in addition to colloidal gold-labeled antibodies (Au-NPs@detection mAb), conventionally labeled antibodies from existing technologies can also be used. The labeled antibody is composed of a label and a molecule for specific binding to AQP4-IgG; the label is selected from at least one of colloidal gold nanoparticles, colored latex microspheres, fluorescent microspheres, and magnetic microspheres. The molecule for specific binding to AQP4-IgG is selected from at least one of monoclonal antibodies for binding to AQP4-IgG, polyclonal antibodies for binding to AQP4-IgG, antibody fragments for binding to AQP4-IgG, genetically engineered antibodies for binding to AQP4-IgG, and nanobodies for binding to AQP4-IgG. Monoclonal antibodies, polyclonal antibodies, antibody fragments, genetically engineered antibodies, and nanobodies are all common antibody forms in existing technologies and are widely used in the conjugation pad 3 of lateral flow immunochromatographic test strips. These antibodies are typically covalently coupled to markers (such as colloidal gold, fluorescent microspheres, etc.) to specifically recognize and bind to target molecules (e.g., AQP4-IgG) in the sample, thereby imparting a labeling signal to the target molecule. This labeling signal can then be visualized on the detection line 7 (T line) and the control line 8 (C line), used to determine the presence of the target molecule and to verify that the detection system is functioning correctly, respectively.

[0073] (3) Reaction pad 5 The reaction pad 5 is made of nitrocellulose membrane (NC membrane, Sartorius, CN140, unbacked). The upper surface of the reaction pad 5 is marked with a test line 7 (T line) and a control line 8 (C line). Gel-coated HEK-293T cells (G-AQP4-293T, 10 cells) overexpressing AQP4 protein were used. 7 cells / mL; selectable range is 10. 6 -10 8A test line 7 is drawn using goat anti-mouse IgG (secondary antibody, 2 mg / mL; optional range 1-2 mg / mL); a control line 8 is drawn using goat anti-mouse IgG (secondary antibody, 2 mg / mL; optional range 1-2 mg / mL); the amount used for both lines is 0.8 μL / cm (optional range 0.1-2.0 μL / cm, further preferred 0.7-0.9 μL / cm). The AQP4 protein overexpressing gelled HEK-293T cells serves as the antigen, effectively binding to the target molecule AQP4-IgG in the sample, thus retaining the AQP4-IgG-colloidal gold-labeled antibody complex on test line 7. The presence of the target molecule AQP4-IgG in the sample is indicated by the color development of the colloidal gold on test line 7. The goat anti-mouse IgG on control line 8 is an antibody produced by goats that specifically binds to mouse IgG antibodies, used to bind to the colloidal gold-labeled antibody (Au-NPs@detection mAb) that has not bound to test line 7. The molecules used for marking the control line 8 can be any molecules that bind to the labeled antibody. More specifically, the control line 8 of the reaction pad 5 of the lateral flow immunochromatographic test strip is coated with molecules for binding to the labeled antibody. These molecules bind to molecules on the labeled antibody that specifically bind to AQP4-IgG (e.g., various antibodies), immobilizing the labeled antibody flowing out of the binding pad 3 and not captured by the detection line 7 onto the control line 8, thus verifying whether the detection system is working properly. Depending on the different molecules on the labeled antibody that specifically bind to AQP4-IgG, the molecules on the control line 8 that bind to the labeled antibody can be: rabbit anti-mouse IgG, donkey anti-mouse IgG, etc., all of which are conventional forms in the prior art.

[0074] Nafion-CNF hybrid hydrogel is a composite hydrogel material constructed from Nafion and CNF solutions. The Nafion solution is a perfluorosulfonic acid ionomer solution (Aladdin P400486) with a solute volume percentage of 5% (v / v). The CNF solution is a cellulose nanofibrils dispersion (Xianfeng Nano 102201) with a solute mass percentage of 1% (w / w). The Nafion-CNF hybrid hydrogel contains 0.2% (v / v) Nafion and 0.1% (w / w) CNF, and is prepared as follows (10 mL system): 0.4 mL of Nafion solution and 1 mL of CNF solution are added to 8.6 mL of ultrapure water, and the mixture is shaken and mixed at room temperature for 30 min to obtain the desired concentration of Nafion-CNF hybrid hydrogel solution. When preparing Nafion-CNF hybrid hydrogels, the final concentrations that can be used are: Nafion 0.1-1% (v / v, more preferably 0.2-0.3%) and CNF 0.1-1% (w / w, more preferably 0.1-0.2%). Both can enhance the T-line signal intensity, maintain good gel cell morphology, improve the sensitivity of AQP4-IgG detection, and extend the shelf life of portable detection devices.

