Bimodal engineering cell based on cell transfection and intracellular gelling and construction method thereof

Abstract

The invention relates to the technical field of engineering cells, in particular to a bimodal engineering cell based on cell transfection and intracellular gelling and a construction method of the bimodal engineering cell. Target cell membrane protein is overexpressed on the cell surface of the bimodal engineering cell, and gelation is carried out in the cell. The preparation process comprises the following steps: preparing a cell transfected with a target cell membrane protein gene; and resuspending the cell transfected with the target cell membrane protein gene in a hydrogel solution, and carrying out freeze thawing treatment and photocuring treatment to obtain the bimodal engineering cell. According to the technical scheme, the technical problem that the consistency of the content, the type and the space structure of the membrane protein and source cells is difficult to accurately maintain while the metabolic activity of the cells is deprived in the prior art can be solved. According to the scheme, a cell transfection and intracellular gelation synergistic strategy is utilized, and the product can be used for drug delivery or selective biological electron affinity sensing, can adapt to different types of target membrane proteins, and is compatible with the requirements of multiple fields such as cell therapy, disease biomarker detection and targeted drug delivery.

Description

Technical Field

[0001] This invention relates to the field of engineered cell technology, specifically to a dual-modal engineered cell based on cell transfection and intracellular gelation, and a method for constructing the same. Background Technology

[0002] In the fields of cell therapy and disease biomarker detection, maintaining cell membrane integrity and the native conformation of surface proteins is a core prerequisite for ensuring their functional activity and specific recognition efficacy. This requirement stems from the unique properties of the cell membrane as a biological interface. Its lipid bilayer not only constitutes a physical barrier but also enables signal transduction and molecular recognition through the precise spatial arrangement of membrane proteins (such as receptors and channel proteins). Under current technology, biomimetic nanovesicles prepared based on ultrasound or membrane extrusion techniques have been widely used in drug delivery systems and the construction of bioelectronic sensing interfaces. However, their separation and preparation process faces significant technical bottlenecks: the loss of membrane proteins (typically reaching 40-60% of the original content) and conformational changes caused by mechanical separation severely weaken the specific recognition ability of biosensors. This problem is particularly prominent in the detection of disease biomarkers based on membrane proteins (such as AQP4-IgG detection). For example, when AQP4 protein (aquaporin 4) is separated from the astrocyte membrane, the orthogonal particle array (OAP) conformation formed by its tetramers is easily disrupted, leading to the loss of antibody binding sites and directly affecting detection sensitivity.

[0003] Further analysis reveals that the non-physiological distribution of membrane proteins caused by random recombination of membrane fractions significantly affects the targeting efficiency of drug delivery systems. Taking integrin αvβ3 as an example, it exhibits a periodic clustered distribution on the natural cell membrane, and this spatial arrangement is directly related to the binding affinity of ligands (such as RGD peptides). Experimental data show that when nanovesicles are prepared using traditional membrane extrusion methods, the disordered spatial distribution of integrins can lead to a 67% decrease in the binding efficiency of targeting ligands and a 52% reduction in tumor tissue accumulation. This loss of targeting is even more pronounced in the delivery of therapeutic antibodies (such as anti-EGFR antibodies). The change in the free energy (ΔG) of antibody-receptor binding decreases from -9.8 kcal / mol to -6.3 kcal / mol, severely limiting the therapeutic effect.

[0004] "Cellular backpack" technology, as an emerging strategy in cell therapy, endows cells with new therapeutic functions by anchoring functional materials (such as immunomodulators and gene carriers) on the surface of living cells. This technology has shown clinical potential in scenarios such as CAR-T cell enhancement and directed migration of mesenchymal stem cells. However, a key technical challenge it faces is how to avoid the structural disintegration of the carrier-cell complex caused by endocytosis. Studies have shown that when the particle size of the material is >200 nm, cells take it into their cells via clathrin-mediated endocytosis. This process not only causes the functional material to become ineffective but also induces lysosomal stress, leading to a decrease in carrier cell survival rate to below 30% within 48 hours. Furthermore, endocytosis-induced cytotoxicity can cause a decrease in mitochondrial membrane potential (ΔΨm decreases by 58%) and a burst of reactive oxygen species (ROS) (increases by 3.2 times), further accelerating cell apoptosis.

[0005] The core contradiction of the above problems lies in the fact that existing technologies cannot precisely maintain the consistency of the content, type, and spatial structure of membrane proteins with those of the source cells while depriving cells of metabolic activity. Summary of the Invention

[0006] The present invention aims to provide a method for constructing dual-modal engineered cells based on cell transfection and intracellular gelation, in order to solve the technical problem that existing technologies are unable to accurately maintain the consistency of membrane protein content, type and spatial structure with the source cells while depriving cells of metabolic activity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A bimodal engineered cell based on cell transfection and intracellular gelation, wherein the cell surface overexpresses a target cell membrane protein; and the cell gels intracellularly.

