A chromatographic membrane with a fluid diode structure and a multi-index immunochromatographic test strip

Abstract

This invention provides a chromatographic membrane with a fluid diode structure and a multi-index immunochromatographic test strip. The surface of the chromatographic membrane is composed of a series of continuously distributed, parallel barbed arrow-shaped grooves, formed by continuous barbed arrow-shaped groove units connected end-to-end; each barbed arrow-shaped groove unit is composed of axisymmetric arc-shaped curved walls. The chromatographic membrane provided by this invention has a faster flow rate, enabling faster, better, and more uniform water transport, avoiding the decrease in liquid flow velocity with increasing flow distance; it can flow in a directional manner, with reverse blocking, a higher rectification coefficient, fast and efficient mass transfer, a longer flow distance, reduced reagent consumption and residue, and the advantage of transporting trace samples. Using the chromatographic membrane for multi-index immunochromatographic detection can also accelerate the detection speed, reduce differences in contact / reaction time with different detection bands, and reduce the amount of sample used in the detection process.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to a chromatography membrane with a fluid diode and a multi-index immunochromatographic test strip. Background Technology

[0002] Immunochromatography is a novel membrane detection technology based on antigen-antibody specific immune reactions. The method involves immobilizing antibodies or antigens labeled with various markers in the conjugate pad of a test strip; using strip-shaped fiber chromatographic material (coated with antibody or antigen) as the stationary phase and the sample and reaction solution as the mobile phase; and using the capillary action of the mobile phase on the chromatographic membrane to move the analyte released from the conjugate pad to the detection line and achieve immunodetection.

[0003] Chromatographic membranes are the site of liquid flow and the endpoint of specific immune reactions during immunochromatography. They are also the result interpretation components in immunochromatographic detection. Their inherent properties, processing technology, and product quality directly affect the reliability of immunochromatographic results. Currently, most chromatographic membranes are fibrous membranes, constructed by tightly stacking various fibrous materials, such as nitrocellulose membranes, cellulose acetate membranes, and cellulose acetate butyrate membranes. Due to the random orientation of the fibrous fibers, the reagent size usually needs to be increased. Due to their high productivity, high throughput, and low cost, they have become the preferred choice for chromatographic test strips. When the sample and reaction solution wet this type of fibrous membrane, the tiny gaps between the fibers provide space for capillary flow of the liquid; however, due to the random orientation of the fibers, the flow performance of this capillary flow is poor, leading to differences between batches of fibrous membranes. Furthermore, because the tiny gaps collapse after being wetted, the flow rate of the mobile phase is further slowed down, thus requiring the immunochromatographic test strips to be reduced in size to achieve rapid and accurate detection. In addition, the development of multi-index immunochromatographic test strips based on fiber filament chromatography membranes is also limited, as the slow flow rate on the membrane requires an increase in the amount of reagent to achieve liquid spreading during use.

[0004] Therefore, in order to overcome the problems of existing fiber filament chromatography membranes and the flowability issues of multi-index immunochromatographic assays, it is urgent to develop a new type of immunochromatographic membrane. This membrane needs to have controllable flow orientation, high transport efficiency, and excellent and uniform flow performance to replace traditional fiber filament chromatography membranes and become a new core component in immunochromatographic test strips, thus providing a guarantee for immunochromatographic technology and reliable immunochromatographic assays. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the above-mentioned problems and provide a chromatographic membrane with a fluid diode structure and a multi-index immunochromatographic test strip. This invention can solve the problems of the chromatographic speed slowing down with the increase of chromatographic distance on the fiber filament chromatographic membrane, the non-specific adsorption of material components on the membrane and the retention of sample solution, and the limited fluidity and development of the multi-index immunochromatographic test strip. It can enhance the efficiency of liquid transport, reduce the consumption of detection reagents, and thus achieve rapid detection.

[0006] The purpose of this invention is to provide a chromatography membrane with a fluid diode structure.

