Disposable electrochemical biosensor detection kit

Abstract

The invention discloses a disposable electrochemical biosensor detection kit, and belongs to the technical field of in-vitro diagnosis. The kit is a disposable in-vitro diagnosis product and is divided into two product systems, namely a molecular detection system and an immunodetection system; the core components of the molecular detection kit are an integrated disposable H-type electrolytic cell, an isothermal amplification buffer solution, a positive control solution, a negative control solution and a blank control solution. The core components of the immunodetection kit are an integrated disposable H-type electrolytic cell, a detection reagent, a substrate reagent, a calibrator I, a calibrator II, a quality control product I and a quality control product II. The kit takes a functionalized nuclear track membrane as a core, the nuclear track membrane modifies corresponding target recognition probes in advance according to different detection targets, and the target recognition probes are pre-sealed and fixed in an H-shaped electrolytic cell to form a non-detachable integrated detection unit with the electrolytic cell; transmembrane transmission of a sample is realized through pressurization of a single inflation port, and high-precision detection can be realized by combining isothermal amplification or a double-antibody sandwich technology and an electrochemical sensing technology.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic technology, specifically relating to a single-use, full-component detection kit based on functionalized nuclear pore membranes and electrochemical sensing technology. Background Technology

[0002] In vitro diagnostics (IVD) is a core supporting tool for early screening, accurate diagnosis, dynamic monitoring of disease progression, and personalized medication guidance in clinical practice, accounting for over 70% of information sources in clinical diagnosis and treatment decisions. However, current mainstream IVD technologies still face several intractable industry challenges: 1. In the field of nucleic acid molecular detection, quantitative real-time PCR, as the clinical gold standard, heavily relies on high-precision temperature-switched amplification instruments. These instruments are costly, lack portability, and are difficult to popularize in primary healthcare institutions and point-of-care testing scenarios. Furthermore, the operation process is cumbersome, with the entire testing cycle typically taking over 2 hours. 1. For tests lasting over 24 hours, the current methods cannot meet the needs of emergency and rapid on-site screening, and suffer from problems such as easy contamination of amplified products and high false positive rates; 2. In the field of protein immunoassay, chemiluminescence and enzyme-linked immunosorbent assays are the current mainstream technologies, but they generally suffer from problems such as large sample volume, long reaction incubation time, multiple operation steps, and weak anti-interference ability; the test results are easily affected by environmental and operational factors, have poor batch-to-batch stability, and rely on large-scale testing equipment, making it impossible to achieve rapid point-of-care quantitative detection; 3. Electrochemical biosensors, as a cutting-edge technology integrating biorecognition and electrochemical signal conversion, have core advantages such as fast response, simple operation, detectability of trace samples, and real-time output results, and are a key development direction in the field of in vitro diagnostics. However, existing electrochemical biosensor technologies still have significant shortcomings: Firstly, the sensing carriers mostly use traditional glassy carbon electrodes, First, screen-printed electrodes suffer from poor biocompatibility and limited specific surface area, resulting in low immobilization efficiency and poor binding stability of biorecognition elements, leading to low signal-to-noise ratio and insufficient repeatability. Second, existing nuclear pore membrane-based electrochemical sensing devices use nuclear pore membranes with low pore density and poor channel uniformity, making it impossible to achieve high-density immobilization of biorecognition elements, and the detection sensitivity and detection limit are insufficient to meet the clinical needs for micro-target detection. Third, existing technologies can only realize single-molecule or immunoassay detection systems, which cannot be compatible with the two core detection scenarios, resulting in poor versatility and limited clinical application scenarios. Fourth, there is a lack of complete and supporting disposable reagent kit systems, and the core detection units cannot be fully enclosed for single use, posing a risk of cross-contamination. Furthermore, there are no supporting reference standards, calibrators, and quality control products, which cannot meet the full-process quality control requirements of clinical testing and hinder industrialization.

