Immunochromatography detection device and preparation method thereof

Abstract

The invention discloses an immunochromatography detection device and a preparation method thereof. The immunochromatography detection device comprises a bottom plate, a sample pad, a combination pad, a cellulose membrane and a water absorption pad, and the sample pad, the combination pad, the cellulose membrane and the water absorption pad are sequentially pasted to the same side of the bottom plate in the chromatography direction; the immunochromatography system further comprises a functional treatment layer, the functional treatment layer is arranged in at least one preset area of the immunochromatography channel, the functional treatment layer is made of a high polymer material which can be dissolved in an alcohol solvent and can form a dry film in a spraying mode, and the dry film swells or rehydrates to form a high-viscosity layer after making contact with a water-based sample in the detection process. A functional treatment layer is arranged in at least one preset area of an immunochromatography channel, and a material of the functional treatment layer forms a local flow blocking area through molecular chain hydration and volume expansion after meeting water, so that the controllable delay of the chromatography flow velocity is realized on the premise of not changing the basic structure and operation steps of immunochromatography.

Description

An immunochromatographic detection device and its preparation method Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to an immunochromatographic detection device and its preparation method. Background Technology

[0002] Lateral flow immunochromatography (LFA) is a point-of-care testing (POCT) method that uses capillary flow to drive the sample through a test strip and achieve specific binding and color development on fixed test (T) and control (C) lines. LFA offers advantages such as ease of operation, speed, and low cost; however, its inherently short capture window and limited reaction time result in less than ideal sensitivity in many clinical and environmental scenarios. Common strategies to improve LFA sensitivity include: improving label design (increasing label particle size), optimizing antibody affinity and immobilization, and extending reaction time in the capillary channel (e.g., through flow rate control or constructing retention zones).

[0003] Currently, representative solutions for the "flow rate reduction / increased retention" technical approach include:

[0004] Paper-hydrogel hybrid structures: Choi et al. reported in Adv. Healthc. Mater. (2017) that they combined paper-based materials with hydrogels to construct "hydrogel lines" or "hydrogel pads" in LFAs, achieving flow rate control and significantly improving sensitivity for dengue virus detection. This method typically involves introducing hydrogel components (such as natural polysaccharides or synthetic gel materials) locally into the paper or membrane, causing the flowing liquid to be partially captured or its release delayed in that area, thereby prolonging the binding time. The advantages are that the retention zone is directly constructed using aqueous materials and it is biocompatible; the disadvantages are that this technology requires strict temperature control to ensure the material switches between the aqueous and gel phases to complete the coating process and achieve functionality, making it suitable for small-scale studies but difficult to truly apply to industrial production.

[0005] Dispensing or printing "gel / blocking zones" on membranes: Some literature / technical bulletins mention creating localized blocking zones by dripping or streaking low-melting-point agarose or other gels onto NC membranes or upstream of them. This method allows for relatively quick flow rate reduction and verification of its impact on sensitivity at the laboratory level, but the drip method is difficult to homogenize and industrialize, and the hot-melt dispensing method requires careful control of protein stability (if conjugated / antibody exposed).

[0006] Physical / chemical modification of membrane materials or membrane surfaces: Capillary flow rates can be altered by selecting cellulose membranes (NC membranes) with different pore sizes / hydrophilicities, mechanically compressing the membrane, or forming a barrier layer with an insoluble coating (such as ethyl cellulose). These methods are simple and reproducible, but often come at the cost of reduced overall flow rate and lack the characteristic of "swelling and reactivating upon contact with water," thus presenting a trade-off between improving binding efficiency and maintaining label release. Film-forming agents such as ethyl cellulose and eudranitrile can form water-resistant membranes to slow permeation, but may lead to particle aggregation in front of the membrane or long-term protein adsorption.

[0007] Adding thickeners to samples or buffers: Adding polymeric thickeners directly to sample buffers (such as adding a small amount of polyvinylpyrrolidone (PVP), carboxymethyl cellulose, etc.) can temporarily slow down the flow rate, but this method has a significant impact on the release and binding kinetics of the label, is not conducive to consistency, and may change the antibody binding behavior; it also has limitations in terms of ease of use for users.