[0075] Before streaking the C and T lines, the NC membrane is modified with Nafion-CNF hybrid hydrogel. The back side of the NC membrane is modified with Nafion-CNF hybrid hydrogel (especially the position corresponding to detection line 7). The process of treating the NC membrane with Nafion-CNF hybrid hydrogel solution is as follows: Using a streaking instrument, an appropriate amount of the prepared Nafion-CNF hybrid hydrogel solution is drawn and streaked on the back side of the NC membrane at the position corresponding to the T line with a parameter of 0.8 μL / cm (optional range 0.1-2.0 μL / cm, more preferably 0.7-0.9 μL / cm). The membrane is then dried overnight (8-12 h) at room temperature (optional range 20-30℃).

[0076] (4) G-293T cell pretreatment samples and detection methods Take 50-100 μL of the sample to be tested and mix it thoroughly with the prepared G-293T cell pellet until the final cell density is 1 × 10⁻⁶. 7 Approximately 10 cells / mL (selectable range 1×10⁻⁶) 6 -1×10 8(Cells / mL), incubate at room temperature for 5 min (selectable range 20-30℃, 5-10 min) to form sample 9 (i.e., the sample to be tested needs to be sealed before loading). Pipette 50 μL of solution through the sample well and add it to sample pad 1, let stand for 5-10 min to allow color development to complete. The sample to be tested can be serum, plasma, cerebrospinal fluid, urine, etc. (AQP4-IgG clinical samples are generally serum). The entire device can complete the process from sample loading to visual qualitative interpretation of the result within 10 min, with a detection limit as low as 2 ng / mL. For details on the method of interpreting test results, please refer to [link to documentation]. Figure 5 B.

[0077] Example 3: Modification of NC membranes with Nafion-CNF hybrid hydrogel and its effects Add 0.4 mL of Nafion solution and 1 mL of CNF solution to 8.6 mL of ultrapure water, and mix by shaking at room temperature for 30 min to obtain the Nafion-CNF hybrid hydrogel solution of the desired concentration. Take 8 μL of the prepared Nafion-CNF hybrid hydrogel solution and drop it onto a 1 cm × 1 cm unbacked NC membrane to ensure uniform coverage of the membrane surface without leakage from the edges. Place it at room temperature (20-30℃) to dry overnight (8-12 h). Commercially available hydrogel solutions, including Gel-MA (Gelatin Methacryloyl; Aladdin M398240), SA-MA (Methacrylated Sodium Alginate; Aladdin M306010), and CMCS-MA (Carboxymethyl Chitosan Methacryloyl; Aladdin M398219), were purchased and prepared at a concentration of 10% (w / v). The same procedure was performed on the NC membrane. In addition, the NC membrane of the control group was not treated in any way.

[0078] Invert the modified NC membrane and add 5 μL of colloidal gold probe solution (colloidal gold-labeled antibody, Au-NPs@detection mAb, 10 μg / mL) to the other side. Also add 5 μL of colloidal gold probe solution to a control NC membrane of the same size. After 5 minutes, compare and observe the color development, and take pictures. Use ImageJ software to extract the color intensity for analysis. The experimental results are as follows: Figure 6 As shown, the Nafion-CNF hybrid hydrogel can generate tangential nanoelectrodynamic forces to aggregate probes onto the NC membrane surface, while the control group or the treatment group using other hydrogels did not show this effect. Some probes were encapsulated into the deep structure of the NC membrane and thus could not produce a color signal visible to the naked eye.