[0003] Therefore, developing an electrochemical biosensor detection kit that combines high detection performance, dual-system compatibility, complete supporting components, single-use of all components, and standardized mass production capability has become a core issue that the industry urgently needs to address. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects of the prior art and solve the core problems of existing in vitro diagnostic technologies, such as high dependence on equipment, long detection cycle, cumbersome operation, poor versatility, risk of cross-contamination from repeated use, and insufficient detection performance. The invention provides a disposable electrochemical biosensor detection kit that is compatible with both molecular detection and immunoassay systems. It uses a universal integrated disposable H-type electrolytic cell as the core detection unit to achieve rapid, accurate, standardized, and pollution-free detection of multiple targets and scenarios in clinical settings.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: The present invention provides a disposable electrochemical biosensor detection kit, which is an in vitro diagnostic product for single use. It is divided into two major product systems: molecular detection and immunoassay. The two systems share the same standardized disposable H-type electrolytic cell structure design. For the detection requirements of different detection targets, it is only necessary to replace the pre-labeled target recognition probe on the functionalized nuclear pore membrane during the prefabrication stage, and then pre-seal and fix the modified functionalized nuclear pore membrane in the H-type electrolytic cell to make an integrated disposable H-type electrolytic cell detection unit for the corresponding target. There is no need to adjust the overall structure and core components of the kit. The prepared detection unit is used once with the whole kit and cannot be disassembled or reused.

[0006] The core components of the molecular detection kit include a disposable H-type electrolytic cell, isothermal amplification buffer, positive control, negative control, and blank control solution. Nucleic acid extraction reagents, washing solutions, and other auxiliary components can be added according to detection needs. This kit is suitable for in vitro qualitative detection of various nucleic acid targets, such as tumor gene methylation, microRNA, pathogenic fungi, and viral / bacterial nucleic acids. Only the specific primers in the isothermal amplification buffer and the pre-modified specific nucleic acid probes on the functionalized nuclear pore membrane need to be replaced to prepare disposable kits for different target detections. The immunoassay... The core components of the test kit include a disposable H-type electrolytic cell, detection reagents, substrate reagents, calibrator 1, calibrator 2, quality control 1, and quality control 2. Auxiliary components such as sample diluents and washing solutions can be added according to testing requirements. This kit is suitable for the in vitro quantitative detection of various protein / small molecule targets, including Alzheimer's disease biomarkers, myocardial injury biomarkers, biogenic amine neurotransmitters, tumor markers, and inflammatory factors. Simply replacing the labeled antibody in the detection reagent and the pre-modified specific capture antibody on the functionalized nuclear pore membrane allows for the preparation of disposable kits for different target detections.

[0007] Furthermore, the disposable H-type electrolytic cell is the core detection unit of the kit. It is an integrated, non-reusable H-type electrolytic cell structure, containing two independent and symmetrical sample chambers and a detection chamber. A functionalized nucleopore membrane is pre-sealed between the two chambers, forming an inseparable closed electrolytic cell system. The entire system is discarded after use. The functionalized nucleopore membrane is not only the physical separating medium between the two chambers, but also the core reaction region for target recognition and electrochemical signal conversion. During the prefabrication stage, the corresponding target recognition probe is modified and sealed within the electrolytic cell. It is used once and for all with the electrolytic cell.

[0008] The functionalized nuclear pore membrane has target recognition probes labeled on its inner walls. These probes include various biorecognition elements such as nucleic acid complementary probes, monoclonal antibodies, aptamers, and peptides, which can be arbitrarily replaced during the prefabrication stage according to the detection target. After pre-modification, the membrane is sealed and assembled in a disposable H-type electrolytic cell. The disposable H-type electrolytic cell is equipped with 2-3 electrodes, all prefabricated and fixed in the sample chamber and the detection chamber, and is used once with the entire electrolytic cell. The electrolytic cell has 1-3 injection ports for the injection and discharge of samples and reagents in the corresponding chambers. It also has one gas inlet connected to a controllable pressurization device to pressurize and inflate the electrolytic cell chambers. The pressure changes in the chambers create a pressure interaction that drives the sample solution to be transported from one end chamber of the electrolytic cell across the functionalized nuclear pore membrane to the other end chamber. This significantly improves the binding efficiency of the target and the recognition probes on the nuclear pore membrane, shortens the reaction time, and ensures sufficient contact between the target and the recognition probes, thereby improving detection sensitivity.

