A lateral flow chromatographic test strip based on electrochemical detection of immune signal replacement, preparation method and application thereof

Abstract

The application discloses a lateral flow chromatographic test strip based on an immune signal replacement of electrochemical detection, a preparation method and application thereof. The test strip comprises a sample pad, a capture antibody pad, a solid-phase protein film, a detection electrode and a signal amplification pad. The capture antibody pad is fixed with a capture antibody with a label, can combine an antigen in a sample and form an antigen-antibody complex. The solid-phase protein film is fixed with a solid-phase protein, which can combine the antigen in the antigen-antibody complex. The signal amplification pad is fixed with a target substance which can combine the label of the capture antibody. The application can reflect the antigen amount of the antibody with a label to generate an electrical signal at the end of the lateral flow or to generate a strong electrochemical signal through label catalysis amplification, can obviously improve the detection accuracy and sensitivity, and obviously reduces the interference signal of the electrochemical detection based on the immune reaction.

Description

Technical Field

[0001] This invention belongs to the field of in vitro detection technology, and relates to an integrated detection method based on immune lateral flow and electrochemical detection. Specifically, it relates to a lateral flow chromatography test strip based on electrochemical detection and immune signal replacement, its preparation method, and its application. Background Technology

[0002] Existing immunoassay-based detection technologies have been widely used in point-of-care testing due to their advantages such as ease of operation, rapid response, and low cost, especially suitable for rapid on-site screening scenarios. However, this technology mainly relies on visual inspection or simple equipment to judge the color intensity of the test line, thus it can usually only achieve qualitative or semi-quantitative analysis and is difficult to provide accurate concentration values, limiting its application in fields requiring high-precision quantitative detection.

[0003] In contrast, electrochemical detection methods offer significant advantages such as high sensitivity, low detection limits, and ease of quantitative measurement. Currently, many electrochemical immunoassay methods still employ a sandwich immunoassay mode similar to lateral flow techniques, relying on the formation of a "sandwich" structure between the capture antibody and the detection antibody on the target analyte. Detection is then achieved by generating an in-situ signal on the sensor surface or an amplified signal through a label. However, electrochemical methods face challenges in practical applications: they are susceptible to interference from non-specific adsorption and electrode surface contamination in complex sample matrices, resulting in significant background noise and affecting the signal-to-noise ratio. Furthermore, the limited efficiency of immunoreactions at solid-phase interfaces can lead to incomplete or uneven immunobinding. These issues necessitate further improvements in signal sensitivity for electrochemical methods. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a lateral flow chromatography test strip based on electrochemical detection and immune signal displacement, its preparation method, and its application. This invention solves the technical problems of insufficient detection accuracy in existing immune lateral flow-based detection technologies, and the need for further improvement in sensitivity of electrochemical detection due to susceptibility to impurities and insufficient antigen-antibody reactions.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A lateral flow chromatography test strip based on electrochemical detection and immune signal displacement includes a base plate, on which a sample pad, an antibody capture pad, a solid protein membrane and a detection electrode are arranged sequentially along the direction of sample flow, and the surface of the detection electrode is covered with a signal amplification pad.

[0007] The capture antibody pad is immobilized with tagged capture antibodies that can bind to antigens in the sample and form antigen-antibody complexes.

[0008] The solid protein membrane is immobilized with solid protein, which can bind antigens in antigen-antibody complexes;

[0009] The solid-phase protein and the capture antibody are two different specific antibodies against the same antigen.

[0010] The signal amplification pad has a target material fixed on it that can bind to a tag that captures antibodies.

[0011] The present invention also has the following technical features:

[0012] Specifically, the tags for capturing antibodies include: catalytically active markers, aptamers, receptors, and lectins; correspondingly, the targets for signal amplification pads include: substrates of markers, conjugates of electroactive substances, target molecules of aptamers, ligands of receptors, and target sugars of lectins.

[0013] Specifically, the base plate is made of insulating material; the insulating material is selected from polyethylene terephthalate, polyethylene, polypropylene, polystyrene or acrylonitrile-butadiene-styrene copolymer.

[0014] Specifically, both the sample pad and the capture antibody pad are made of a material with a capillary structure, and the material is pre-soaked in a sample pad treatment solution. The sample pad treatment solution is a 0.01-0.1M neutral buffer containing 0.5-5 wt% blocking protein, 0.5-2.5 wt% water-soluble polymer and 0.1-2.0 vt% surfactant.

[0015] Specifically, the blocking protein is selected from one or more of bovine serum albumin, skim milk powder, cell protein, and bacterial protein; the water-soluble polymer is selected from one or more of polyvinylpyrrolidone, polyvinyl alcohol, and polyethylene glycol; the surfactant is selected from one or more of Tween, Span, and Triton; and the neutral buffer is selected from phosphate buffer or Tris-HCl buffer.