[0079] Similarly, the modified NC membrane was flipped over, and a gelled cell suspension (10 μL, density 10) was dropped onto its other side. 4 Cells / mL; because only the cell morphology observation after gelation was involved, no distinction was made between bimodal engineered cell suspension and gelled cell suspension alone. An equal volume of gelled cell suspension was also added to the same size control NC membrane and stored at room temperature (25℃, 40%RH). The cell morphology retention ability of each group was continuously observed, and the percentage of intact cells was statistically analyzed (counting the total number of cells in 5 fields of view, with a total >200). The experimental results are shown in Table 1: Nafion-CNF hybrid hydrogel can provide a suitable environment for gelled cell preservation on the NC membrane substrate, avoiding the collapse and destruction of the cell membrane structure due to excessive water loss from the internal gel; while other treatment groups showed similar effects within 0-3 days, they could not maintain this effect long-term.

[0080] Table 1: Percentage of cells with intact morphology (Experimental results are the average of 5 replicate experiments)

[0081] The experimental data above clearly demonstrate that the Nafion-CNF hybrid hydrogel plays a key role in optimizing the detection system in the application scenario of this technical solution: on the one hand, it can effectively enhance the intensity of the color signal. This effect not only significantly improves the visual recognition of the detection results, but also reduces signal errors caused by background interference, making the target signal clearer and easier to quantify; on the other hand, the hybrid hydrogel can stabilize the cell morphology in the gel state in the complex paper-based material environment of the test strip, avoiding problems such as cell shrinkage and deformation.

[0082] Maintaining stable gel cell morphology is a prerequisite for ensuring detection sensitivity: abnormal changes in cell morphology often lead to conformational distortion of the AQP4 protein on its cell membrane surface, thereby obscuring or altering the protein's binding sites. Supported by Nafion-CNF hybrid hydrogels, gel cell morphology is well maintained, allowing the AQP4 protein to retain its native spatial conformation, thus fully exposing its specific binding sites for AQP4-IgG. This directly improves the binding efficiency and stability of both. The portable detection device in this protocol can maintain good gel cell morphology even after extended storage, ensuring detection effectiveness over longer periods.

[0083] It is the synergy of these two aspects that ultimately achieves high-sensitivity detection of AQP4-IgG: not only does it lower the detection limit, but it also makes the signal response of low-concentration target analytes more stable and reliable, and greatly extends the shelf life of the portable detection device in this scheme, providing a more solid experimental foundation for subsequent quantitative analysis.

[0084] Example 4: Optimization of Coating Concentration Conditions for Colloidal Gold-Labeled Antibody (Au-NPs@detection mAb) A portable device for point-of-care detection of AQP4-IgG was prepared and sample added for detection according to the methods described in Examples 1 and 2. Except for the coating concentration of the colloidal gold-labeled antibody, all other procedures were performed optimally. The sample to be tested was a commercially available AQP4-IgG solution at a loading concentration of 100 ng / mL (prepared using a commercially available universal antibody diluent). A portion of the sample to be tested underwent a blocking process to obtain the loading sample. The blocking process was as follows: 100 μL of the sample to be tested was aspirated and mixed with a pre-prepared G-293T cell pellet to achieve a cell density of 102. 7 Cells / mL, incubated at room temperature for 5 min. Some samples to be tested were not blocked and were directly added to the portable device of this protocol for detection, with a sample volume of 50 μL. In this example, different concentrations of colloidal gold-labeled antibody (Au-NPs@detection mAb) were tested: 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL. The experimental results are as follows. Figure 7 As shown, when the coating concentration is 30 μg / mL, the positive results are clearly visible, and no non-specific reactions occur after blocking. The experimental results indicate that if the samples to be tested are not blocked (G-293T cells are not used for incubation with the samples), false positives will occur. If the concentration of the colloidal gold-labeled antibody is too high, false positive results will still occur even with blocking; if the concentration of the colloidal gold-labeled antibody is too low, the detection results of positive samples will be weak after blocking, which is not conducive to experimental observation.