[0009] Further, the specific preparation method of the functionalized core-pore membrane is as follows: 1. Substrate pretreatment: Using a polymer membrane bombarded by heavy ions as the substrate, the membrane is ultrasonically cleaned alternately with anhydrous ethanol and deionized water to ensure that the substrate surface is free of impurities; 2. Chemical etching: The pretreated polymer membrane bombarded by heavy ions is placed in an etching solution and etched at a constant temperature of 40-95℃ for 2-60 min. The etching solution is a sodium hydroxide solution with a concentration range of 3-10 mol / L. During the etching process, the membrane is stirred at a uniform speed to ensure etching uniformity. After etching, the membrane is immediately rinsed with deionized water until neutral to prepare a porous membrane containing multiple uniform nanopores; 3. Surface activation treatment: The etched porous membrane is placed in an activation solution and activated at room temperature for 0.5-3 h. An EDC mixture is used; after activation, the membrane is rinsed with the corresponding buffer solution to remove residual activation solution; 4. Probe fixation: The activated membrane is then... The porous membrane is then placed in a target recognition probe solution and incubated at 2-8℃ for 2-6 hours to allow the probe to be stably fixed to the active groups on the inner wall of the pores via covalent bonding. After incubation, it is rinsed with washing solution to thoroughly remove unbound free probes and avoid non-specific binding. 5. Active site blocking: The membrane with fixed probes is placed in a blocking solution and blocked at room temperature for 0.5-3 hours to block the remaining unreacted active sites on the inner wall of the pores and avoid non-specific adsorption during the detection process. The blocking solution is bovine serum albumin solution or glycine solution. After blocking, it is rinsed with washing solution to obtain the modified functionalized nuclear pore membrane. The prepared functionalized nuclear pore membrane is pre-sealed and fixed in a disposable H-type electrolytic cell to form an integrated disposable detection unit. The whole unit is for single use and cannot be disassembled or reused. The unassembled functionalized nuclear pore membrane is sealed and dried at 2-8℃ for later use.

[0010] Furthermore, the standard composition of each component of the kit is as follows, which can be adapted and adjusted according to the requirements of the detection target and reaction system: 1. Isothermal amplification buffer: This is a dedicated reaction system for loop-mediated isothermal amplification (LAMP). The core components include Bst DNA polymerase, isothermal amplification-specific primer set, thermostable uracil DNA glycosidase, Tris-HCl buffer, MgCl2, dNTPs, and amplification promoters. The amplification promoters include betaine, dimethyl sulfoxide, formamide, and glycerol. It can achieve efficient and specific amplification of target nucleic acids under isothermal conditions of 63℃±1℃, without primer dimers or non-specific amplification; 2. Detection reagent: The core is a target-specific labeled antibody solution. The labels include alkaline phosphatase, horseradish peroxidase, and glucose oxidase. The antibody is preferably a monoclonal antibody, which, along with the capture antibody immobilized on the nuclear pore membrane, targets different antigenic epitopes of the target protein and has no cross-reactivity. The system contains PBS buffer, protective protein (bovine serum albumin), and the preservative ProClin. 300; 3. Substrate reagent: Contains chromogenic substrate components and buffer systems matched with the label. The substrate system for alkaline phosphatase labeling contains 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitrotetrazole blue chloride (NBT). The substrate system for horseradish peroxidase labeling contains TMB or OPD chromogenic substrate, along with Tris-HCl buffer or citrate-disodium hydrogen phosphate buffer. It can specifically react with the labeled enzyme to form an insoluble precipitate, causing blockage of the nuclear pore membrane channels and triggering a detectable change in the current signal; 4. Positive control: A buffer system containing target substances of known concentration and activity. Nucleic acid targets correspond to plasmid DNA containing the target sequence or artificially synthesized nucleic acid fragments. Protein targets correspond to recombinant target antigens. The system contains TE or PBS buffer, protective protein, and the preservative ProClin. 300, used to verify the effectiveness of the amplification / immunoassay system and electrochemical detection system, and to investigate the risk of detection system failure; 5. Negative control: a blank matrix buffer system without the target substance, using a blank matrix homologous to clinical test samples, including human genomic DNA and human sputum matrix, containing PBS buffer, protective protein and preservative ProClin 300, used to investigate system contamination and non-specific amplification / binding reactions; 6. Blank control solution: a blank buffer system without the target substance, the core of which is nuclease-free water or sterile PBS buffer, containing preservative ProClin 300, used to replace the sample to construct the blank reaction system, and to help investigate exogenous contamination from the environment, consumables, and operation process; 7.Calibrator 1 and Calibrator 2: These are buffer systems containing recombinant target antigens at different concentration gradients. The concentrations of the two calibrators cover the lower and upper limits of the linear range for clinical testing, respectively. The systems contain PBS buffer, protective proteins, and the preservative ProClin 300. Target values ​​are provided on the kit's target value sheet and are used to construct a standard curve for accurate quantitative calculation of the target concentration in the sample. Quality Control 1 and Quality Control 2: These are buffer systems containing recombinant target antigens at fixed concentration ranges, divided into low-value and high-value controls, covering clinically critical levels. The systems contain PBS buffer, protective proteins, and the preservative ProClin 300. Target ranges are provided on the kit's target value sheet and are used to monitor the stability of the detection system and the accuracy of the results, ensuring reliable clinical test results.