[0007] Another object of the present invention is to provide applications of the chromatographic membrane.

[0008] Another object of the present invention is to provide a multi-index chromatography test paper having a fluid diode structure.

[0009] Another object of the present invention is to provide applications for multi-index chromatography test strips.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention designs a fluid diode-structured chromatography membrane capable of enabling rapid directional flow of liquid. The surface of this membrane is composed of a series of continuously distributed, parallel barbed arrow-shaped grooves. These barbed arrow-shaped grooves are formed by continuous barbed arrow-shaped groove units connected end-to-end. Each barbed arrow-shaped groove unit is composed of an axisymmetric arc-shaped curved wall surface, including a first side wall and a second side wall. The width L6 of the first and second side walls from the axis is the same. The tangents of the outer arc surfaces at the tail ends of the first and second side walls intersect to form a barbed angle α. The tangent of the outer arc surface at the head end of the second side wall intersects to form a sharp angle with the head end of the first side wall of the adjacent barbed arrow-shaped groove unit. The tangent of the outer arc of the first side wall surface at the beginning and the line parallel to the axis form an angle β between the wall surface axis and the axis.

[0012] This fluid diode structure enables directional liquid flow without external energy input and achieves liquid surface pinning in the opposite direction of flow; the arc-shaped curved walls in its groove are hydrophilic sidewalls, which can guide the rapid flow of liquid. The sharp angle... The angle β between the barbed and wall axis enables unidirectional flow in the continuous barbed arrow-shaped groove structure. The design is based on the Laplace pressure theory of bent liquids, surface wetting theory, and surface tension analysis, achieving rapid directional flow of the liquid. The barb angle α primarily provides stronger capillary action to the liquid within the barbed arrow-shaped groove unit, allowing the liquid to flow more quickly towards the arrow's tail.

[0013] Further, Where γGS is the surface energy of the gas-solid interface, γSL is the surface energy of the solid-liquid interface, γGL is the surface energy of the gas-liquid interface, and θc is the limiting contact angle at which the liquid remains stable at a sharp angle boundary; sharp angle The angle is defined as ranging from 0° to 90°, preferably from 5° to 20°, and more preferably...

[0014] Further, Wherein, θ is the inherent contact angle of the wall (reflecting the degree of wetting of the solid surface by the liquid, which is only related to the inherent properties of the liquid and the solid), L3 is the distance between the top and bottom of the first end of the axisymmetric first side wall, and H is the height of the groove; the included angle β of the wall axis is defined in the range of 5° to 40°, and more preferably β = 30°.

[0015] Furthermore, the barb angle α is defined as ranging from 0° to 90°, preferably from 5° to 40°, and more preferably α = 26°.

[0016] This fluid diode structure guides the rapid directional flow of liquid in two parts. First, after the liquid enters the barbed, arrow-shaped groove unit, the hydrophilic groove sidewalls with their curved surfaces increase the pressure difference between the liquid inside the groove and near the surface, thus driving the liquid to spread and flow. Second, the grooves prevent the liquid from spreading outwards in the axial direction, creating sharp angles. This makes it more difficult for the liquid to wet the inner wall of the barbed arrow-shaped groove unit structure, while the barb angle α provides a stronger capillary effect on the liquid within the barbed arrow-shaped groove unit, causing the liquid within the barbed arrow-shaped groove unit structure to flow faster towards the arrow's tail. The specific flow principle is as follows:

[0017] 1) When the liquid flows towards the arrow's tail, just as it enters a barbed arrow-shaped groove unit 120, the hydrophilic sidewalls within the barbed arrow-shaped groove unit cause the liquid to form a meniscus-shaped concave liquid surface. At this point, the internal pressure of the liquid near the arrowhead is greater than the pressure near the arrow's tail, and the liquid will spread and flow towards the arrow's tail under the influence of the pressure difference. When the liquid's leading surface reaches the sharp angle at the connection between adjacent barbed arrow-shaped groove units... When positioned, the liquid will split, flowing to the barbed wings and the next barbed arrow-shaped groove unit respectively; then repeating in this way, the liquid achieves continuous rapid flow within the structure based on the pressure difference.