[0016] Specifically, if the target is a large protein with multiple epitopes, a double-antibody sandwich mode is used. The stationary phase can be antibodies against cardiovascular markers (such as troponin antibodies and NT-proBNP antibodies), specific monoclonal antibodies against inflammatory markers (such as mouse monoclonal antibodies against CRP), or antibodies against infectious diseases (such as mouse monoclonal antibodies against SARS-CoV-2, Mycoplasma pneumoniae antibodies, Coxsackievirus A16 antibodies, influenza virus antibodies, and Toxoplasma gondii antibodies). If the target is a small molecule metabolite with a single antigenic epitope, a blocking mode is used. The stationary phase can be conjugated antigens to the metabolite to be detected (such as salbutamol conjugated antigen, ractopamine conjugated antigen, and melamine conjugated antigen used for food safety, and trimethylamine oxide conjugated antigen, kynurenine conjugated antigen, and homocysteine ​​conjugated antigen used for metabolite detection).

[0017] Specifically, solid-phase protein membranes can be made from nitrocellulose membranes, polyvinylidene fluoride membranes, or nylon membranes.

[0018] Specifically, the detection electrode includes a working electrode, a reference electrode, a counter electrode, and a conductive wire. The working electrode is a carbon electrode or a modified multi-walled or single-walled carbon nanotube, the reference electrode is silver chloride, the conductive wire is copper paste or carbon paste, and the counter electrode is a carbon electrode. The detection electrode is used to detect the electrical signal generated when the tag reacts with its substrate, or to detect the electrical signal generated when the tag reacts on the surface of the detection electrode.

[0019] Specifically, the signal amplification pad is made of a water-absorbing material, which is pre-soaked in a signal amplification pad treatment solution. This treatment solution contains a target substance that can react with the substance labeled with the capture antibody. If the capture antibody is labeled with horseradish peroxidase, the signal amplification pad treatment solution includes solution A and solution B. Solution A is an aqueous solution containing 0.5–30 mM hydrogen peroxide urea, 5–20 mM citric acid, 0.1–1.0 wt% polyethylene glycol 6000, and 5–20 mM disodium hydrogen phosphate; solution B is an organic solution containing 0.5–30 mM TMB. If the capture antibody is labeled with alkaline phosphatase, the signal amplification pad treatment solution contains 0.5–30 mM p-nitrophenyl phosphate. If the capture antibody is labeled with glucose oxidase, the signal amplification pad treatment solution contains a series of substrates for its cascade reaction.

[0020] This invention also protects a method for preparing a lateral flow chromatography test strip based on electrochemical detection and immune signal displacement as described above, the method specifically comprising the following steps:

[0021] Step 1, Preparation of detection electrode: Conductive paste is screen-printed on the substrate and dried at 120 degrees Celsius for 30 minutes to obtain the detection electrode; then the detection electrode is cut into strips.

[0022] Step 2, preparation of solid protein membrane: attach the membrane to the substrate, dissolve the antibody or conjugated antigen in antibody buffer; then spray the prepared liquid onto the solid protein membrane in an environment with a humidity of 30% to 55%, and dry it at 37°C for 1 hour.

[0023] Step 3, Preparation of capture antibody pad: Dissolve the labeled capture antibody in antibody buffer, and then spray the capture antibody protein onto the glass cellulose membrane at a certain spray rate in an environment with a humidity of 20% to 30%, and then dry it at 37°C for 1 hour; after drying, cut it and then stick it on the capture antibody pad area of ​​the base plate, with one side of the capture antibody pad pressed on the solid protein membrane, and the pressing edge width is 1 mm;

[0024] Step 4, prepare the sample pad: Immerse the sample pad in the sample pad treatment solution for 10 minutes, then remove it and dry it at 37°C; after drying, cut it and then paste it onto the sample pad area of ​​the base plate, with one side of the sample pad pressing on the capture antibody pad, and the pressing edge width is 1mm;

[0025] Step 5, prepare the signal amplification pad: prepare solution A and solution B; then immerse the signal amplification pad in solution A for 1 minute and dry at 37°C; then immerse the signal amplification pad in solution B and dry at 37°C; after drying, cut the pad and then attach it to the electrode with conductive adhesive, ensuring that it completely covers the detection electrode. One side of the signal amplification pad is pressed onto the solid protein membrane with a pressing edge width of 1 mm.

[0026] Specifically, in steps two and three, the antibody buffer is a 0.01M neutral phosphate buffer containing 1 wt% bovine serum albumin and 0.5 wt% sucrose.

[0027] Specifically, in steps two and three, the concentration of the solid-phase protein is 0.1–2 mg / mL; the concentration of the tagged capture antibody is 0.1–1 mg / mL.

[0028] This invention also protects the use of the electrochemical detection-based immune signal displacement lateral flow chromatography test strips described above for detecting cardiovascular markers (such as troponin, NT-proBNP), inflammatory markers (such as CRP), infectious diseases (such as SARS-CoV-2, Mycoplasma pneumoniae, Coxsackievirus A16, influenza virus, Toxoplasma gondii, etc.); and small molecule metabolites (such as salbutamol, ractopamine, melamine, etc. used for food safety, and trimethylamine oxide, kynurenine, homocysteine, etc. used for metabolites).