[0011] Compared with existing technologies, this invention has the following significant and outstanding advantages: 1. Dual-system compatibility, extremely strong versatility and adaptability: This invention adopts a standardized structure of a universal disposable H-type electrolytic cell, which is compatible with both nucleic acid molecular detection and protein immunoassay, two core in vitro diagnostic systems. Only the pre-modified recognition probes and matching reagents on the nuclear pore membrane need to be replaced in the prefabrication stage to cover the detection needs of hundreds of targets in various clinical scenarios such as early tumor screening, infection diagnosis, emergency detection of cardiovascular diseases, and screening for neurodegenerative diseases. There is no need to redevelop the detection device, which greatly reduces the cost of research and development, mass production and clinical application, and solves the core pain points of poor versatility and limited application scenarios of existing technologies; 2. High-pore-density functionalized nuclear pore membrane, achieving a qualitative improvement in detection performance: This invention clarifies the standardized preparation process of functionalized nuclear pore membranes. Through a controllable process of heavy ion bombardment-chemical etching, a pore density of 1×10⁻⁶ is obtained. 4 ~1×10 13 pcs / cm 2The nanoporous membrane, with its ultra-high pore density resulting in an ultra-large specific surface area, enables ultra-high density immobilization of biorecognition elements. The number of target binding sites is increased by more than two orders of magnitude compared to traditional nuclear pore membranes, significantly improving the signal-to-noise ratio and sensitivity of the detection signal, meeting the stringent requirements of clinical micro-target detection. Simultaneously, the standardized preparation process ensures batch-to-batch pore uniformity deviation of the nuclear pore membrane ≤3% and batch-to-batch difference in probe immobilization efficiency ≤2%, providing a core guarantee for standardized clinical detection. 3. Breakthrough improvements in detection efficiency and convenience: The molecular detection of this invention... The kit integrates LAMP isothermal amplification and electrochemical sensing technology, eliminating the need for a precision temperature-controlled PCR instrument. Amplification can be completed using only a standard constant-temperature metal bath, and the entire detection process from sample to result can be completed within 30-50 minutes, requiring only 5-10 μL of nucleic acid sample. The immunoassay kit utilizes nanopore-blocked electrochemical sensing combined with a double-antibody sandwich method, enabling full-process quantitative detection within 5-20 minutes. The sample volume requires only 50-100 μL, and the entire operation is extremely simple, requiring only 3-5 steps and no professional expertise. Personnel training is all that's needed, completely eliminating reliance on large, precision instruments and perfectly adapting to various application scenarios such as primary hospitals, emergency rooms, bedside testing, and on-site screening; 4. Integrated disposable structural design significantly improves detection safety, stability, and binding efficiency: The disposable H-type electrolytic cell of this invention adopts an integrated, non-separable design. After the functionalized nuclear pore membrane is pre-assembled, it is used once as a whole with the electrolytic cell, fundamentally eliminating the risk of sample cross-contamination and test result distortion caused by reuse, and greatly improving the safety and compliance of clinical testing; At the same time, it adopts an optimized flow path design with a single injection port + a single air inlet, and drives sample transmembrane transmission through the pressure interaction of controllable pressurization. Compared with the passive diffusion mode, the binding efficiency of the target and the recognition probe on the nuclear pore membrane is increased by more than 6 times, and the reaction time is shortened by more than 50%, while avoiding sample residue and dead volume, ensuring the repeatability of test results; The pre-fabricated fixed compatible design with 2-3 electrodes ensures the accuracy and stability of current signal acquisition, with intra-batch coefficient of variation ≤5% and inter-batch coefficient of variation ≤8%, which is far superior to similar products in the industry. Attached Figure Description