[0008] The main drawbacks of existing technologies lie in the inconvenience of construction and industrialization processes. Most hydrogel-based "paper-hydrogel" or membrane dispensing (such as agarose dripping) methods are prepared using aqueous systems, leading to stability and consistency issues during drying, storage, and mass production. Precise and uniform batch coating is difficult to achieve with dripping / stretching operations. Insufficient compatibility with conjugated particles / antibodies is also a concern: some materials that form water-resistant films (such as high-concentration ethyl cellulose or Eudragit) can adsorb proteins on or inside the membrane surface, or prevent the passage of colloidal labels, resulting in decreased sensitivity or background signals and clogging risks. Furthermore, balancing controllable delay and label release is challenging: directly adding viscosity modifiers to the sample or creating irreversible barriers on the membrane can slow down the process, but often results in incomplete label release or blocked binding sites, affecting reliability and reproducibility. Summary of the Invention

[0009] To address the aforementioned problems in existing technologies, this invention proposes an immunochromatographic detection device, comprising a base plate, a sample pad, a conjugation pad, a cellulose membrane, and an absorbent pad. The sample pad, conjugation pad, cellulose membrane, and absorbent pad are sequentially adhered to the same side of the base plate in the chromatography direction. The device also includes a functional processing layer disposed in at least one predetermined area of ​​the immunochromatographic channel. The functional processing layer is made of a polymeric material soluble in alcohol solvents and capable of forming a dry film through spraying. Upon contact with an aqueous sample during detection, the dry film swells or rehydrates to form a high-viscosity layer. High viscosity refers to significantly impeded flow, with a viscosity >10,000 mPa·s (10 Pa·s). When the functional processing layer encounters water / sample solution, it can rehydrate and exhibit controllable water absorption and swelling or viscosity increase, forming a high-viscosity layer, thereby flexibly controlling the chromatography time to improve binding efficiency.

[0010] Furthermore, the functional processing layer is applied to a predetermined area by spraying, the predetermined area including: the side of the cellulose membrane away from the base plate, and / or the side of the sample pad away from the base plate, and / or the side of the bonding pad away from the base plate.

[0011] Furthermore, the functional processing layer is disposed at the end of the binding layer and / or the front end of the cellulose membrane. Specifically, in relation to the chromatography direction, the end of the binding layer refers to the end closest to the cellulose membrane; the front end of the cellulose membrane is the end closest to the binding layer.

[0012] Furthermore, it also includes a delay pad, which is adhered to the base plate, with one end of the bonding pad adhered to the side of the base plate near the base plate and the other end adhered to the side of the cellulose membrane away from the base plate, wherein the predetermined area is the side of the delay pad away from the base plate.

[0013] Furthermore, the material of the functional treatment layer includes a swelling agent selected from one or more of hydroxypropyl cellulose, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.

[0014] Furthermore, the material of the functional treatment layer includes a nonionic surfactant, which is selected from one or both of Tween-20 and Triton X-100. Tween-20 (polysorbate-20) is a nonionic surfactant with the chemical name polyoxyethylene (20) dehydrated sorbitan monolaurate, which is prepared by condensation of sorbitan monolaurate and ethylene oxide under alkaline conditions. Triton X-100 (polyethylene glycol octylphenyl ether) is an organic polymer compound with the structural formula C 14 H22O(C2H4O) n It is a colorless or nearly colorless transparent viscous liquid. It is soluble in water, toluene, xylene, and ethanol, but insoluble in petroleum ether.

[0015] Furthermore, the material of the functional treatment layer includes a protein blocking agent, which is selected from one or both of gelatin and casein.

[0016] Furthermore, the alcohol solvent is selected from one or both of ethanol and isopropanol.

[0017] A method for preparing an immunochromatographic detection device includes the following steps: S1, dissolving the material of the functional treatment layer in an alcohol solvent to form a blocking agent solution; in the blocking agent solution, the mass fraction of a swelling agent is 0.1-5%, the mass fraction of a nonionic surfactant is 0.1-2%, and the mass fraction of a protein blocking agent is 0.1-1%; exemplary, the mass fraction of the swelling agent is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 5%. The mass fraction of nonionic surfactants is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or any two of these; the mass fraction of protein blocking agents is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, or any two of these.

[0018] S2. The inhibitor solution is sprayed onto a predetermined area to form a functional treatment layer in the predetermined area.