[0029] Compared with the prior art, the present invention has the following technical effects:

[0030] The detection signal of this invention is based on the displacement signal of the immune lateral flow between antigen and antibody. The concentration of the solid-phase protein must be greater than or equal to the amount of the capturing antibody to ensure that the capturing antibody can fully react with the solid-phase protein after binding to the antigen. The detection electrode is set at the end of the lateral flow chromatography and is used to detect the electrical signal generated by the enzyme-catalyzed signal amplification substance labeled with the capturing antibody. This enables highly sensitive and quantitative electrical detection of antigen-antibody binding events during immunochromatography. This invention employs a double-antibody sandwich structure or blocking mechanism and is not limited to traditional antigen-antibody interactions. Its sensing molecular system can be extended to any molecular pair with high specificity and high affinity binding characteristics, including but not limited to aptamers and their target molecules, enzymes and specific substrates, receptors and ligands, lectins and sugars, etc. It can fully utilize the directional affinity reactions of various biorecognition elements to achieve sensitive and specific detection of a wider range of targets.

[0031] In summary, this invention utilizes the displacement signal of the immune lateral flow between antigen and antibody to enable labeled antibodies that reflect antigen levels to generate an electrical signal at the end of the lateral flow or to generate a strong electrochemical signal through label catalysis and amplification. This can significantly improve detection accuracy and sensitivity, and significantly reduce interference signals in electrochemical detection based on immune reactions. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the screen-printed electrode structure in this detection system.

[0033] Figure 2 This is a schematic diagram of the base plate partitions of a lateral flow chromatography test strip based on electrochemical detection and immune signal displacement.

[0034] Figure 3 This is a photograph of the lateral flow chromatography test strip based on electrochemical detection and immune signal displacement, as described in Example 2.

[0035] Figure 4 This is a schematic diagram of the electrical signal substitution detection results. Figure 4 In the middle: (a) is a schematic diagram of the electrical signal displacement detection results based on the double-antibody sandwich method; (b) is a schematic diagram of the electrical signal displacement detection results based on the blocking method.

[0036] Figure 5 This is a superimposed graph of the electrical signal curves of different concentrations of antigen in Example 1, where the horizontal axis represents voltage (in volts), silver chloride is the reference electrode, and the vertical axis represents current (in amperes).

[0037] Figure 6 The graph shows the concentration of the detectable substance versus the electrical signal in Example 1, where the horizontal axis represents the concentration of MP antigen (in nanomoles per liter) and the vertical axis represents the current signal (in amperes).

[0038] Figure 7 This is a photograph of the colloidal gold test strip used in Comparative Example 1.

[0039] Figure 8 This is a photograph of the lateral flow chromatography test strip based on electrochemical detection and immunoassay for melamine detection, as described in Example 2.

[0040] Figure 9 This is a superimposed graph of electrical signal curves for the detection of melamine antigen at different concentrations in Example 2. The horizontal axis represents voltage (in volts), and the reference electrode used is a silver chloride electrode. The vertical axis represents current (in amperes).

[0041] Figure 10 The curve showing the relationship between melamine and electrical signal in Example 2 is shown, where the horizontal axis represents the concentration of melamine (in nanomoles per liter) and the vertical axis represents the current signal (in amperes).

[0042] Figure 11 This is a photograph of the colloidal gold test strip used in Comparative Example 2.

[0043] Figure 12 This is a schematic diagram illustrating the detection principle of a lateral flow chromatography test strip based on electrochemical detection and immunoassay signal displacement.

[0044] The specific content of the present invention will be further explained in detail below with reference to the embodiments. Detailed Implementation

[0045] It should be noted that, unless otherwise specified, the antigens, antibodies and raw materials mentioned in this invention are all conventional antigens, antibodies and raw materials known in the art.

[0046] The detection principle of this invention is based on electrical signal substitution using a double-antibody sandwich structure. The principle and results are as follows: Figure 4 As shown in (a), the higher the antigen concentration, the weaker the electrical signal. Specifically (using horseradish peroxidase-tagged antibodies as an example):

[0047] When a sample droplet containing antigen is added to the sample pad, the high molecular weight substances in the sample pad dissolve rapidly, providing a suitable buffering environment for the antigen and promoting the cleavage of the antigen complex. Large particles in the sample are filtered and retained by the glass fiber membrane, while the liquid containing antigen flows laterally along the chromatography membrane under capillary action.

[0048] The liquid flow first reaches the capture pad area, which is pre-embedded with a capture antibody labeled with horseradish peroxidase (HRP). The antigen and the enzyme-labeled capture antibody specifically bind here, forming an antigen-antibody complex. This complex continues to move forward with the laminar flow into the detection area of ​​the nitrocellulose (NC) membrane.