[0012] Figure 1 This is a schematic cross-sectional view of the disposable H-type electrolytic cell in the electrochemical biosensor detection kit of the present invention;

[0013] Figure 2 This is a schematic diagram illustrating the preparation of the functionalized nuclear pore membrane of the present invention;

[0014] Figure 3 This is a flowchart illustrating the detection principle of the molecular detection kit of the present invention;

[0015] Figure 4 This is a flowchart illustrating the detection principle of the immunoassay kit of the present invention.

[0016] The markings in the diagram are as follows: 1-Electrolytic cell body, 2-Sample chamber, 3-Detection chamber, 4-Functionalized nucleopore membrane, 5-Injection port, 6-Gas inlet, 7-Working electrode, 8-Reference electrode, 9-Electrolyte. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are only used to explain the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0018] Example 1: Preparation and Pre-assembly of a Functionalized Nuclear Pore Membrane Adapted for Human Septin9 Gene Methylation Detection: This example describes the preparation of a functionalized nuclear pore membrane adapted for human Septin9 gene methylation detection, and its integrated pre-assembly with a disposable H-type electrolytic cell. The specific steps are as follows: 1. Substrate Pretreatment: Take a PET membrane bombarded by a heavy ion accelerator and ultrasonically clean it three times alternately with anhydrous ethanol and deionized water; 2. Chemical Etching: Place the pretreated PET membrane in a 6 mol / L sodium hydroxide etching solution and etch it at a constant temperature of 55°C for 8 minutes. After etching, immediately rinse it with deionized water until neutral; 3. Surface Activation Treatment: Place the etched porous membrane in a solution containing 5 mg / mL sodium hydroxide... 4. Probe immobilization: The activated membrane was placed in a 10 μmol / L Septin9 gene methylation-specific complementary probe solution and incubated for 4 hours to allow the amino terminus of the probe to covalently bind to the carboxyl group in the membrane pores. After incubation, the membrane was washed three times with washing solution to remove unbound free probes. 5. Active site blocking: The membrane with immobilized probes was placed in a 10% glycine blocking solution and blocked at room temperature for 1 hour to block unreacted active sites. After blocking, the membrane was washed three times with washing solution to obtain a functionalized nuclear pore membrane adapted for human Septin9 gene methylation detection. 6. Integrated pre-assembly: The prepared functionalized nuclear pore membrane was pre-sealed and fixed between the two chambers of a disposable H-type electrolytic cell to form an integrated disposable detection unit for single use. The unassembled functionalized nuclear pore membrane was sealed and dried at 4°C for later use.

[0019] Example 2: Human Septin9 Gene Methylation Detection Kit (Molecular Detection): This kit is used for in vitro qualitative detection of Septin9 gene methylation levels for early non-invasive screening of colorectal cancer. It is available in kits of 50 samples and is a single-use product. The core components include: 50 disposable H-type electrolytic cells (pre-assembled with functionalized nuclear pore membranes prepared in Example 1), 220 μL / tube × 5 tubes of isothermal amplification buffer, 0.5 mL / bottle of positive control, 0.5 mL / bottle of negative control, and 1 mL / bottle of blank control solution.

[0020] The disposable H-type electrolytic cell uses a high-purity polytetrafluoroethylene (PTFE) tank and is an integrated, non-reusable structure. The functionalized nuclear pore membrane prepared in Example 1 is pre-sealed between the two chambers, forming an inseparable disposable detection unit. The electrolytic cell is equipped with two pre-fabricated electrodes: a glassy carbon working electrode and an Ag / AgCl reference electrode, fixed in the sample chamber and detection chamber respectively. The tank has one injection port and one gas inlet, with the gas inlet connected to a micro-controllable pressurization device. The entire electrolytic cell is for single use with the kit and cannot be disassembled repeatedly. The isothermal amplification buffer contains Bst DNA polymerase, Septin9 gene methylation-specific LAMP primers, thermostable uracil DNA glycosidase, Tris-HCl buffer, MgCl2, dNTPs, and betaine. The positive control contains a known methylated Septin9 gene plasmid fragment, TE buffer, bovine serum albumin, and 0.05% ProClin. 300; Negative control contained human genomic DNA without methylated Septin9 gene, PBS buffer, bovine serum albumin, and 0.05% ProClin 300; Blank control contained nuclease-free water, PBS buffer, bovine serum albumin, and 0.05% ProClin 300.