[0019] Furthermore, in the spraying liquid, the mass fraction of the swelling agent is 0.3-3 wt%, the mass fraction of the nonionic surfactant is 0.2-2%, and the mass fraction of the protein sealant is 0.1-0.5%.

[0020] Furthermore, the spraying amount of the inhibitor solution is 4-6 μL / cm, and the number of sprayed strips is 1-7.

[0021] The beneficial effects of this invention are: 1) By setting a functional processing layer in at least one predetermined region of the immunochromatographic channel, this processing layer is composed of a polymeric material soluble in alcohol solvents and capable of forming a dry film by spraying. The dry film undergoes controlled swelling or rehydration upon contact with an aqueous sample during detection, forming a high-viscosity layer. The material of the functional processing layer is a hydrophilic polymeric material that swells upon contact with water rather than completely dissolves. This material can form a continuous thin film structure in a dry state, and upon contact with water, it forms localized flow-blocking regions through molecular chain hydration and volume expansion. Thus, the chromatographic flow rate can be controlled and slowed down without altering the basic structure and operating steps of immunochromatography.

[0022] 2) The functional processing layer maintains structural stability under dry storage conditions and undergoes rehydration and swelling triggered by the sample liquid during detection. This swelling process is spatially limited to the spraying area and temporally synchronized with the sample liquid flow process, thereby creating a "retention-slow release" effect on the sample liquid and labeled particles, achieving dynamic control of the chromatographic flow rate rather than static blockage.

[0023] 3) The degree of chromatographic flow rate delay can be adjusted by controlling the spraying position, spraying load, or treatment area length of the functional treatment layer. The functional treatment layer is preferably located in the connection area between the sample pad and the cellulose membrane, the upstream region of the cellulose membrane, or the region before the detection line, to extend the effective antigen-antibody reaction time without affecting the release of labeled particles.

[0024] 4) By introducing surfactants and / or blocking agents into the spraying solution, the non-specific adsorption of labeled particles such as colloidal gold and latex microspheres by the functional treatment layer is reduced, ensuring that the labeled particles can still migrate with the sample solution and participate in the immune reaction in the swelling area.

[0025] 5) The functional processing layer is formed by spraying with an alcohol solvent system. The polymer material is dissolved in ethanol, isopropanol or a mixture thereof to form a spraying solution, which is applied to a predetermined area of ​​the cellulose membrane, sample pad or conjugate pad by spraying or atomizing. After the solvent evaporates, a uniform dry film is formed to adapt to the industrial preparation process of immunochromatographic test strips. Attached Figure Description

[0026] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 is a schematic diagram of an immunochromatographic detection device in one embodiment of the present invention; Figure 2 is a schematic diagram of an immunochromatographic detection device in another embodiment of the present invention.

[0029] 1. Base plate; 2. Sample pad; 3. Binding pad; 4. Cellulose membrane; 5. Absorbent pad; 6. Delay pad; 7. Functional treatment layer. Detailed Implementation

[0030] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0031] Unless otherwise shown or indicated in the operational embodiments, all figures used to represent the amounts, physicochemical properties, etc., of ingredients in the specification and claims are to be understood to be adjusted by the term "about" in all cases. Therefore, unless stated to the contrary, the numerical parameters listed in the foregoing specification and appended claims are approximations, and those skilled in the art can appropriately modify these approximations to obtain the desired characteristics by utilizing the teachings disclosed herein. The use of numerical ranges indicated by endpoints includes all numbers within that range and any range within that range; for example, 1 to 5 includes 1, 1.1, 1.3, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.

[0032] In existing technologies, most paper-hydrogel or membrane dispensing (such as agarose dripping) hydrogels are prepared using aqueous solutions, which presents stability and consistency issues during drying, storage, or mass production. The dripping / stretching process makes precise and uniform batch coating difficult. Insufficient compatibility with conjugated particles / antibodies is also a concern: some hydrogels that form water-resistant films (such as high-concentration ethyl cellulose or Eudragit) can adsorb proteins on or inside the membrane, or prevent the passage of colloidal labels, leading to decreased sensitivity or background signals and clogging risks. Furthermore, balancing controllable delay and label release is challenging: directly adding viscosity modifiers to the sample or creating irreversible barriers on the membrane can slow down the release, but often results in incomplete label release or blocked binding sites, affecting reliability and reproducibility.