[0085] Example 5: Optimization of gelled cell (G-AQP4-293T) coating concentration conditions A portable device for point-of-care AQP4-IgG detection was prepared and tested according to the methods described in Examples 1 and 2. Except for the gelatin cell coating concentration, all other parameters were performed optimally to investigate the effect of gelatin cell coating concentration on detection results. The sample to be tested was a commercially available AQP4-IgG solution with a loading concentration of 100 ng / mL (prepared using a commercially available universal antibody diluent). A portion of the sample to be tested underwent a blocking process to obtain the loading sample. The blocking process was as follows: 100 μL of the sample to be tested was aspirated and mixed with a pre-prepared G-293T cell pellet to achieve a cell density of 102. 7 Cells / mL, incubated at room temperature for 5 min. Some samples to be tested were not blocked and were directly added to the portable device of this protocol for detection, with a sample volume of 50 μL. The gel cell coating concentrations were: 10... 6 cells / mL, 10 7 cells / mL, 10 8 Cells / mL. Experimental results are as follows: Figure 8 As shown: the coating concentration is 10. 7 When the cell count is 100 cells / mL, the positive result is clearly visible, and no non-specific reaction occurs after blocking.

[0086] Example 6: Specificity Verification A portable device for point-of-care detection of AQP4-IgG was prepared and tested according to the methods described in Examples 1 and 2, following the optimal procedure. The samples to be tested were the following solutions, with solutes of AQP4-IgG (aquaporin 4 antibody), AQP1-IgG (aquaporin 1 antibody), MOG-IgG (myelin oligodendrocyte glycoprotein antibody), RF (rheumatoid factor), and HAS (human serum albumin), at concentrations of 50 ng / mL, 50 ng / mL, 50 ng / mL, 100 IU / mL, and 50 ng / mL, respectively. The samples to be tested were subjected to a blocking treatment as described in Example 4 before being loaded for detection. The experimental results are as follows: Figure 9 As shown: the T line showed color after adding AQP4-IgG solution, but did not show color after adding AQP1-IgG, MOG-IgG, RF, and HSA, indicating that these substances do not cause interference and that the detection platform has good specificity.

[0087] Example 7: Sensitivity Verification A portable device for point-of-care testing of AQP4-IgG was prepared and tested according to the methods described in Examples 1 and 2, following the optimal procedure. The samples to be tested were AQP4-IgG solutions with concentrations of 0 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, and 4 ng / mL, respectively. The samples were subjected to a blocking treatment as described in Example 4 before being loaded for testing. The experimental results are as follows: Figure 10 As shown, when the concentration is as low as 2 ng / mL, the T line can still be observed, indicating that the detection limit of this detection platform is 2 ng / mL.

[0088] Example 8: Stability Verification A portable device for point-of-care detection of AQP4-IgG was prepared according to the methods described in Examples 1 and 2, and carried out in the optimal manner. After storage for 1, 3, 5, 10, 15, and 30 days under the same conditions (25°C, 40% RH), samples were added for detection. The samples to be tested were AQP4-IgG solutions (after blocking treatment as described in Example 4) at low, medium, and high concentrations of 10 ng / mL, 50 ng / mL, and 100 ng / mL, respectively. The experimental results are as follows: Figure 11 As shown, the experimental results demonstrate that the portable device prepared in this scheme has good stability and can still maintain a relatively ideal detection effect after being placed for a long time.

[0089] Example 9: Clinical Sample Validation A portable device for point-of-care testing of AQP4-IgG was prepared and tested according to the methods described in Examples 1 and 2, following the optimal procedure. The clinical sample was specifically serum, which was sealed using the method described in Example 4 before testing. The experimental results are shown in Table 2: This method yielded results consistent with the gold standard method used clinically, with an accuracy of 100%.

[0090] Among them, CBA (Cell-Based Assay) is currently the gold standard method for detecting AQP4-IgG because it preserves the native conformation of AQP4 protein on the cell membrane, thus achieving high sensitivity and high specificity. The process is as follows: The human AQP4-M23 gene is transfected into cells such as HEK293, where the AQP4 protein is expressed and localized on the cell membrane surface. Simultaneously, control cells without AQP4 transfection are prepared (to exclude non-specific binding). Human serum (which may contain AQP4-IgG) is added to the cells. If AQP4-IgG is present in the serum, it will specifically bind to the AQP4 protein on the cell membrane; negative serum will not bind. Then, fluorescently labeled anti-human IgG antibody is added. This secondary antibody binds to the human AQP4-IgG already bound to AQP4, generating a fluorescent signal. Under a fluorescence microscope, a positive result is indicated by bright fluorescence in the cell membrane of AQP4-transfected cells, while untransfected control cells show no fluorescence; a negative result is indicated by no specific membrane fluorescence in transfected cells. Furthermore, only membrane-specific fluorescence is considered positive; intracellular or diffuse fluorescence is usually a false positive.