[0049] Another specific antibody against the same antigen (the stationary phase antibody) is immobilized on the NC membrane. The amount of the stationary phase antibody is optimized to ensure that it is not less than the amount of the enzyme-labeled capture antibody. When the antigen-enzyme-labeled antibody complex flows through this area, the stationary phase antibody captures the antigen in the complex, forming a "sandwich" type immune complex structure, thereby immobilizing the enzyme-labeled antibody on the surface of the NC membrane.

[0050] The liquid continues to flow forward, immersing the signal amplification area (electrode area). If the antigen concentration in the sample is high, all enzyme-labeled capture antibodies are immobilized on the NC membrane. At this time, because there is no free HRP enzyme present in the signal amplification area, only the background level TMB substrate electrochemical reaction occurs, generating a specific signal.

[0051] If the antigen content in the sample is low and fails to bind to all the enzyme-labeled capture antibodies, the unbound free HRP-labeled antibodies migrate with the liquid flow to the signal amplification region. In this region, HRP catalyzes the oxidation of the TMB substrate to generate TMBox, producing a significantly enhanced electrical signal.

[0052] If the sample does not contain the target antigen, all enzyme-labeled capture antibodies are not captured on the NC membrane, but migrate to the signal amplification region, catalyzing the conversion of all TMB substrates into TMBoxes, generating the strongest electrical signal.

[0053] When the antigen concentration in a sample exceeds the binding capacity of the enzyme-labeled capture antibody, the "Hook effect" occurs due to antigen excess, which can actually amplify the detection signal, resulting in a false signal. To address this, the high-concentration sample can be appropriately diluted and retested, or the amount of enzyme-labeled capture antibody can be set higher than the maximum physiological concentration of antigen that may be present in the sample, thus avoiding this effect.

[0054] The detection principle of this invention can also be based on electrical signal substitution using the blocking method, as described below. Figure 4 As shown in (b), the higher the antigen concentration, the stronger the electrical signal. Specifically (using horseradish peroxidase-tagged antibodies as an example):

[0055] When sample droplets containing small-molecule antigens are added to the sample pad, the high-molecular-weight substances in the sample pad dissolve rapidly, providing a suitable buffering environment for the antigens. Large particles in the sample are filtered and retained by the glass fiber membrane, while the liquid containing antigens flows laterally along the chromatography membrane under capillary action.

[0056] The liquid flow first reaches the capture pad area, which is pre-embedded with a capture antibody labeled with horseradish peroxidase (HRP). The antigen and the enzyme-labeled capture antibody specifically bind here, forming an antigen-antibody complex. This complex continues to move forward with the laminar flow into the detection area of ​​the nitrocellulose (NC) membrane.

[0057] The coupled antigen (solid phase protein) is immobilized on the NC membrane, and its coating amount is optimized to ensure that it is not less than the amount of enzyme-labeled capture antibody. When the antigen-enzyme-labeled antibody complex flows through this area, the solid phase protein cannot react with the antigen-enzyme-labeled antibody complex, thereby allowing the enzyme-labeled antibody-antigen complex to flow forward with the lateral flow to the signal amplification pad, impregnate the signal amplification area (built-in detection electrode), and the HRP on the enzyme-labeled antibody catalyzes the TMB substrate to generate an electrical signal.

[0058] If the antigen content in the sample is very high, all enzyme-labeled capture antibodies will react with the antigen. The antibodies will be blocked and unable to react with the solid phase protein. The enzymes on the antibodies cannot be fixed and will migrate to the signal amplification region with the lateral flow, catalyzing all TMB substrates to be converted into TMBoxes, generating the strongest electrical signal.

[0059] If the antigen content in the sample is low, the enzyme-labeled capture antibody may not completely bind to the antigen. The unbound enzyme-labeled antibody reacts with the solid-phase protein and is immobilized. Meanwhile, the antigen-bound enzyme-labeled antibody migrates to the signal amplification region via lateral flow, catalyzing the conversion of all TMB substrates into TMBoxes, thus generating a specific signal.

[0060] If the sample does not contain the target antigen, all enzyme-labeled capture antibodies will react with the fixed antigen. At this time, since there is no free HRP enzyme in the signal amplification region, only the background level TMB substrate electrochemical reaction will occur.

[0061] When the antigen concentration in the sample exceeds the binding capacity of the enzyme-labeled capture antibody, the excess antigen will cause all the enzyme-labeled antibody to be released to the signal amplification pad, resulting in only the maximum signal and no increase in signal strength.

[0062] Following the above technical solutions and concepts, specific embodiments of the present invention are given below. It should be noted that the present invention is not limited to the following specific embodiments, and all equivalent modifications made based on the technical solutions of this application fall within the protection scope of the present invention.

[0063] Example 1:

[0064] This embodiment provides a lateral flow chromatography test strip based on electrochemical detection and immune signal displacement, including a base plate. The base plate is provided with a sample pad, an antibody capture pad, a solid protein membrane and a detection electrode arranged sequentially along the direction of sample flow. The surface of the detection electrode is covered with a signal amplification pad.

[0065] As a specific embodiment, the lateral flow chromatography test strip based on electrochemical detection and immune signal replacement has a length of 5-15 cm and a width of 4-15 mm.