[0033] Based on the problems of existing technology, the purpose of this invention is to extend the effective residence time of the marker in the detection zone, improve the antigen-antibody reaction window, and thus significantly improve the detection sensitivity (lower the detection limit) in a rapid detection strip based on lateral flow immunoassay (LFA) by forming a treatment layer on the chromatography channel that can be formed into a dry film by alcohol spraying and can be controlled to swell / rehydrate into a viscous / gel layer when exposed to water. This also avoids or minimizes the damage to the conjugated release of particles and the integrity of the membrane channel. The technical problem includes the following specific challenges: 1) How to select or prepare film-forming polymers (or mixtures) soluble in alcohol solvents to form a uniform dry film on NC membranes or sample pads / binding pads using a spraying / coating process; 2) How to rehydrate the dry film upon contact with water / sample solution and exhibit controllable water absorption and swelling or viscosity increase (i.e., "controllable swelling retention"), thereby flexibly controlling the chromatography time to improve binding efficiency; 3) While achieving the above-mentioned deceleration and sensitivity improvement, how to avoid irreversible capture of labeled substances such as colloidal gold or latex microspheres, avoid unacceptable background or blockage on the membrane, and ensure consistency and stability between batches.

[0034] This invention proposes an immunochromatographic detection device, comprising a base plate 1, a sample pad 2, a conjugate pad 3, a cellulose membrane 4, and an absorbent pad 5. The sample pad, conjugate pad, cellulose membrane, and absorbent pad are sequentially attached to the same side of the base plate in the chromatographic direction. The sample pad holds the sample solution, the cellulose membrane (NC membrane) holds the captured antibody and is a crucial area for the immune reaction, the conjugate pad holds the labeled antibody, and the absorbent pad provides the kinetic energy for chromatography, allowing the solution to flow upwards through the chromatographic process.

[0035] The immunochromatographic detection device also includes a functional processing layer 7, which is disposed in at least one predetermined area of ​​the immunochromatographic channel. The sample will inevitably pass through the predetermined area during the detection process, that is, it will inevitably pass through the functional processing layer 7.

[0036] The material of the functional processing layer 7 can be dissolved in alcohol / alcohol solvents or form a uniform solution or dispersion. A uniform dry film is formed in a predetermined area through industrial-friendly processes such as spraying / coating. When the dry film is exposed to water / sample loading, it can be controllably rehydrated and swell or its viscosity increases, thereby prolonging the effective residence time of the sample and the label in the detection area, improving the antigen-antibody binding efficiency and detection sensitivity, while ensuring the releaseability of the label (such as colloidal gold or latex microspheres), avoiding irreversible adsorption or membrane blockage, and possessing good dry storage stability and mass production capability.

[0037] The technical solution includes at least the following technical elements: 1) a functional material system that can be sprayed to form a film and swells when exposed to water; 2) a membrane surface spraying and dry membrane construction process based on alcohol solvents; 3) a controllable swelling-retention mechanism triggered during immunochromatography; and 4) compatibility design with labeled particles, membrane materials and immunoreaction systems.

[0038] Technical Element 1: Selection of Swellable Film-Forming Polymer Material To achieve controllable retardation of the chromatographic flow rate, this invention selects or constructs a polymer material system that can absorb water and swell upon contact with water, significantly increasing local liquid phase resistance. The material meets the following technical characteristics: it is a continuous thin film or continuous coating layer under dry conditions, without damaging the mechanical integrity of the film; it undergoes volume expansion, viscosity increase, or local gelation upon contact with water or sample liquid; the swelling process is reversible or quasi-reversible, without forming an impermeable dense barrier layer.

[0039] Research has revealed that the materials used in the functional treatment layer include swelling agents selected from one or more of hydroxypropyl cellulose (HPC), polyvinylpyrrolidone (PVP), and hydroxypropyl methylcellulose (HPMC). Upon contact with water, these materials can form locally high-viscosity or gel-like regions through molecular chain hydration, chain segment extension, and enhanced intermolecular interactions, thereby significantly improving capillary flow resistance.