[0008] Furthermore, a bimodal engineered cell based on cell transfection and intracellular gelation, wherein the gel material used for intracellular gelation includes at least one of methacrylamide hyaluronic acid, methacrylamide gelatin, and methacrylamide dextran.

[0009] Furthermore, a bimodal engineered cell based on cell transfection and intracellular gelation is described. The intracellular gelation method is as follows: a hydrogel solution containing cells transfected with the target cell membrane protein gene, gel material, and dimethyl sulfoxide is prepared, and after being treated by standing at 2-8℃ for 15-30 min, it is frozen at -75 to -85℃ for >8 h; next, it is thawed in a water bath at 37-40℃ for 15-30 min; then, the cells are centrifuged, washed, resuspended, and photocured to obtain the bimodal engineered cell.

[0010] Furthermore, the gel material has a mass-volume percentage of 2-3%; the dimethyl sulfoxide has a volume percentage of 5-10%. The photocuring process involves irradiating the cells resuspended in the buffer solution with 405nm ultraviolet light for 1-2 minutes.

[0011] Furthermore, cells transfected with the target cell membrane protein gene were obtained by lentiviral transfection, liposome transfection, or electroporation transfection. Preferably, the cells used are human embryonic kidney epithelial cell lines; Preferably, when using the lentiviral transfection method, a lentiviral expression vector integrating the target cell membrane protein gene is first constructed, and then packaged to obtain viral fluid; the viral fluid is used to transfect cells to obtain cells transfected with the target cell membrane protein gene.

[0012] This technical solution also provides a method for preparing dual-modal engineered cells based on cell transfection and intracellular gelation, characterized by comprising the following steps performed sequentially: S1: Prepare cells transfected with the target cell membrane protein gene; S2: Prepare a hydrogel solution containing cells transfected with the target cell membrane protein gene, gel material, and dimethyl sulfoxide. After standing at 2-8℃ for 15-30 min, freeze at -75 to -85℃ for >8 h. Next, thaw in a water bath at 37-40℃ for 15-30 min. Then, centrifuge to collect cells, wash the cells, resuspend the cells, and obtain bimodal engineered cells by photopolymerization.

[0013] Furthermore, in S2, the mass-volume percentage of the gel material is 2-3%; the volume percentage of dimethyl sulfoxide is 5-10%; and the photocuring is performed by irradiating the cells resuspended in the buffer solution with 405nm ultraviolet light for 1-2 minutes. The gel material includes at least one of methacrylamide hyaluronic acid, methacrylamide gelatin, and methacrylamide dextran.

[0014] Furthermore, in S1, cells transfected with the target cell membrane protein gene are obtained by lentiviral transfection, liposome transfection, or electroporation transfection.

[0015] This technical solution also provides the application of bimodal engineered cells based on cell transfection and intracellular gelation in the preparation of lateral flow immunochromatographic test strips, wherein the detection lines of the reaction pad of the lateral flow immunochromatographic test strip are coated with bimodal engineered cells; The lateral flow immunochromatographic test strip, combined with a sample blocking reagent, forms a detection kit. The sample blocking reagent is gelled cells. The gelled cells are prepared by the following method: untransfected cells, gel material, and dimethyl sulfoxide are mixed to form a hydrogel solution. After freezing and thawing, the cells are washed and resuspended, and then photocured to obtain gelled cells.

[0016] Furthermore, the lateral flow immunochromatographic test strip was used to detect AQP4-IgG; The bimodal engineered cells coated on the detection line of the reaction pad are gelled cells overexpressing AQP4 protein; 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.

[0017] Among them, the rapid and accurate detection of aquaporin 4 antibody (AQP4-IgG, anti-aquaporin 4 immunoglobulin G) is a key step in the clinical diagnosis and treatment of neuromyelitis optica spectrum disorders (NMOSD).

[0018] 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 cryopreservation and thawing, they were washed and resuspended again, and then photocured to obtain gelled cells overexpressing AQP4 protein. The AQP4 protein on the surface of the gelled cells overexpressing AQP4 protein can bind to the target substance AQP4-IgG.

[0019] Engineered cells transfected with the AQP4 gene were prepared by the following method: constructing a lentiviral expression vector for 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 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.

[0020] Furthermore, gelled cells are prepared by the following method: untransfected cells, methacrylamide hyaluronic acid, and dimethyl sulfoxide are mixed to form a hydrogel solution. After freezing and thawing, the cells are washed and resuspended, and then photocured to obtain gelled cells.

[0021] 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℃.