[0066] As a specific embodiment, the base plate is made of insulating material, such as thin sheets of polyethylene terephthalate (PET), polyethylene (PE), polypropylene (PP), polystyrene (PS), acrylonitrile-butadiene-styrene copolymer (ABS), etc.

[0067] As a specific embodiment, the sample pad is made of a material with a capillary structure and requires pretreatment by soaking and drying in a sample pad treatment solution to filter and retain impurities in the sample. The sample pad can be made of glass cellulose membrane, polyester membrane, cellulose filter paper, or non-woven fabric. The sample pad treatment solution is a 0.01–0.1 M neutral buffer containing 0.5–5 wt% blocking protein, 0.5–2.5 wt% water-soluble polymer, and 0.1–2.0 wt% surfactant. The blocking protein can be bovine serum albumin (BSA), skim milk powder, broken cell protein, or bacterial protein. The water-soluble polymer can be polyvinylpyrrolidone (PVP) with a number average molecular weight of 8000–20000, polyvinyl alcohol (PVA) with a number average molecular weight of 20000–80000, or polyethylene glycol (PEG) with a number average molecular weight of 1000–10000. The surfactant can be Tween 20, Tween 80, Span 20, or Triton X-100. The neutral buffer can be phosphate buffer or Tris-HCl buffer.

[0068] As a specific embodiment, the capture antibody pad is also made of a material with a capillary structure. It requires pretreatment by soaking and drying with sample pad treatment solution, and the capture antibody with a tag is fixed on it. It can specifically bind to the antigen in the sample. The tag can have its own electrical signal or catalytic signal amplification of the substance in the pad to generate an electrical signal.

[0069] As a specific and optional scheme in this embodiment, the tag for the capture antibody is a catalytically active marker, such as horseradish peroxidase (HRP), iron-based nanozyme, or alkaline phosphatase (AP). These enzymes catalyze the reaction of corresponding substrates (such as TMB, OPD, or ABTS) to generate electroactive products (such as TMBox), which then undergo electrochemical changes on the electrode surface, producing a measurable signal. As another specific and optional scheme in this embodiment, the tag for the capture antibody is an electroactive substance that can directly generate an electrical signal, such as biotin, methylene blue, colloidal gold, ferrocene, or malachite green. These substances undergo redox reactions on the electrode surface, directly causing changes in current or potential, thereby outputting a detection signal.

[0070] As a specific and optional approach in this embodiment, the tag for the capture antibody is HRP. The corresponding signal amplification pad contains the enzyme substrate TMB.

[0071] As a specific embodiment, the stationary phase membrane can be a nitrocellulose membrane (NC membrane), a polyvinylidene fluoride membrane (PVDF membrane), or a nylon membrane. A different type of specific antibody (different from the capture antibody) capable of binding to antigens in the sample (referred to as the stationary phase antibody) is immobilized on the membrane. The stationary phase antibody can bind to the antigen in the antigen-antibody complex. The stationary phase protein can be an antibody against cardiovascular markers (such as troponin antibody, NT-proBNP antibody), a specific monoclonal antibody against inflammatory markers (such as a mouse monoclonal antibody against CRP), or an antibody against infectious diseases (such as a mouse monoclonal antibody against SARS-CoV-2, Mycoplasma pneumoniae antibody, Coxsackievirus A16 antibody, influenza virus antibody, Toxoplasma gondii antibody, etc.). As a specific embodiment, the signal amplification pad is made of a water-absorbing material, such as absorbent paper, cellulose filter paper, glass fiber, or non-woven fabric. The material needs to be pretreated by soaking in a signal amplification pad treatment solution and then drying to enhance the signal and further improve detection sensitivity. The signal amplification pad processing solution includes solution A and solution B. Solution A is an aqueous solution containing 0.5–30 mM hydrogen peroxide urea, 5–20 mM citric acid, 0.1–1.0 wt% polyethylene glycol 6000, and 5–20 mM disodium hydrogen phosphate. Solution B is a solution containing 0.5–30 mM TMB.

[0072] As a specific and optional solution in this embodiment, the solvent of solution B is selected from one or more of ethanol, water, methanol, propanol, and acetonitrile.

[0073] As a specific embodiment, the detection electrode is a three-electrode detection system, which can be a screen-printed electrode. The working electrode is a carbon electrode, the reference electrode is silver chloride, the counter electrode is a carbon electrode, and the conductive wire is copper paste. In this embodiment, the detection electrode can be integrated with the lateral flow system on the same backing substrate, or it can be used as an independent module. The detection electrode is attached to the bottom of the absorbent pad (signal amplification pad) at the end of the detection card and between it and the backing using conductive adhesive, thereby achieving effective integration with the detection system.

[0074] This embodiment provides a method for preparing a lateral flow chromatography test strip based on electrochemical detection and immune signal displacement for detecting Mycoplasma pneumoniae antigen (MP). The method specifically includes the following steps:

[0075] Step 1, Preparation of the detection electrode: Conductive paste is screen-printed onto a PET substrate, dried at 120 degrees Celsius for 30 minutes, and the detection electrode is obtained; for example... Figure 1 As shown, the detection electrode is cut into strips 4 mm wide.