[0040] Technical Element 2: Alcohol-Based Spray Coating Film Formation System. A functional treatment layer is formed using an alcohol-based spray coating film formation system. To achieve uniform distribution and industrial-scale preparation of functional materials on immunochromatographic membranes, this invention employs an alcohol-based solvent system as the carrier solvent. The technical solution includes: dissolving the functional material in ethanol, isopropanol, or a mixture thereof to form a homogeneous spray coating solution. The swelling agent mass fraction of the solution is preferably 0.1-5 wt%, more preferably 0.3-3 wt%; applying the solution to a predetermined area using spraying, pneumatic spraying, or micro-coating methods; and removing the solvent after spraying by natural evaporation or low-temperature drying, allowing the polymer (swelling agent) to form a continuous dry film on the membrane surface or within the pores.

[0041] By using an alcohol-based solvent system, pre-swelling or uneven deformation of the membrane structure caused by aqueous coating can be avoided, while significantly improving the consistency and repeatability of the coating.

[0042] Technical Element 3: Dry Film – Rehydration upon Contact with Water – Controllable Swelling Mechanism. The key technical effect of this invention is achieved through the following dynamic process: Under dry storage conditions, the functional material exists in the form of a dense or semi-dense film, without affecting the integrity of the test strip; after the sample liquid enters the treatment area, the film rapidly absorbs water and undergoes rehydration; the rehydrated material undergoes limited volume swelling or viscosity increase, forming a local flow resistance zone; this resistance zone creates a "retention-slow release" effect on the liquid phase, prolonging the residence time of the sample liquid and labeled particles in the detection line area. By controlling the polymer type, molecular weight, spraying load, and treatment zone length, the extent of flow rate delay can be precisely adjusted, avoiding complete blockage of capillary channels.

[0043] This invention achieves the following through the aforementioned flow rate delay mechanism: extending the contact time between the target molecule and the capture antibody at the detection line; increasing the probability of low-concentration target molecules being captured; thereby reducing the detection limit without increasing the antibody dosage or changing the labeling system.

[0044] Technical Element 4: Material-Particle-Membrane Material Compatibility System To avoid irreversible adsorption of functional materials on colloidal gold, latex microspheres and other labeled particles, the present invention further introduces the following technical measures: a low concentration of nonionic surfactant is introduced into the spraying solution. The nonionic surfactant is selected from any one or two of Tween-20 and Triton X-100.

[0045] A protein sealant is introduced into the spraying solution to seal the treated area after spraying, maintaining continuous pore channels even after swelling. The protein sealant is selected from one or both of gelatin and casein.

[0046] In some embodiments, the functional processing layer 7 is applied to a predetermined area by spraying, the predetermined area including: the side of the cellulose membrane 4 away from the base plate 1, and / or the side of the sample pad 2 away from the base plate 1, and / or the side of the bonding pad 3 away from the base plate 1.

[0047] Without altering the basic structure of immunochromatography or adding user operation steps, it achieves controllable slowing of sample flow rate, prolonging the effective reaction time between target molecules and capture antibodies, thereby improving detection sensitivity and lowering the detection limit.

[0048] In some embodiments, the functional processing layer is disposed at the end of the binding layer and / or the front end of the cellulose membrane.

[0049] In some embodiments, a delay pad 6 is also included, which is adhered to the base plate 1, with one end adhered to the side of the bonding pad 3 near the base plate and the other end adhered to the side of the cellulose membrane 4 away from the base plate. The predetermined area is the side of the delay pad 6 away from the base plate. The effect of designing the delay pad 6 is similar to that of spraying a retardant onto the end of the bonding pad or the front end of the NC membrane.

[0050] In some embodiments, the alcohol solvent is selected from one or both of ethanol and isopropanol.

[0051] A method for preparing an immunochromatographic detection device includes the following steps: S1, dissolving the material of the functional treatment layer in an alcohol solvent to form a blocking agent solution; in the blocking agent solution, the mass fraction of the swelling agent is 0.1-5 wt%, the mass fraction of the nonionic surfactant is 0.1-2%, and the mass fraction of the protein blocking agent is 0.1-1%.

[0052] Preferably, in the spraying liquid, the mass fraction of the swelling agent is 0.3-3 wt%, the mass fraction of the nonionic surfactant is 0.2-2%, and the mass fraction of the protein sealant is 0.1-0.5%.

[0053] S2. The inhibitor solution is sprayed onto a predetermined area to form a functional treatment layer in the predetermined area.

[0054] The spraying amount of the inhibitor solution is 4-6 μL / cm, and the number of spraying strips is 1-7.