[0018] 2) When the liquid flows in the direction of the arrow, it will flow along the gradually narrowing channel, filling and reaching the sharp angle at the connection of adjacent barbed arrow-shaped groove units. At this location, due to surface tension, the liquid will gradually form a meniscus-shaped convex surface. Only when the liquid continuously accumulates at this point, and the curvature of the convex surface continuously increases until the liquid surface contacts the second sidewall of the next adjacent barbed arrow-shaped groove unit, can it break through the restriction and flow into the next adjacent barbed arrow-shaped groove unit, wetting the inner wall of the structure. Because the barb angle α provides a stronger capillary effect on the liquid within the barbed arrow-shaped groove unit, and the liquid will spread and flow more rapidly towards the arrow tail direction according to the pressure gradient, it is difficult for the liquid at this location to accumulate in large quantities and thus break through the restriction to flow into the next barbed arrow-shaped groove unit. Therefore, the liquid surface flowing in the arrow direction is pinned, which is a control of the directional flow of the liquid.

[0019] Preferably, the axial length L1 from the first end of the first sidewall to the first end of the second sidewall of the barbed arrow-shaped groove unit is 100-2000 μm; the axial length L2 of the second sidewall is 1-2 times L1; the distance L3 between the upper and lower ends of the first sidewall of the axisymmetric first sidewall is 10-200 μm; the distance L4 between the upper and lower ends of the second sidewall of the axisymmetric second sidewall is 1-10 times L3; and the height H of the groove unit is 5-500 μm.

[0020] Preferably, the minimum distance L5 between the parallel-distributed adjacent fluid diode grooves is 5 to 500 μm.

[0021] The fluid diode structure chromatography membrane provided by this invention, with the assistance of the hydrophilic arc-shaped curved groove sidewalls of the fluid diode structure enhancing the internal pressure difference of the liquid, possesses the following flow behavior control capabilities: 1) It has the ability to allow rapid liquid flow; 2) Due to the fluidity and continuity of the liquid, the chromatography membrane can draw liquid from other stationary phases stacked on the membrane surface into the fluid diode structure on the membrane surface through capillary action; 3) With the assistance of the special morphology of the fluid diode structure, the chromatography membrane has the ability to lock the liquid on the membrane, preventing the liquid from flowing in the opposite or tangential direction to the specified flow direction, thus achieving directional flow of the liquid. Furthermore, the chromatography membrane provided by this invention has a higher flow coefficient, a longer flow distance, higher mass transfer efficiency during detection, and less reagent consumption, reducing the sample volume required for detection and enabling the transfer of trace samples; the flow of this chromatography membrane is controlled, preventing ineffective diffusion and overflow of the liquid on the chromatography membrane; simultaneously, it prevents the liquid from flowing back towards the flow initiation direction to these hygroscopic media, reducing liquid flow loss. On the other hand, after the chromatographic membrane is formed, it undergoes hydrophilization and functionalization treatment, which can prevent non-specific adsorption of substances caused by hydrophobic interactions, thereby reducing the flow loss of substances carried in the liquid based on the above-mentioned beneficial effects.

[0022] The present invention also provides a method for preparing a chromatography film having a fluid diode structure, comprising the following steps:

[0023] S1. Using technologies such as laser cutting, precision micromachining, photolithography, and 3D printing, the surface structure of the above-mentioned chromatographic membrane is directly processed and printed on the flat surface of the prepared material to obtain a chromatographic membrane with a fluid diode structure.

[0024] Alternatively, a reverse structure of the above-mentioned surface structure of the chromatographic membrane can be manufactured on a flat surface of the preparation material to obtain a manufacturing mold; then, the fluid diode structure can be replicated and molded by means of hot pressing, injection molding, molding, etc., to obtain a chromatographic membrane with a fluid diode structure.