[0022] Furthermore, the conjugation pad of the lateral flow immunochromatographic test strip is coated with 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.

[0023] In addition to the preferred embodiments described above, the binding pad of the lateral flow immunochromatographic test strip may be coated with a labeled antibody. The labeled antibody is composed of a marker and a molecule specifically 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.

[0024] 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.

[0025] In addition to the preferred embodiments described above, the control line of the reaction pad of the lateral flow immunochromatographic test strip may be 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 labeled antibody that has flowed out of the binding pad and was not captured by the detection line on the control line, thus verifying the proper functioning of the detection system. Depending on the different molecules on the labeled antibody that specifically bind to target molecules such as AQP4-IgG, the molecules on the control line for binding 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.

[0026] Furthermore, the method for preparing the lateral flow immunochromatographic test strip for the point-of-care detection of AQP4-IgG is as follows: 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.

[0027] The technical principle of this technical solution is as follows: The core of this invention lies in constructing a dual-modal synergistic system of "surface functionalization-intracellular gelation." Through the synergistic effect of cell transfection and intracellular gelation, it solves the technical problem that existing technologies cannot precisely maintain the consistency of membrane protein content, type, and spatial structure with the source cell while depriving cells of metabolic activity. The specific technical principle is as follows: (2) Surface functionalization: Using mature transfection techniques such as lentiviral transfection, liposome transfection, or electroporation transfection, the target membrane protein gene (such as the AQP4 gene) is precisely introduced into host cells (such as human embryonic kidney epithelial cells HEK-293T). Through gene expression regulation, the target membrane protein is stably overexpressed on the cell surface. This process endows engineered cells with a specific functional basis, providing high-affinity specific recognition sites for target biomarkers in disease biomarker detection scenarios; and providing precise binding targets for targeted ligands in drug delivery scenarios.

[0028] (2) Construction of intracellular gelling support system: Photocurable gelling materials with excellent biocompatibility, such as methacrylamide hyaluronic acid and methacrylamide gelatin, were selected to prepare hydrogel solutions containing transfected cells, gel materials, and dimethyl sulfoxide (DMSO). Utilizing the freeze-thaw process (deep freezing-warm water thawing), DMSO was used to avoid ice crystal formation during freeze-thaw cycles that could damage the cell membrane, while also promoting efficient penetration of the hydrogel solution into the cell interior. Subsequently, a short-term irradiation with ultraviolet light of a specific wavelength was used to induce a photocrosslinking reaction, causing the intracellular hydrogel to form a three-dimensional network framework. This framework can simulate the spatial support function of natural cytoplasm, providing stable internal support for the cell membrane.

[0029] (3) Dual-modal synergistic mechanism: Surface overexpression of target membrane proteins ensures the specific functions of engineered cells (marker recognition / targeted binding). The intracellular three-dimensional gel scaffold, after depriving cells of metabolic activity, continuously maintains the overall morphological stability of the cell, preventing cell membrane collapse due to cell structure disintegration, thereby ensuring that the physiological spatial arrangement of membrane proteins is not disrupted. Among them, the introduction of DMSO forms a synergistic effect: it acts as a cryoprotectant to reduce the damage to the cell membrane and membrane protein conformation caused by freeze-thaw cycles, and also improves the intracellular permeation efficiency of the hydrogel solution, providing a guarantee for the uniform construction of the intracellular gel system.

[0030] (4) Application of the extended adaptation principle (taking lateral flow immunochromatographic test strips as an example): Bimodal engineered cells overexpressing target membrane proteins (such as AQP4) are coated onto the test strip's detection line. Detection is achieved by utilizing the specific binding of the target membrane protein's natural conformation to the markers in the sample (such as AQP4-IgG). Simultaneously, Nafion-CNF hybrid hydrogel is modified on the back of the detection line. The properties of this hybrid hydrogel prevent the gelled cells from shrinking and deforming due to the paper-based loading environment, while simultaneously enhancing signal enrichment and reducing non-specific diffusion. Using untransfected gelled cells as a sample blocking reagent can specifically block non-specific binding sites, reducing the risk of false positives.

[0031] Compared with existing technologies (such as ultrasonic / membrane extrusion for preparing biomimetic nanovesicles, traditional cell backpack technology, single-cell transfection technology, etc.), this invention has the following significant advantages: (1) Precisely maintain the integrity of membrane protein content, conformation and spatial arrangement: It completely solves the problems of membrane protein loss and conformational damage caused by traditional membrane separation technologies (ultrasound, membrane extrusion). By transfecting and overexpressing to ensure sufficient content of target membrane proteins, combined with the spatial support of the intracellular gel scaffold, it can effectively maintain the native conformation and physiological spatial arrangement of membrane proteins, ensure the integrity of their binding sites, and significantly improve the sensitivity of biomarker detection and the targeted specificity of drug delivery.