[0076] Step 2, preparation of solid-phase protein membrane: Nitrocellulose membrane (NC membrane) is attached to the reaction area of ​​the substrate. Mycoplasma pneumoniae antibody (MP antibody 5) is dissolved in buffer (specifically: 0.01M neutral phosphate buffer containing 1 wt% bovine serum albumin and 0.5 wt% sucrose) to a concentration of 0.1-2 mg / mL. Then, this solution is sprayed onto the NC membrane at a certain spray rate in an environment with a humidity of 30%-55%, and then dried at 37°C for 1 hour.

[0077] Step 3, Preparation of the capture antibody pad: Dissolve horseradish peroxidase-labeled MP antibody-19 in antibody buffer (specifically, 0.01M neutral phosphate buffer containing 1 wt% bovine serum albumin, 0.5 wt% polyvinylpyrrolidone, and 0.5 vt% Tween 20), with an antibody concentration of 0.1–1 mg / mL; then spray the capture antibody onto the glass cellulose membrane at a certain spray rate in an environment with a humidity of 20%–30%, and then dry it at 37°C for 1 hour; after drying, cut the glass cellulose membrane to an appropriate size and attach it to the capture pad position on the base plate, with the side in contact with the NC membrane pressed against the NC membrane, with a pressing edge width of 1 mm.

[0078] Step 4, prepare the sample pad: Immerse the glass cellulose membrane in the sample pad treatment solution (specifically, 0.01M neutral phosphate buffer containing 1 wt% bovine serum albumin, 0.5 wt% polyvinylpyrrolidone and 0.5 vt% Tween 20) for 10 minutes, then remove it and dry it at 37°C; then cut the glass cellulose membrane to a suitable size and attach it to the capture pad position on the base plate, with the side in contact with the capture antibody pad pressed against the capture antibody pad, with a pressing edge width of 1 mm.

[0079] Step 5, prepare the signal amplification pad: Prepare solution A containing 20mM hydrogen peroxide urea, 10mM citric acid, 0.5% polyethylene glycol 6000 and 10mM disodium hydrogen phosphate; prepare a 20mM tetramethyl-o-aniline ethanol solution B; immerse absorbent paper in solution A for 1 minute and dry at 37°C; then immerse the absorbent paper in solution B and dry at 37°C; cut the absorbent paper to a suitable size to prepare the signal amplification pad, and then attach it to the electrode area with conductive adhesive to cover the detection electrode, wherein the side in contact with the NC film needs to be pressed onto the NC film with a pressing edge width of 1mm.

[0080] In this embodiment, the structure of the final electrochemical test strip is as follows: Figure 12 As shown, each area is compressed by 1mm, thus forming a capillary-linked whole through compression. Then, perpendicular to the lateral flow direction, it is cut into strips with a width of 4-12mm. A physical image is shown below. Figure 3 As shown.

[0081] In this embodiment, the final electrochemical test strip is used to detect Mycoplasma pneumoniae. The detection process is as follows: 240 μL of sample is added to the sample pad of the test strip, allowing it to undergo a lateral flow reaction. Simultaneously, the electrochemical workstation is turned on, the DPV program is selected, and the parameters are set as follows: initial potential 0.6 V, endpoint potential 0 V, potential increment 0.004 V, amplitude 0.05 V, pulse width 0.0167 s, pulse period 0.5 s, rest time 2 s, and sensitivity 10. -4 The electrical signal value is detected after the liquid flows laterally onto the signal amplification pad and reacts for 5 minutes.

[0082] Test results as follows Figure 5 and Figure 6 As shown. From Figure 5 The image shows the superimposed electrical signals generated by samples containing different concentrations of MP antigen after lateral flow. Figure 5 The horizontal axis represents electrical potential, and the vertical axis represents current. Different colored lines represent the electrical signals detected from samples of different concentrations. As can be seen from the graph, there is a significant TMBox reduction peak at the 0.25V position (consistent with literature reports). The electrical signal becomes weaker as the MP content increases. Figure 6 The graph shows the relationship between the concentration of Mycoplasma pneumoniae in the sample and the current. The horizontal axis represents the concentration of Mycoplasma pneumoniae antigen, and the vertical axis represents the current intensity. The graph shows that the electrical signal weakens as the concentration of Mycoplasma pneumoniae increases. The strongest electrical signal can be detected when the MP concentration is 0.4 nM, therefore the sensitivity is 0.4 nM.

[0083] Comparative Example 1:

[0084] This comparative example provides a method for preparing a colloidal gold test strip. The antigen and antibody are exactly the same as in Example 1, and the preparation method of the colloidal gold test strip is a conventional method known in the prior art. In this comparative example, the sensitivity for detecting MP antigen is 20 nM. Figure 7 As shown, the detection principle is based on the double-antibody sandwich method, so the higher the antigen concentration, the stronger the signal.