[0021] The detection process of this kit is as follows: 1. Sample processing: Take 2 mL of genomic DNA and transform the extracted DNA using a sulfite conversion reagent. Unmethylated cytosine undergoes deamination to generate uracil sulfonate, while methylated cytosine retains its chemical structure. 2. Isothermal amplification: Take 5 μL of the transformed DNA sample and mix it with 20 μL of isothermal amplification buffer to construct a 25 μL reaction system. Simultaneously set up positive control, negative control, and blank control systems. Place each reaction system in a 63℃ isothermal metal bath for amplification for 30 min. 3. Electrochemical detection: Add the amplification product to a disposable H-type electrolytic capacitor through the injection port. The sample chamber of the electrolytic cell is pressurized to 0.03 MPa through the gas inlet, driving the amplification product to be transported across the functionalized nuclear pore membrane to the detection chamber. This ensures that the amplification product fully binds to the specific probes on the nuclear pore membrane. The electrochemical workstation is then activated to record the current response signal I2, while simultaneously detecting the initial current signal I1 of the nuclear pore membrane that has not bound the amplification product. 4. Result interpretation: Calculate the I2 / I1 ratio. A ratio > 0.98 indicates a negative result, a ratio < 0.94 indicates a positive result, and a ratio between 0.94 and 0.98 is considered a gray area, requiring re-extraction of the sample for testing. 5. After the test is completed, the disposable H-type electrolytic cell is discarded along with the entire kit and cannot be reused.

[0022] Example 3: Human Phosphorylated Tau-217 Protein Assay Kit (Immunoassay): This kit is used for the in vitro quantitative detection of phosphorylated Tau-217 protein concentration for early auxiliary diagnosis of Alzheimer's disease. It is a single-use product with 50 test kits per kit. The core components include: 50 disposable H-type electrolytic cells, 2.5 mL / bottle of assay reagent, 2.5 mL / bottle of substrate reagent, 0.5 mL / bottle of calibrator 1, 0.5 mL / bottle of calibrator 2, 0.5 mL / bottle of quality control sample 1, and 20.5 mL / bottle of quality control sample 2.

[0023] The disposable H-type electrolytic cell uses a high-purity polytetrafluoroethylene (PTFE) tank and is an integrated, non-reusable structure. A functionalized nucleopore membrane is pre-sealed between the two cavities, forming an inseparable disposable detection unit. This nucleopore membrane is prepared according to the method in Example 1, except that the substrate pore density is 1×10⁻⁶. 7 pcs / cm 2 The immobilized probe is a phosphorylated Tau-217 protein-specific monoclonal capture antibody. The electrolytic cell comes pre-installed with two electrodes: a glassy carbon working electrode and an Ag / AgCl reference electrode, fixed in the sample chamber and detection chamber respectively. The cell has one injection port and one gas inlet. The electrolytic cell is a single-use kit and cannot be disassembled repeatedly. The detection reagent is an alkaline phosphatase-labeled phosphorylated Tau-217 protein-specific detection antibody solution containing PBS buffer, bovine serum albumin, and 0.05% ProClin. 300; substrate reagents contain BCIP, NBT, and Tris-HCl buffer; calibrator one contains 5 pg / mL recombinant phosphorylated Tau-217 antigen, calibrator two contains 20 pg / mL recombinant phosphorylated Tau-217 antigen, quality control one contains 3 pg / mL recombinant phosphorylated Tau-217 antigen, quality control two contains 10 pg / mL recombinant phosphorylated Tau-217 antigen, all containing PBS buffer, bovine serum albumin, and 0.05% ProClin 300.