[0032] (2) Avoiding endocytosis failure and cytotoxicity, and improving functional efficacy and stability: This invention overcomes the technical defects of traditional cell backpack technology, which relies on "exogenous material anchoring to induce clathrin-mediated endocytosis." This invention eliminates the need to anchor exogenous materials on the cell surface, achieving function through the overexpression of target membrane proteins on the cell's own surface, thus avoiding carrier disintegration and functional material failure caused by endocytosis. Simultaneously, it avoids cytotoxicity caused by endocytosis, such as lysosomal stress, decreased mitochondrial membrane potential, and reactive oxygen species bursts, significantly improving the functional stability and application safety of engineered cells.

[0033] (3) Excellent biocompatibility, strong process controllability and suitability for large-scale production: The selected photocurable gel material can be degraded into natural biomolecules in vivo without any exogenous toxic residues. The amount of DMSO is strictly controlled within the safe concentration range of 5-10%, further ensuring biosafety. The entire preparation process (transfection, freeze-thaw, photocuring, centrifugation and washing) is highly standardized. The concentration of gel material, transfection efficiency, freeze-thaw parameters, photocuring conditions, etc. can all be precisely controlled, avoiding the uncertainty of random recombination of membrane fractions in traditional technologies, and is suitable for large-scale industrial production.

[0034] (4) Wide range of applications and value as a general-purpose technology platform: The dual-modal engineered cells of this invention can be adapted to various fields of needs, such as the detection of biomarkers for different diseases (e.g., AQP4-IgG detection for the diagnosis of neuromyelitis optica spectrum disorders), targeted drug delivery (e.g., anti-EGFR antibody delivery), and enhancement of cell therapy, by changing the target membrane protein gene. Especially in the application of lateral flow immunochromatographic test strips, it can replace the traditional antigen protein coated detection line. Combined with Nafion-CNF hybrid hydrogel modification and gelation cell blocking technology, it significantly improves the specificity and signal clarity of the detection, providing general-purpose technical support for the clinical translation of engineered cells.

[0035] (5) Reduce false positives and improve diagnostic reliability: In biological detection applications, the sample is sealed by untransfected gelled cells, which can specifically block the non-specific binding of impurities in the sample to engineered cells in the detection line, greatly reducing false positives and solving the diagnostic error problem caused by non-specific binding in traditional detection methods, thus improving the reliability of clinical detection. Attached Figure Description

[0036] 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).

[0037] 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).

[0038] Figure 3 The results show the morphological observations of the bimodal engineered cells and normal cells in Example 1.

[0039] 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).

[0040] Figure 5 The results show the microscopic morphological observation of the dual-modal engineered cells in Example 2.

[0041] Figure 6 The results of the confocal microscopy characterization experiment of the dual-modal engineered cells in Example 2 are as follows: (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 the target structure; The statistical graph compares the "fluorescence integral density / area of ​​HAMA" with and without DMSO, i.e., the fluorescence signal intensity of HAMA per unit area; Statistical results are expressed as mean ± standard deviation, n=5; *** indicates P < 0.0001).

[0042] Figure 7 Comparative images of gelled cell morphology prepared by different methods in Example 3.

[0043] Figure 8 The results show the targeting ability of different cells in Example 3.

[0044] Figure 9 A schematic diagram of the paper-based chip layer structure for application examples (A: Paper-based chip layer structure; Sample pad 1, Buffer pad 2, Binding pad 3, PVC backing plate 4, Reaction pad 5, Absorbent pad 6, Detection line 7, 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 control line 8 indicates a positive result, the appearance of a band only on control line 8 indicates a negative result).

[0045] Figure 10 The color intensity statistics of colloidal gold after treating NC membranes with different hydrogels are shown in the following application examples (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).

[0046] Figure 11 The results of the dual-modal engineered cell coating concentration condition optimization experiment are shown in the example (N represents negative sample; P represents positive sample).

[0047] Figure 12 The results of the verification experiment on the detection limit of the lateral flow immunochromatographic test strip for application examples were presented. Detailed Implementation

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] PEG-it TMThis 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] SA-MA (Methacrylated Sodium Alginate): By modifying the hydroxyl groups in the sodium alginate molecule with methacrylamide, it is endowed with photocrosslinking ability.

[0061] CMCS-MA (Carboxymethyl Chitosan Methacryloyl): A photocrosslinkable derivative obtained by introducing methacryloyl groups into water-soluble carboxymethyl chitosan (CMCS).