[0055] The following examples illustrate the disclosure of this invention in more detail. These examples are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of this disclosure. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are based on weight, and all reagents used in the examples are commercially available or synthesized by conventional methods and can be used directly without further processing. The instruments used in the examples are also commercially available.

[0056] Example 1: Conventional preparation process for colloidal gold immunochromatographic test strips for direct determination of saliva. Preparation of colloidal gold solution: Take 100 ml of 0.01% chloroauric acid aqueous solution and heat to boiling. Add 0.7 ml of 1% trisodium citrate aqueous solution while stirring. The golden yellow chloroauric acid aqueous solution turns purple-red within 2 minutes. Continue boiling for 15 minutes. After cooling, add distilled water to restore the original volume. The gold sol prepared in this way has a highest absorption peak in the visible light region of 535 nm, and the measured value OD535 is about 1.1.

[0057] Colloidal gold-antibody labeling and purification: Adjust the pH of 1 mL of colloidal gold solution to 8.0 with 0.1 M K₂CO₃ solution. Then, add 1 mg of the antibody to be labeled dropwise to the colloidal gold solution and mix at low speed at room temperature for 60 minutes. Add 1 mL of blocking agent (100 mM Tris-HCl (pH 8.5), 1% BSA, 5% skim milk, 0.5% PEG 8000, 0.1% Proclin 300), and continue mixing by rotation for 30 minutes. Centrifuge at 4 °C / 14,000 rpm for 5 minutes, discard the supernatant, and resuspend the precipitate in reconstitution solution (50 mM Tris-HCl (pH 8.0), 0.1% BSA, 7.5% trehalose, 0.1% Tween 20). Repeat twice. Resuspend the precipitate in 1 mL of reconstitution solution and store at 4 °C for later use.

[0058] Treatment of various chromatography-related membranes: Immerse the glass fiber membrane in sample processing solution (standard saliva sample formulation: 50mM Tris-HCl (pH 8.0), 100mM NaCl, 1% Triton X-100, 0.5% sodium caseinate, 0.5% N-acetyl-L-cysteine), then dry at 37°C for 12 hours. Resuspend the labeled and purified gold-labeled antibody solution, fix it on the polyester fiber membrane using a quantitative spraying method at 2μL / cm, and then dry at 37°C for 12 hours. Detection line (T line): Spray another monoclonal antibody (1mg / mL) against the target analyte; control line (C line): Spray a secondary antibody against the labeled antibody (e.g., goat anti-mouse IgG, 1mg / mL), fix it on the cellulose membrane (NC membrane) using a quantitative streaking method at 1μL / cm, and then dry at 37°C for 12 hours.

[0059] Attaching the chromatography membrane and cutting and assembling the test strips: On the PVC backing board, attach the dried components in sequence with overlap: NC membrane → gold label pad → sample pad → absorbent pad, overlapping each component by approximately 1-2 mm to ensure smooth chromatography without gaps. Cut the assembled plate into test strips of uniform width of 4 mm using a strip cutter. Stack the cartridge, saliva swab, and test strips in order and press them together using a cartridge press, then close the cartridge cap.

[0060] Example 2: Preparation process of colloidal gold immunochromatographic test strips according to the method of the present invention. This example of the present invention uses hydroxypropyl cellulose (HPC) as a swelling agent, Tween 20 as a surfactant, and gelatin as a blocking agent. The blocking agent solution was prepared according to the following mass-volume fractions: HPC: 0.3–3 wt%, Tween 20: 0.2–2 wt%, gelatin: 0.1–0.5 wt%; anhydrous ethanol or a >70% (v / v) aqueous ethanol solution was used as the solvent. The solution was magnetically stirred at room temperature until a homogeneous transparent or semi-transparent solution was formed, and then set aside.

[0061] This invention is illustrated by adding a delay pad and spraying a retardant solution onto it. Spraying the retardant at the end of the bonding pad or the front of the NC film has a similar effect; any retardant structure in this manner is within the scope of this invention. The retardant is uniformly sprayed onto the delay pad using the gold spraying function of a gold spraying apparatus, with the spraying amount controlled at 5 μL / cm. The number of consecutive spraying lines is set according to different research needs. After spraying, the delay pad is dried at 25-40°C to allow the ethanol to completely evaporate, forming a continuous and uniform thin film on and inside the delay pad.