[0085] Example 2:

[0086] This embodiment provides a method for preparing a lateral flow chromatography test strip based on electrochemical detection and immunoassay for detecting small molecule metabolites (melamine). The method specifically includes the following steps:

[0087] Step 1, Preparation of the detection electrode: Conductive paste is screen-printed onto a PET substrate, dried at 120 degrees Celsius for 30 minutes, and the detection electrode is obtained; for example... Figure 1 As shown, the detection electrode is cut into strips 4 mm wide.

[0088] Step 2, preparation of solid-phase protein membrane: Nitrocellulose membrane (NC membrane) is attached to the solid-phase protein membrane area of ​​the substrate. Melamine-conjugated antigen is dissolved in buffer solution (specifically: 0.01M neutral phosphate buffer containing 1 wt% bovine serum albumin and 0.5 wt% sucrose), with a conjugated antigen concentration of 0.1-1 mg / mL. Then, melamine-conjugated protein is sprayed onto the NC membrane at a certain spray rate in an environment with a humidity of 30%-55%, and then dried at 37°C for 1 hour.

[0089] Step 3, Preparation of the capture antibody pad: Dissolve horseradish peroxidase-labeled melamine antibody-5 in antibody buffer (specifically: 0.01M neutral phosphate buffer containing 1 wt% bovine serum albumin, 0.5 wt% polyvinylpyrrolidone and 0.5 vt% Tween 20), with an antibody concentration of 0.1–1 mg / mL; then spray the capture antibody protein onto the glass cellulose membrane at a certain spray rate in an environment with a humidity of 20%–30%, and then dry it at 37°C for 1 hour; after drying, cut the glass cellulose membrane to an appropriate size and attach it to the capture pad position on the base plate, with the side in contact with the NC membrane pressed against the NC membrane, with a pressing edge width of 1 mm.

[0090] Step 4, prepare the sample pad: Immerse the polyester membrane in the sample pad treatment solution (specifically, 0.01M neutral phosphate buffer containing 1 wt% bovine serum albumin, 0.5 wt% polyvinylpyrrolidone and 0.5 vt% Tween 20) for 10 minutes, then remove it and dry it at 37°C; then cut the membrane to a suitable size and attach it to the capture pad position on the base plate, with the side in contact with the capture antibody pad pressed against the capture antibody pad, with a pressing edge width of 1 mm.

[0091] Step 5, prepare the signal amplification pad: Prepare solution A containing 20mM hydrogen peroxide urea, 10mM citric acid, 0.5% polyethylene glycol 6000 and 10mM disodium hydrogen phosphate; dissolve tetramethyl-o-aniline (TMB) in ethanol to prepare solution B with a TMB concentration of 20mM; immerse absorbent paper in solution A for 1 minute and dry at 37°C; then immerse the absorbent paper in solution B and dry at 37°C; cut the absorbent paper to a suitable size to prepare the signal amplification pad, and then paste it onto the electrode area of ​​the PET sheet to cover the detection electrode, wherein the side in contact with the NC film needs to be pressed onto the NC film with a pressing edge width of 1mm.

[0092] In this embodiment, the structure of the final electrochemical test strip is as follows: Figure 2 As shown, each area is compressed by 1mm, thus forming a capillary-linked whole through compression. Then, perpendicular to the lateral flow direction, it is cut into strips with a width of 4mm. A physical image is shown below. Figure 3 As shown.

[0093] In this embodiment, the final electrochemical test strip is used to detect melamine molecules. The detection process is as follows: The electrochemical workstation is turned on, the DPV program is selected, and the parameters are set as follows: initial potential is 0.6 V, endpoint potential is 0 V, potential increment is 0.004 V, amplitude is 0.05 V, pulse width is 0.0167 seconds, pulse period is 0.5 seconds, rest time is 2 seconds, and sensitivity is 10. -4 The electrical signal value was detected after the lateral flow reached the signal amplification pad and reacted for 5 minutes. The sample loading volume was 200 μL.

[0094] Test results as follows Figure 9 and Figure 10 As shown. From Figure 9 The image shows the superimposed electrical signals generated after lateral flow of samples containing different concentrations of melamine. Figure 9 The horizontal axis represents electrical potential, and the vertical axis represents current. Different colored lines represent the electrical signals detected from samples of different concentrations. A significant TMBox reduction peak can be observed at the 0.25 volt position (consistent with literature reports). The higher the melamine content, the stronger the electrical signal. Figure 10 The graph shows the relationship between melamine concentration and current in the sample, with the horizontal axis representing melamine concentration and the vertical axis representing current intensity. The graph shows that the electrical signal strengthens with increasing melamine concentration. The experimental results indicate that the sensitivity is more than 0.4 nM.