[0062] DexMA (Methacryloyl Dextran): A photocrosslinking derivative obtained by introducing methacryloyl groups onto the hydroxyl groups of the dextran molecule. DexMA can form hydrogels under ultraviolet or visible light initiation.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] Example 1: Construction of bimodal engineered cells (1) Targeted modification of cell membrane surface This method involves introducing genes encoding target proteins (cell membrane proteins) into target cells, enabling their stable expression on the cell membrane surface, thereby achieving targeted functional modification of the target cell membrane surface. This type of transgenic manipulation is a routine technique in molecular biology and cell engineering, widely adopted and highly standardized in basic research and preclinical applications. Currently, several mature and efficient gene delivery strategies can be used to achieve stable expression of exogenous genes in target cells. For example, lentiviral vector systems have advantages such as high infection efficiency and integration into the host genome, and are often used to construct stable expression cell lines; liposome-mediated transfection is simple to operate and has low toxicity, making it suitable for cell transfection; electroporation uses transient electrical pulses to create reversible micropores in the cell membrane, allowing exogenous DNA to efficiently enter the cell, making it suitable for cell transfection. All of these methods have been validated in numerous literature studies and commercial experimental protocols, effectively introducing genes encoding target proteins (cell membrane proteins) into target cells and reliably achieving functional expression of the target gene on the cell membrane surface. Therefore, the cell membrane surface modification strategy adopted in this study is entirely based on existing mature technology platforms and possesses good reproducibility and scalability. The specific operating procedure is briefly described below: Lentiviral / liposome transfection method: Seed cells into culture plates to achieve a cell density of 70%-90% at transfection. Replace the medium with serum-free medium 2 hours before transfection. Then, replace the original medium with pre-prepared serum-free cell culture medium containing the transfection complex (liposomes or lentivirus), incubate for 4-8 hours, aspirate the medium, add fresh serum-containing medium, and continue culturing for 24-48 hours. If the virus contains an resistance gene, begin screening for stable transfected cells after 3-4 days.

[0069] Electroporation transfection: Collect cells and suspend them in electroporation buffer; mix cells with nucleic acid and transfer them to an electroporation cup; set parameters for electroporation; transfer cells to a culture dish containing culture medium for further culture.

[0070] Through the above methods, engineered cells transfected with the target gene are obtained for subsequent intracellular gelation treatment.

[0071] More specifically, we will use AQP4 protein (aquaporin 4) as a target protein expressed on the cell surface as an example to illustrate the specific process of targeted modification of the cell membrane surface. In addition to AQP4 protein, other target proteins expressed on the cell surface can also be used according to actual application needs, thereby constructing engineered cells with target cell membrane proteins overexpressed on the cell surface to achieve targeted modification of the cell membrane surface.

[0072] The cell line used for gel cell preparation in this study was HEK-293T (human embryonic kidney epithelial cells), a highly efficient tool cell for transfection. The mRNA sequence of the human AQP4-M23 gene (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. Specifically, the synthesized human AQP4 gene CDS fragment was inserted into the multiple cloning site of the pCDH-MSCV-MCS-EF1-puro plasmid (System Biosciences, SBI) as the vector backbone to form the target gene expression plasmid. The human AQP4 gene CDS fragment (SEQ ID NO.1) is as follows:

[0073] 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.

[0074] 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 Reagents, mix and incubate overnight (≥ 8 h) at 4°C. Centrifuge at 1500×g for 30 min at 4°C, discard the supernatant, and resuspend the virus precipitate 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.

[0075] 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.

[0076] (2) Intracellular gelation treatment Prepare a cell hydrogel solution containing the engineered cells transfected with the target gene described above (final density 1×10⁻⁶). 6 -1×10 8 The hydrogel solution contains 2-3% (w / v) of gel material and 5-10% (v / v) of dimethyl sulfoxide (DMSO). The gel material can be at least one of methacryloyl hyaluronic acid (HAMA), methacryloyl gelatin (Gel-MA), and methacryloyl dextran (DexMA). The hydrogel solution is incubated at 2-8°C (preferably 4°C) for 15-30 min, then overnight at -75 to -85°C (preferably -80°C) for >8 h (preferably 8 h-30 d, more preferably 8 h-12 h; if inconvenient to handle, it can be stored for up to 30 days under these conditions). Then, the cells are incubated in a water bath at 37-40°C for 15-30 min (preferably 30 min) to obtain the hydrogel-treated engineered cells in a freeze-thawed cell-hydrogel mixture. The freeze-thawed cell-hydrogel mixture is centrifuged at 1200-1500 rpm for 3-5 min. Resuspend the lower cell pellet in 5-8 mL of PBS, centrifuge at 800-1000 rpm for 3-5 min, and repeat the washing process 2-3 times. After washing, resuspend in PBS with an equal volume of the original cell hydrogel solution. Finally, cure under light at 405 nm for 1-2 min. Add the protease inhibitor mixture at a 1:100-200 volume ratio and store at 4°C for later use. Following these steps, the bimodal engineered cells of this protocol are obtained.