[0062] The process of pasting the chromatographic membrane and cutting and assembling the test strips according to Example 1 is followed. The prepared delay pad is pasted under the binding pad and on the NC membrane. When pasting, ensure that the sprayed inhibitor strip is fully exposed.

[0063] Example 3: Sensitivity Assessment Targets and Sample Preparation for Direct Saliva Immunochromatographic Assay To better evaluate the effectiveness of the present invention's technical approach on samples, this example selected two representative targets in saliva: pepsin (PEP) and human chorionic gonadotropin (HCG). Clinically high-concentration positive saliva samples were used as samples, diluted with negative saliva samples to prepare concentration gradient sensitivity reference standards for performance testing of various technical methods. The concentrations of the reference standards are shown in Table 1: Table 1

[0064] Example 4: Effect of Different Concentrations of Swelling Agent on Detection Results. The effectiveness of the present invention was evaluated using hydroxypropyl cellulose (HPC) as an example of a swelling agent. The inhibitor was prepared according to the following swelling agent concentration gradient, with each concentration designed to have three consecutive stripes of width. The test card was then prepared according to the method described in Example 2. The specific components of the test card are shown in Table 2.

[0065] Table 2

[0066] The prepared detection card was used to test the sensitivity reference disk prepared in Example 3 to evaluate the detection performance of the blocker described in this invention. The statistical results of the positive and negative results of the samples are shown in Table 3: Table 3

[0067] The chromatography completion times for both the PEP-0 and HCG-0 experimental groups were recorded simultaneously, as shown in Table 4: Table 4

[0068] Based on the above test results, the retardant effect of the HPC-based swelling agent prepared according to this invention on the chromatography process gradually increases with the increase of the swelling agent concentration. Within a certain range, the retardant can significantly improve the detection sensitivity of the target. However, when the concentration of the swelling agent is too high, it will also seriously affect the chromatography process, leading to blockage of colloidal gold particles and chromatography failure.

[0069] Example 5: Effect of Different Spray Widths (Number of Continuous Spray Strips) on Detection Results. The effectiveness of this invention was evaluated using hydroxypropyl cellulose (HPC) as an example of a swelling agent. The inhibitors 1% HPC, 1% Tween 20, and 0.25% gelatin were prepared according to the following swelling agent concentrations. Different numbers of continuous spray strips (corresponding to different inhibition widths) were designed, as shown in Table 5. Then, the test card was prepared according to the method described in Example 2: Table 5

[0070] The prepared detection card was used to test the sensitivity reference disk prepared in Example 3 to evaluate the detection performance of the blocker described in this invention. The statistical results of positive and negative results for the samples are shown in Table 6: Table 6

[0071] The chromatography completion time for both the PEP-0 and HCG-0 experimental groups was recorded, and the results are shown in Table 7: Table 7

[0072] The test results show that as the width of the inhibitor increases (i.e., the number of consecutive spray strips), the time required to complete chromatography gradually increases, the inhibitory effect becomes stronger, and the corresponding detection sensitivity gradually improves. However, when the number of spray strips exceeds seven, a performance decline trend appears, possibly due to greater retention of colloidal gold particles. Adjusting the number of spray strips can, to some extent, regulate the chromatography time and detection performance.

[0073] Example 6: Effect of Different Surfactant Concentrations in the Restrictor on Detection Efficacy. The effectiveness of the restrictor of this invention was evaluated using surfactant Tween20 as an example. The restrictor was prepared according to the Tween20 concentration gradient in Table 8, with each concentration designed to have three consecutive bands of width. The detection card was then prepared as described in Example 2: Table 8

[0074] The prepared detection card was used to test the sensitivity reference disk prepared in Example 3 to evaluate the detection performance of the blocker described in this invention. The statistical results of the positive and negative results of the samples are shown in Table 9: Table 9

[0075] The chromatography completion time for both the PEP-0 and HCG-0 experimental groups was recorded, and the results are shown in Table 10: Table 10

[0076] Based on the above test results, the content of Tween20 in the blocking agent has a significant impact on the blocking effect. When the concentration is too low, it may change the hydrophilic and hydrophobic structure on the surface of the swelling agent, which may completely block the colloidal gold particles and cause chromatography failure. When the concentration is too high, it may affect the antigen-antibody binding effect and thus fail to effectively improve the sensitivity.