[0095] Comparative Example 2

[0096] This comparative example provides a method for preparing a colloidal gold test strip. The antigen and antibody are exactly the same as in Example 2, and the preparation method of the colloidal gold test strip is a conventional method known in the prior art. In this comparative example, the detection principle is based on the blocking method, such as... Figure 11 As shown, the sensitivity for detecting melamine is 100 nM. The detection principle is based on the blocking method; 100 nM completely blocks the melamine, while below 100 nM, the melamine is not blocked, and a detection band appears.

Claims

1. A lateral flow chromatography test strip based on electrochemical detection and immunoassay signal displacement, comprising a substrate, characterized in that, The base plate is arranged sequentially along the direction of sample flow, including a sample pad, a capture antibody pad, a solid protein membrane, and a detection electrode. The surface of the detection electrode is covered with a signal amplification pad. The capture antibody pad is immobilized with tagged capture antibodies that can bind to antigens in the sample and form antigen-antibody complexes. The solid protein membrane is immobilized with solid protein, which can bind antigens in antigen-antibody complexes; The signal amplification pad has a target material fixed on it that can bind to a tag that captures antibodies.

2. The lateral flow chromatography test strip based on electrochemical detection and immunoassay as described in claim 1, characterized in that, The tags for capturing antibodies include: catalytically active markers, aptamers, receptors, and lectins; correspondingly, the targets of the signal amplification pad include: substrates of markers, target molecules of aptamers, ligands of receptors, and target sugars of lectins.

3. The lateral flow chromatography test strip based on electrochemical detection and immunoassay as described in claim 1, characterized in that, Both the sample pad and the capture antibody pad are made of a material with a capillary structure, and the material is pre-soaked in a sample pad treatment solution. The sample pad treatment solution is a 0.01-0.1M neutral buffer containing 0.5-5 wt% blocking protein, 0.5-2.5 wt% water-soluble polymer and 0.1-2.0 vt% surfactant.

4. The lateral flow chromatography test strip based on electrochemical detection and immunoassay as described in claim 1, characterized in that, The detection electrode includes a working electrode, a reference electrode, a counter electrode, and a conductive wire; the detection electrode is used to detect the electrical signal generated when the tag reacts with the target, or to detect the electrical signal generated when the tag reacts on the surface of the detection electrode.

5. The lateral flow chromatography test strip based on electrochemical detection and immunoassay as described in claim 1, characterized in that, The signal amplification pad is made of a water-absorbing material, which is pre-soaked in a signal amplification pad treatment solution. The signal amplification pad treatment solution consists of solution A and solution B. Solution A is an aqueous solution containing 0.5–3 mM hydrogen peroxide urea, 5–20 mM citric acid and 5–20 mM disodium hydrogen phosphate. Solution B is a solution containing the enzyme-labeled substrate TMB.

6. A method for preparing a lateral flow chromatography test strip based on electrochemical detection and immunoassay as described in any one of claims 1 to 5, characterized in that, The method specifically includes the following steps: Step 1, Preparation of detection electrode: Conductive paste is screen-printed on the substrate and dried at 120 degrees Celsius for 30 minutes to obtain the detection electrode; then the detection electrode is cut into strips. Step 2, Preparation of solid protein membrane: Attach the solid protein membrane to the substrate and dissolve the solid protein in antibody buffer; Then, the solid protein was sprayed onto the solid protein film in an environment with a humidity of 30% to 55%, and then dried at 37°C for 1 hour. Step 3, preparation of capture antibody pad: Dissolve the tagged capture antibody in antibody buffer, then spray the capture antibody protein onto glass cellulose membrane in an environment with a humidity of 20% to 30%, and then dry at 37°C for 1 hour; after drying, cut and then stick it on the base plate, with one side of the capture antibody pad pressed on the solid protein membrane. Step 4, prepare the sample pad: Immerse the sample pad in the sample pad treatment solution for 10 minutes, then remove it and dry it at 37°C; after drying, cut it and then stick it on the base plate, with one side of the sample pad pressing on the capture antibody pad; Step 5, prepare the signal amplification pad: prepare solution A and solution B; then immerse the signal amplification pad in solution A for 1 minute and dry at 37°C; then immerse the signal amplification pad in solution B and dry at 37°C; after drying, cut it and then stick it to the electrode detection end, ensuring that it completely covers the detection electrode, with one side of the signal amplification pad pressed on the solid protein membrane.

7. The method for preparing the lateral flow chromatography test strip based on electrochemical detection and immunoassay as described in claim 6, characterized in that, In steps two and three, the antibody buffer is a 0.01M neutral phosphate buffer containing 1 wt% bovine serum albumin and 0.5 wt% sucrose.

8. The method for preparing the lateral flow chromatography test strip based on electrochemical detection and immunoassay as described in claim 6, characterized in that, In steps two and three, the concentration of the solid-phase protein is 0.1–2 mg / mL; the concentration of the tagged capture antibody is 0.1–1 mg / mL.

9. The application of the lateral flow chromatography test strip based on electrochemical detection and immune signal displacement as described in any one of claims 1 to 5 for the detection of cardiovascular markers, inflammatory indicators, infectious disease pathogens, and small molecule metabolites.

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