[0077] The gel materials used in this technical solution, such as methacrylated hyaluronic acid (HAMA; EFL Tech, model EFL-HAMA-150K), methacrylated gelatin (Gel-MA; Aladdin, M398240), and methacrylated dextran (DexMA; Aladdin, M768078), possess excellent biocompatibility and photo-initiated cross-linking properties, which can reduce the number of freeze-thaw cycles and mitigate cell damage. Existing technologies typically use polyethylene glycol diacrylate (PEG-DA) as the gel material for preparing gelled cells. Besides photo-initiated cross-linking, other factors, especially temperature, can also induce cross-linking in this type of hydrogel. However, the freeze-thaw cycle in this method involves complex temperature changes, therefore conventional gel materials from existing technologies cannot be used.

[0078] More specifically, taking the preparation of bimodal engineered cells that overexpress AQP4 protein on the cell surface as an example, we will explain in detail: gelled HEK-293T cells (G-AQP4-293T) that overexpress AQP4 protein.

[0079] A cell hydrogel solution was prepared, containing engineered cells transfected with the AQP4 gene, 2% (w / v) methacrylamide hyaluronic acid (HAMA), and 5% (v / v) dimethyl sulfoxide (DMSO), which were then co-incubated. The cell density was controlled at 1×10⁻⁶ cells / year. 7 Approximately 10 cells / mL were collected, resulting in a total of 2-5 mL of cell hydrogel solution. The solution was incubated at 4°C for 30 min, then placed at -80°C overnight (approximately 10 h), and then thawed at 37°C for 30 min to obtain the hydrogel-treated engineered cells in the freeze-thawed cell hydrogel solution. Next, the freeze-thawed cell hydrogel solution was centrifuged at 1200 rpm for 5 min. 5 mL of PBS was added to resuspend the lower cell pellet, and the mixture was centrifuged at 800 rpm for 5 min. This washing process was repeated three times. After washing, an equal volume of PBS was added to resuspend the cells. Finally, the cells were photocured at 405 nm for 2 min. Finally, a protease inhibitor mixture was added at a 1:200 volume ratio, and the cells were stored at 4°C for later use. Through these steps, the bimodal engineered cells of this protocol were obtained; more specifically, these are bimodal engineered cells overexpressing AQP4 protein on their cell surface, i.e., the gelled HEK-293T cells (G-AQP4-293T) overexpressing AQP4 protein obtained in this embodiment.

[0080] 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.

[0081] 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 a gelling material added simultaneously, allowing the gelling material to penetrate the cells and form hydrogel-treated engineered cells. Finally, the cells are photocured to form the finished bimodal engineered cell product.

[0082] Following the method described in this embodiment, engineered cells transfected with the target gene (HEK-293T cells overexpressing AQP4 protein but not subjected to intracellular gelation) and dual-modality engineered cells (cells expressing AQP4 protein and subjected to gelation) were prepared, and then further analysis was 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 .

[0083] 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”.

[0084] 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 .

[0085] Example 2: The Dual Effect of DMSO as a Cell Cryoprotectant and Permeability Enhancer 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 5 This 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.

[0086] 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 6 .

[0087] 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.

[0088] Example 3: Comparison of morphology of gel cells prepared by different methods The bimodal engineered cells with surface overexpression of AQP4 protein prepared in Example 1 were used as gel cells ①.

[0089] Reference method (Cheng Gao, Targeted therapies of inflammatory diseases with intracellularly gelated macrophages in mice and rats, Nat Commun. 2024 Jan 6;15(1):328.) Preparation of gelled cells ②: HEK-293T cells were incubated with 2% Phe-CS solution (phenylalanine-modified chitosan solution, preparation method detailed in the literature), and then the culture dishes were frozen at -80℃ for 15 min. Afterwards, the culture dishes were removed from the freezer and placed in a 37℃ water bath for thawing. After thawing, the cells were quickly rinsed with PBS to remove residual Phe-CS on the cell surface. Then, 50 μM CD8 was added to the treated cell culture dishes and frozen at -80℃ for 15 min. Afterwards, the culture dishes were removed from the freezer and placed in a 37℃ water bath for thawing, and then rinsed with PBS to remove residual CD8 on the cell surface, obtaining gelled cells ②.

[0090] Referring to the method for preparing dual-modal engineered cells with surface overexpression of AQP4 protein in Example 1, the freeze-thaw process was changed from "standing at 4°C for 30 minutes, then placing at -80°C overnight for about 10 hours, and then thawing in a water bath at 37°C for 30 minutes" to "standing at 4°C for 30 minutes, then placing at -80°C overnight for about 10 hours, and then thawing in a water bath at 37°C for 30 minutes, repeating the above operation 3 times." This yielded gelled cells ③.