[0024] The detection process of this kit is as follows: 1. Immunoreaction: Take 50 μL of the test sample, calibrator, and quality control, and mix them with 50 μL of the detection reagent. Incubate at room temperature for 15 min. Add the mixture to the sample chamber of the disposable H-type electrolytic cell through the injection port. Apply a pressure of 0.02 MPa through the gas inlet to drive the mixture to transport across the membrane, so that the target antigen in the sample forms a "capture antibody-antigen-enzyme-labeled antibody" sandwich complex with the capture antibody and enzyme-labeled detection antibody on the nuclear pore membrane. Wash three times with washing solution to remove unbound free substances. 2. Substrate reaction: Add substrate reagent to the disposable H-type electrolytic cell and incubate at room temperature for 3 min. The base on the enzyme-labeled antibody reacts with the substrate reagent. 1. Phosphatase reacts specifically with the substrate to form an insoluble blue-purple precipitate, which deposits within the nanopores of the nuclear pore membrane, causing pore blockage and triggering changes in the current signal; 2. Quantitative detection: Turn on the electrochemical workstation and record the current change value I2-I1. Establish a standard curve with the calibrator concentration as the x-axis and I2-I1 as the y-axis. Calculate the concentration of phosphorylated Tau-217 protein in the sample based on the I2-I1 value; 3. Quality control judgment: The test results are valid if the test results of quality control sample 1 and quality control sample 2 are within the target value range indicated in the kit instructions; 4. After the test is completed, the disposable H-type electrolytic cell is discarded along with the entire kit and cannot be reused.

[0025] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A disposable electrochemical biosensor detection kit, characterized in that, The kit is a single-use in vitro diagnostic product, comprising two main product systems: molecular detection and immunoassay. The molecular detection kit includes a disposable H-type electrolytic cell, isothermal amplification buffer, positive control, negative control, and blank control solution. The immunoassay kit includes a disposable H-type electrolytic cell, detection reagents, substrate reagents, calibrator one, calibrator two, quality control one, and quality control two. The disposable H-type electrolytic cell is an integrated, non-reusable H-type electrolytic cell structure. A functionalized nuclear pore membrane is pre-sealed between the two independent chambers of the H-type electrolytic cell, forming an inseparable single-use detection unit that is discarded entirely after use. It is not reusable or re-disassembled; the inner wall of the functionalized nuclear pore membrane is pre-marked with target recognition probes corresponding to the detection targets. For different detection targets, only the pre-marked target recognition probes on the functionalized nuclear pore membrane need to be replaced in the prefabrication stage to complete the preparation of the disposable H-type electrolytic cell for the corresponding target, without adjusting the overall structure of the electrolytic cell and the core components of the reagent kit; the disposable H-type electrolytic cell is equipped with 2-3 electrodes, and 1-3 liquid injection ports and 1 gas filling port are provided in the corresponding chamber; the gas filling port is used to pressurize and fill the chamber of the electrolytic cell, and the sample solution can be driven from one end chamber of the electrolytic cell to the other end chamber through pressure interaction.

2. The disposable electrochemical biosensor detection kit according to claim 1, characterized in that, The functionalized nucleopore membrane is prepared by using a polymer membrane bombarded by heavy ions as a substrate, followed by chemical etching to prepare a thin film containing multiple nanopores, followed by activation treatment, then pre-fixing the target recognition probe corresponding to the detection target, and finally sealing the active sites. The prepared functionalized nucleopore membrane is pre-sealed and fixed between the two chambers of a disposable H-type electrolytic cell, forming an integrated disposable structure with the electrolytic cell, and is used once and for all with the electrolytic cell.

3. The disposable electrochemical biosensor detection kit according to claim 1, characterized in that, The conventional composition of each component of the kit is as follows: (1) Isothermal amplification buffer: containing Bst DNA polymerase, isothermal amplification specific primers, thermostable uracil DNA glycosidase, Tris-HCl buffer, MgCl2, dNTPs and amplification promoter; (2) Detection reagent: containing a buffer system with target-specific labeled antibodies, containing protective proteins and preservatives; (3) Substrate reagent: containing chromogenic substrate components and buffer system; (4) Positive control: A buffer system containing a known concentration of the target antigen, containing protective proteins and preservatives; (5) Negative control: A blank matrix buffer system without the target antigen, containing protective proteins and preservatives; (6) Blank control solution: A blank buffer system without the target antigen, containing preservatives; (7) Calibrator 1 and Calibrator 2: Buffer systems containing different concentration gradients of recombinant target antigen, containing protective proteins and preservatives; (8) Quality control 1 and Quality control 2: Buffer systems containing a fixed concentration range of recombinant target antigen, containing protective proteins and preservatives.

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