[0091] Compared to the existing CBA method, this method can achieve semi-qualitative visual interpretation of results from sample addition within 10 minutes, with a detection limit as low as 2 ng / mL. In contrast, the live cell method requires continuous culture of the required live cells and the use of large instruments such as confocal microscopy and flow cytometry, as well as professional technicians, which takes as long as 2-7 days.

[0092] It is evident that this technical solution is simpler and more time-saving than CBA, while achieving the same detection accuracy, making it a superior detection method compared to existing technical standards.

[0093] Table 2: Clinical Sample Validation Results

[0094] Comparative Example DMSO plays a dual role as a cell cryoprotectant and permeation enhancer in the preparation of gelled cells. A dual-modal engineered cell system was constructed using a synergistic strategy of cell transfection and intracellular gelation. The dual-modal engineered cells (G-AQP4-293T) with surface overexpression of AQP4 protein prepared in Example 1 are used as an example for illustration and results presentation. The control group used HAMA hydrogel solution without DMSO, while the experimental groups were supplemented with 5% and 10% DMSO. Microscopic observation revealed that, compared with the control group (without DMSO), the cells in the experimental groups supplemented with DMSO (5%-10%) had a more rounded and plump cell morphology. Detailed experimental results can be found in [link to relevant documentation]. Figure 12This means that cells are less affected by cell membrane collapse, deformation, and rupture caused by ice crystal formation and thawing during freeze-thaw cycles, thus having less impact on membrane protein conformation and binding sites, and are closer to the state of natural cells, avoiding a decrease in drug targeting or detection specificity.

[0095] The following example illustrates and presents the results using bimodal engineered cells with surface overexpression of AQP4 protein prepared in Example 1. The control group did not have DMSO added to the HAMA hydrogel solution, while the experimental group had 5% DMSO added to the HAMA hydrogel solution. Confocal microscopy characterization was performed using green fluorescence-coupled HAMA hydrogels. Confocal microscopy revealed a significant increase in intracellular fluorescence after DMSO doping, indicating that DMSO helps promote cell entry into the hydrogel during freeze-thaw cycles. Detailed experimental results can be found in [link to relevant documentation]. Figure 13 and Figure 14 .

[0096] The experimental data above demonstrate that DMSO possesses the dual effects of a cryoprotectant and a permeation enhancer, simultaneously maintaining cell integrity and enhancing substance delivery. While the initial intention behind incorporating DMSO into this technique was to improve cell cryoprotection, it was also discovered during the specific gelation process that it promotes the entry of HAMA hydrogel into cells. DMSO protects and maintains the integrity and functional activity of the cell membrane, while simultaneously facilitating the penetration of HAMA hydrogel into the cell.

[0097] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A portable device for point-of-care testing of AQP4-IgG, characterized in that: It includes a lateral flow immunochromatographic test strip and gelled cells for mixing and incubating with the sample to be tested; the detection line of the reaction pad of the lateral flow immunochromatographic test strip is coated with gelled cells that overexpress AQP4 protein; The gelled cells overexpressing AQP4 protein were prepared by the following method: engineered cells transfected with the AQP4 gene were prepared; the cells transfected with the AQP4 protein gene were resuspended in methacryloyl hyaluronic acid solution, and after freezing and thawing, they were washed and resuspended, and then photocured to obtain gelled cells overexpressing AQP4 protein. The gelled cells were prepared by the following method: untransfected cells, methacrylamide hyaluronic acid, and dimethyl sulfoxide were mixed to form a hydrogel solution. After freezing and thawing, the cells were washed and resuspended, and then photocured to obtain gelled cells. The back of the reaction pad where the detection lines are located is modified with Nafion-CNF hybrid hydrogel; Nafion-CNF hybrid hydrogels are used to enhance signal intensity and reduce background interference, as well as to stabilize the morphology of gelled cells overexpressing AQP4 protein on the detection line and prevent the gelled cells from shrinking and deforming; the Nafion-CNF hybrid hydrogels contain 0.1-1% Nafion by volume and 0.1-1% CNF by mass.