[0025] S2. Perform surface hydrophilic functionalization treatment on the chromatography membrane obtained in S1.

[0026] Preferably, the material prepared in step S1 is a polymer, metal, ceramic, semiconductor, glass, thin film, or nonwoven fabric;

[0027] Preferably, the polymer material is not limited to polymers at the technical level, but also includes biodegradable polymers, such as polyphosphazene and polylactic acid (PLAs); and elastomers, such as polydimethylsiloxane (PDMS).

[0028] More preferably, the polymer is selected from one of polymethyl methacrylate (PMMA), high-density polyethylene (HDPE), PLAs, polypropylene (PP), silicone, epoxy resin, hydrogel, polyamide (PA), and polyethylene terephthalate (PET).

[0029] More preferably, the metal is selected from aluminum profiles, copper plates, iron plates, or alloy plates.

[0030] Preferably, the hydrophilic functionalization treatment in step S1 is a physical treatment method or a chemical treatment method.

[0031] More preferably, the physical treatment method includes coating a hydrophilic coating (using water-based acrylic polyurethane topcoat or silica coating), laser oxidation, and sputtering / evaporation treatment (using SiO2, TiO2, or Al2O3).

[0032] More preferably, the chemical treatment method includes plasma treatment, covalent binding modification of hydrophilic groups (using Tween-20, polyethyleneimine), electrostatic adsorption of hydrophilic small molecules, etc.

[0033] Therefore, the present invention provides the application of a chromatography membrane with a fluid diode structure in immunoassay, nucleic acid assay, biological particle assay, or in the preparation of immunochromatographic assay products.

[0034] Preferably, the product is a multi-index immunochromatographic test strip.

[0035] The present invention also provides a multi-index immunochromatographic test strip with a fluid diode structure, comprising the above-mentioned chromatographic membrane with a fluid diode structure, wherein the surface of the chromatographic membrane is provided with a variety of specifically captured antigens / antibodies to form specific immune binding regions.

[0036] Furthermore, the chromatographic test strip also includes other immunochromatographic components: a support base plate, a conjugate pad, an absorbent pad, and a sample pad. The assembly method is as follows: the chromatographic membrane is directly fixed to the surface of the support base plate; absorbent pads and conjugate pads are placed at both ends of the chromatographic membrane, respectively; wherein the absorbent pads and conjugate pads partially overlap with the chromatographic membrane, and the remaining parts are fixed to the surface of the support base plate; at the other end of the conjugate pad, a sample pad is placed, the sample pad partially overlapping with the conjugate pad, and the remaining parts are fixed to the surface of the support base plate.

[0037] Preferably, the overlap length between the absorbent pad, the binding pad, and the chromatography membrane is 2–4 mm; the overlap length between the sample pad and the binding pad is 2–4 mm.

[0038] Preferably, the width of the test paper is about 2.0 to 10.0 mm.

[0039] Preferably, the length of the sample pad is about 1.5 to 2.0 cm.

[0040] Preferably, the length of the conjoint pad is about 1.0 to 1.5 cm.

[0041] Preferably, the length of the chromatography membrane is about 2.0 to 5.0 cm.

[0042] Preferably, the length of the absorbent pad is about 1.5 to 2.5 cm.

[0043] Furthermore, the surface of the chromatographic membrane with a fluid diode structure is provided with a variety of specifically captured antigens / antibodies to form specific immune binding regions. When applied to multi-index immunoassay scenarios, the position of its surface specific immune binding regions can be set in a variety of different arrangements. The formation of immune binding regions includes directly fixing antigens or antibodies to the surface region of the chromatographic membrane, or adding additional three-dimensional substrates to the surface of the chromatographic membrane to form strips.

[0044] In addition, the present invention also provides the application of the multi-index immunochromatographic test strip in liquid flow, chromatography, immunoassay, nucleic acid detection, and biological particle detection.