[0077] Example 7: Effect of Different Blocking Agent Concentrations on Detection Effect. Taking gelatin as an example, the effect of the blocking agent of the present invention was evaluated. The blocking agent was prepared according to the gelatin concentration gradient in Table 11, and each concentration was designed to have a width of three consecutive lines. Then, the detection card was prepared according to the method described in Example 2.

[0078] Table 11

[0079] The prepared test card was used to test the sensitivity reference disk prepared in Example 3 to evaluate the detection performance of the blocker described in this invention. The positive and negative results of the samples are statistically shown in Table 12.

[0080] Table 12

[0081] The complete chromatography time for both the PEP-0 and HCG-0 experimental groups was recorded, and the results are shown in Table 13.

[0082] Table 13

[0083] Based on the above test results, the gelatin content in the retardant has a significant impact on the retardation effect. When the concentration is too low, it may non-specifically adsorb colloidal gold particles, leading to severe chromatography failure. When the concentration is too high, it may cause gelatin molecules to be difficult to dissolve quickly in water, resulting in blockage of the delay pad.

[0084] This invention illustrates the detailed process equipment and process flow through the above embodiments. However, this invention is not limited to the detailed process equipment and process flow described above, meaning that this invention does not necessarily depend on the detailed process equipment and process flow to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials, structural changes, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0085] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0086] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0087] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

[0088] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. An immunochromatographic detection device, comprising a base plate, a sample pad, a conjugate pad, a cellulose membrane, and an absorbent pad, wherein the sample pad, conjugate pad, cellulose membrane, and absorbent pad are sequentially attached to the same side of the base plate in the direction of chromatography, characterized in that, It also includes a functional processing layer disposed in at least one predetermined region of the immunochromatographic channel. The material of the functional processing layer is a polymeric material that is soluble in alcohol solvents and can be sprayed to form a dry film. The dry film swells or rehydrates upon contact with an aqueous sample to form a high-viscosity layer.

2. The immunochromatographic detection device according to claim 1, characterized in that, The functional treatment layer is applied to a predetermined area by spraying. The predetermined area includes: the side of the cellulose membrane away from the base plate, and / or the side of the sample pad away from the base plate, and / or the side of the bonding pad away from the base plate.

3. The immunochromatographic detection device according to claim 2, characterized in that, The functional processing layer is disposed at the end of the binding layer and / or the front end of the cellulose membrane.

4. The immunochromatographic detection device according to claim 1, characterized in that, It also includes a delay pad, which is adhered to the base plate, with one end of the bonding pad adhered to the side of the base plate near the base plate and the other end adhered to the side of the cellulose membrane away from the base plate, wherein the predetermined area is the side of the delay pad away from the base plate.

5. The immunochromatographic detection device according to any one of claims 1-4, characterized in that, The material of the functional treatment layer includes a swelling agent, which is selected from one or more of hydroxypropyl cellulose, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.

6. The immunochromatographic detection device according to claim 5, characterized in that, The material of the functional treatment layer includes a nonionic surfactant, which is selected from Tween-20 and Triton X-100, or both; and / or, the material of the functional treatment layer includes a protein blocking agent, which is selected from gelatin and casein, or both.

7. The immunochromatographic detection device according to claim 1, characterized in that, The alcohol solvent is selected from one or both of ethanol and isopropanol.

8. The method for preparing an immunochromatographic detection device according to any one of claims 1-7, characterized in that, The process includes the following steps: S1, dissolving the material of the functional treatment layer in an alcohol solvent to form a blocking agent solution; in the blocking agent solution, the mass fraction of the swelling agent is 0.1-5%, the mass fraction of the nonionic surfactant is 0.1-2%, and the mass fraction of the protein blocking agent is 0.1-1%; S2, spraying the blocking agent solution onto a predetermined area to form a functional treatment layer in the predetermined area.

9. The method for preparing an immunochromatographic detection device according to claim 1, characterized in that, In the retardant solution, the mass fraction of the swelling agent is 0.3-3 wt%, the mass fraction of the nonionic surfactant is 0.2-2%, and the mass fraction of the protein blocking agent is 0.1-0.5%.

10. The method for preparing an immunochromatographic detection device according to claim 1, characterized in that, The amount of the inhibitor solution sprayed in the predetermined area is 4-6 μL / cm, and the number of sprayed strips is 1-7.

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