[0091] The morphology of the above gel cells was observed under a microscope, and the experimental results are as follows: Figure 7 As shown, the morphology of gel cells ① is significantly better than that of gel cells ② and ③, indicating that the method of reducing the number of freeze-thaw cycles and adding cell cryoprotectants helps to reduce the impact on the cell membrane, thereby maximizing the protection of the natural conformation of its surface proteins.

[0092] Example 4: Binding ability of dual-modal engineered cells to targets The following description and results are presented using bimodal engineered cells with surface overexpression of AQP4 protein prepared in Example 1. Normal cells were HEK-293T cells without AQP4 protein overexpression, and transfected cells were HEK-293T cells overexpressing AQP4 protein but without intracellular gelation treatment. The preparation method is described in Example 1.

[0093] 10 μL of each of the following cell suspensions were aspirated into a cell counting chamber: bimodal engineered cell suspensions (experimental group) stored for 0, 1, 5, 10, 20, and 30 days; fresh transfected cell suspensions without gelation treatment (positive control group); and fresh cell suspensions without transfection or gelation treatment (negative control group). Cell density was obtained by counting the cells. An appropriate amount of PBS was added to adjust the density of each cell suspension to 1 × 10⁻⁶. 7 Cells / mL. Take 1 mL of the above cell suspension and centrifuge at 800 rpm / min for 5 min. Discard the supernatant. Add a solution with a target concentration (antibody against AQP4 protein, AQP4-IgG) of 200 ng / mL to the above cell pellet, mix well and incubate for 1 h. Collect the supernatant by centrifugation, and detect the target concentration in the supernatant using an ELISA kit. Read the detection results using a microplate reader.

[0094] Experimental results are as follows Figure 8 As shown, normal cells, lacking overexpression of the corresponding target membrane protein, lack the ability to bind to the target; therefore, the target concentration in the supernatant is close to the original concentration (200 ng / mL). Meanwhile, although transfected cells overexpressing the corresponding target membrane protein can bind to the target, they have not undergone intracellular gelation and cannot be stored for extended periods. Therefore, after 5 days of storage, due to cell death and membrane structure collapse, they essentially lose their ability to capture the target. In contrast, the dual-modal engineered cells prepared using this method maintained low target concentrations in the supernatant for 30 days, indicating that the cells captured and bound most of the target and possessed long-term stability. See detailed experimental results below. Figure 8 .

[0095] Application Example 1 The bimodal engineered cells in this protocol can be used to prepare lateral flow immunochromatographic test strips. These cells can replace the capture probes coated on the detection line (T line) of the lateral flow immunochromatographic test strip. The capture probes are typically used to specifically bind to a "target-specific probe complex." The binding pad is coated with a specific probe, which usually consists 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 probe can bind to and label the target analyte, forming a "target-specific probe complex."

[0096] More specifically as follows: (1) Structure and preparation method of lateral flow immunochromatographic test strips The following explanation uses a lateral flow immunochromatographic test strip for detecting AQP4-IgG as an example.

[0097] The lateral flow immunochromatographic test strip for detecting AQP4-IgG consists of a shell formed by the interlocking of a top and bottom layer, and a middle paper-based chip layer. The shell is made of modified plastic or polyvinyl chloride. See [link to paper-based chip layer design] for details. Figure 9 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.

[0098] (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.

[0099] 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.

[0100] (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.

[0101] 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.

[0102] 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℃).

[0103] (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 9 B.

[0104] (2) Modification of NC membranes by 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.

[0105] 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 10 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.

[0106] 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.

[0107] Table 1: Percentage of cells with intact morphology (Experimental results are the average of 5 replicate experiments)

[0108] 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.

[0109] 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 the Nafion-CNF hybrid hydrogel, gel cell morphology is well maintained, allowing the AQP4 protein to retain its native spatial conformation. This fully exposes the specific binding sites for AQP4-IgG, directly improving the binding efficiency and stability. The portable detection device in this protocol can maintain good gel cell morphology even after extended storage, thus ensuring detection effectiveness over longer periods.

[0110] 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.

[0111] (3) Optimization of gelling cell (G-AQP4-293T) coating concentration conditions A portable device for point-of-care AQP4-IgG detection was prepared according to the aforementioned method, and samples were added for testing. Except for the coating concentration of gelatinocytes (G-AQP4-293T), all other procedures were performed optimally to investigate the effect of the gelatinocyte (G-AQP4-293T) coating concentration on the 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 the 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 11 As shown: the coating concentration is 10. 7 At a cell / mL ratio, positive results show clear color development, and no non-specific reactions occur after blocking. If the sample to be tested is not blocked (without incubating the sample with G-293T cells), false positives will occur.