2. The portable device for point-of-care testing of AQP4-IgG according to claim 1, characterized in that: Engineered cells transfected with the AQP4 gene were prepared by the following method: constructing a lentiviral expression vector integrating the human AQP4-M23 gene; using the lentiviral expression vector to package the virus and prepare viral fluid; infecting HEK-293T cells with the viral fluid to obtain engineered cells transfected with the AQP4 gene. Prepare a cell hydrogel solution containing 1×10⁻⁶ cells. 6 -1×10 8 Engineered cells transfected with the AQP4 gene were prepared at a concentration of 1 cell / mL, along with 2-3% (w / v) of methacrylamide hyaluronic acid and 5-10% (w / v) of dimethyl sulfoxide. The hydrogel solution was first incubated at 2-8°C for 15-30 min, then frozen at -75 to -85°C for >8 h, followed by thawing at 37-40°C for 15-30 min to obtain the freeze-thawed cell hydrogel solution. The freeze-thawed cell hydrogel solution was then centrifuged and washed to obtain the hydrogel-treated engineered cells. Engineered cells that have been resuspended in hydrogel were photocured with 405nm ultraviolet light for 1-2 minutes to obtain gelled cells that overexpress AQP4 protein.

3. A portable device for point-of-care testing of AQP4-IgG according to claim 2, characterized in that: Gelatinized cells were added to the sample to be tested, and the final density of the gelled cells was 10. 6 -10 8 Cells / mL; the sample to be tested and the gelled cells were incubated together for 5-10 min at a temperature of 20-30℃.

4. A portable device for point-of-care testing of AQP4-IgG according to claim 1, characterized in that: The conjugation pad of the lateral flow immunochromatographic test strip is coated with labeled antibodies; The labeled antibody is composed of a marker and a molecule for specific binding to AQP4-IgG; The marker is selected from at least one of colloidal gold nanoparticles, colored latex microspheres, fluorescent microspheres, and magnetic microspheres; The molecule used for specific binding to AQP4-IgG is selected from at least one of the following: monoclonal antibody for binding to AQP4-IgG, polyclonal antibody for binding to AQP4-IgG, antibody fragment for binding to AQP4-IgG, genetically engineered antibody for binding to AQP4-IgG, and nanobody for binding to AQP4-IgG.

5. A portable device for point-of-care testing of AQP4-IgG according to claim 4, characterized in that: The control line of the reaction pad of the lateral flow immunochromatographic test strip is coated with molecules for binding to the labeled antibody.

6. The use of a portable device for point detection of AQP4-IgG according to any one of claims 1-5 in the preparation of products for diagnosing neuromyelitis optica spectrum disorders.

7. A method for preparing a portable device for point-of-care detection of AQP4-IgG according to any one of claims 1-5, characterized in that: The sample pad, buffer pad, conjugate pad, reaction pad, and absorbent pad are fixed to the back plate in sequence; The method for coating the conjugate pad with colloidal gold-labeled antibody is as follows: a solution of colloidal gold-labeled antibody with a concentration of 10-50 μg / mL is added dropwise to the conjugate pad, and then dried. The method for modifying the reaction pad with Nafion-CNF hybrid hydrogel is as follows: apply Nafion-CNF hybrid hydrogel at a rate of 0.1-2.0 μL / cm on the back of the detection line of the reaction pad, and dry at 20-30℃ for 8-12 hours to obtain the product. The method for drawing detection lines on the reaction pad is as follows: using a dosage of 0.1-2.0 μL / cm, the dispersion of gelled cells overexpressing AQP4 protein is drawn on the back of the modified lines of the Nafion-CNF hybrid hydrogel on the reaction pad, and dried at 20-30℃ for 8-12 hours to obtain the result. The cell density in the dispersion of gelled cells overexpressing AQP4 protein was 10. 6 -10 8 cells / mL; The method for drawing control lines on the reaction pad is as follows: use 0.1-2.0 μL / cm to draw lines of goat anti-mouse IgG solution on the reaction pad, and dry at 20-30℃ for 8-12 hours to obtain the solution. The concentration of sheep anti-mouse IgG in the sheep anti-mouse IgG solution is 1-2 mg / mL.

Images