[0112] (4) Detection limit of lateral flow immunochromatographic test strips A portable device for point-of-care detection of AQP4-IgG was prepared according to the aforementioned method, and samples were added for detection in the optimal manner. 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. The samples were blocked before loading for detection. The blocking method was as follows: 100 μL of the sample to be tested was aspirated and mixed with pre-prepared G-293T cell pellet to achieve a cell density of 102. 7 Cells / mL, incubated at room temperature for 5 min. Experimental results are as follows: Figure 12 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.

[0113] 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 dual-modal engineered cell based on cell transfection and intracellular gelation, characterized in that: Its cell surface overexpresses the target cell membrane protein; its cells gel.

2. The dual-modal engineered cell based on cell transfection and intracellular gelation according to claim 1, characterized in that: The gelling material used for intracellular gelation includes at least one of methacrylamide hyaluronic acid, methacrylamide gelatin, and methacrylamide dextran.

3. The dual-modal engineered cell based on cell transfection and intracellular gelation according to claim 2, characterized in that: The intracellular gelation method is as follows: prepare a hydrogel solution containing cells transfected with the target cell membrane protein gene, gel material and dimethyl sulfoxide, and after treatment at 2-8℃ for 15-30 min, freeze at -75 to -85℃ for >8 h; next, thaw in a water bath at 37-40℃ for 15-30 min; then centrifuge to obtain cells, wash the cells, resuspend the cells, and obtain bimodal engineered cells by photocuring.

4. The dual-modal engineered cell based on cell transfection and intracellular gelation according to claim 3, characterized in that: The gel material has a volume percentage of 2-3%; the dimethyl sulfoxide has a volume percentage of 5-10%. The photocuring process involves irradiating the cells resuspended in the buffer solution with 405nm ultraviolet light for 1-2 minutes.

5. The dual-modal engineered cell based on cell transfection and intracellular gelation according to claim 4, characterized in that: Cells transfected with the target cell membrane protein gene were obtained by lentiviral transfection, liposome transfection, or electroporation transfection. Preferably, the cells used are human embryonic kidney epithelial cell lines; Preferably, when using the lentiviral transfection method, a lentiviral expression vector integrating the target cell membrane protein gene is first constructed, and then packaged to obtain viral fluid; the viral fluid is used to transfect cells to obtain cells transfected with the target cell membrane protein gene.

6. A method for preparing bimodal engineered cells based on cell transfection and intracellular gelation according to any one of claims 1-5, characterized in that: The following steps are performed sequentially: S1: Prepare cells transfected with the target cell membrane protein gene; S2: Prepare a hydrogel solution containing cells transfected with the target cell membrane protein gene, gel material, and dimethyl sulfoxide. After standing at 2-8℃ for 15-30 min, freeze at -75 to -85℃ for >8 h. Next, thaw in a water bath at 37-40℃ for 15-30 min. Then, centrifuge to collect cells, wash the cells, resuspend the cells, and cure them by light to obtain bimodal engineered cells.

7. The method for preparing dual-modal engineered cells based on cell transfection and intracellular gelation according to claim 6, characterized in that: In S2, the mass-volume percentage of the gel material is 2-3%; the volume percentage of dimethyl sulfoxide is 5-10%; the photocuring is performed by irradiating the cells resuspended in the buffer with 405nm ultraviolet light for 1-2 minutes. The gel material includes at least one of methacrylamide hyaluronic acid, methacrylamide gelatin, and methacrylamide dextran.

8. The method for preparing dual-modal engineered cells based on cell transfection and intracellular gelation according to claim 6, characterized in that: In S1, cells transfected with the target cell membrane protein gene are obtained by lentiviral transfection, liposome transfection, or electroporation transfection.

9. The application of a dual-modal engineered cell based on cell transfection and intracellular gelation according to any one of claims 1-5 in the preparation of lateral flow immunochromatographic test strips, characterized in that: The detection line of the reaction pad of the lateral flow immunochromatographic test strip is coated with dual-modal engineered cells; The lateral flow immunochromatographic test strip, combined with a sample blocking reagent, forms a detection kit. The sample blocking reagent is gelled cells. The gelled cells are prepared by the following method: untransfected cells, gel material, and dimethyl sulfoxide are mixed to form a hydrogel solution. After freezing and thawing, the cells are washed and resuspended, and then photocured to obtain gelled cells.

10. The application of a dual-modal engineered cell based on cell transfection and intracellular gelation according to claim 9 in the preparation of lateral flow immunochromatographic test strips, characterized in that: Lateral flow immunochromatographic test strips are used to detect AQP4-IgG; The bimodal engineered cells coated on the detection line of the reaction pad are gelled cells overexpressing AQP4 protein; 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.

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