[0045] The present invention has the following beneficial effects:

[0046] This invention provides a chromatographic membrane with a fluid diode structure and a multi-index immunochromatographic test strip. Through a special morphological design of the chromatographic membrane surface structure, this invention enables directional liquid flow without external energy input and achieves liquid surface pinning in the opposite direction of flow, thereby achieving rapid directional liquid flow. This fluid diode structure is easy to process in batches with high fidelity and replicate onto various substrate surfaces, thus preparing more uniform and stable chromatographic membranes.

[0047] Compared to existing fiber-based chromatography membranes, this method solves the problems of decreased chromatography speed with increasing chromatography distance, non-specific adsorption of components on the membrane, and sample solution retention, thereby enhancing liquid transport efficiency and reducing reagent consumption. It also addresses issues in multi-index immunochromatographic assays such as poor flowability, slow detection speed, variations in contact / reaction time with different detection bands, and high sample volume requirements.

[0048] The preparation of chromatographic membranes based on fluid diode structures is simple and can be mass-produced. The structural mold can be directly replicated, and the membranes can be fabricated on plastic or metal sheets, thus enabling the mass production of chromatographic membranes with this structure. The fabrication conditions are simpler and more uniform than those for existing chromatographic membranes, and the manufacturing process exhibits good stability and reproducibility. Based on these advantages, the chromatographic membrane provided by this invention has the potential to replace existing chromatographic membranes, enhances the flow capacity of liquids on the membrane, improves the problem of insufficient uniformity in flow performance in current chromatographic membrane technology, and reduces the consumption of detection reagents, becoming a breakthrough in next-generation chromatographic technology. Attached Figure Description

[0049] Figure 1 Schematic diagram and physical diagram of the fluid diode structure for rapid directional flow of liquid provided by the present invention (where a is a planar view of the fluid diode structure chromatographic membrane: 100-fluid diode structure chromatographic membrane, 110-fluid diode groove, 120-barbed arrow-shaped groove unit, 121-first sidewall, 122-second sidewall, 123-barbed angle α, 124-sharp angle). 125 - included angle β between the wall and the axis; b is the structural dimension of the fluid diode structure chromatography membrane; c is a physical image of the fluid diode structure chromatography membrane.

[0050] Figure 2 A schematic diagram illustrating the rapid directional flow of liquid in the fluid diode structure provided by the present invention;

[0051] Figure 3 This is a comparison diagram of the chromatographic membrane described in this invention and the filament-based chromatographic membrane in liquid directional flow, as shown in Comparative Example 1.

[0052] Figure 4This is a comparison diagram of the chromatographic membrane described in this invention and the fiber-based chromatographic membrane in terms of rapid liquid flow, as shown in Comparative Example 2.

[0053] Figure 5 This is a comparative diagram showing the results of the chromatography membrane described in this invention and the fiber-based chromatography membrane in terms of non-specificity of material components on the membrane or retention performance of sample solution in Comparative Example 3.

[0054] Figure 6 This is a comparative diagram showing the performance of the chromatography membrane described in this invention and the fiber-based chromatography membrane in terms of micro-liquid transport, as shown in Comparative Example 4.

[0055] Figure 7 The present invention provides a multi-index immunochromatographic test strip with a fluid diode structure (wherein, 210-sample pad, 220-labeled antibody / antigen binding pad, 100-chromatographic membrane with the fluid diode structure, 310-T-line immunobinding zone, 320-C-line immunobinding zone, 230-absorbent pad, 240-adhesive support base plate);

[0056] Figure 8 The present invention provides a multi-index immunochromatographic test strip with a fluid diode structure (wherein, 210-sample pad, 220-labeled antibody / antigen binding pad, 100-chromatographic membrane having the fluid diode structure, 310-T-line immunobinding zone, 320-C-line immunobinding zone, 230-absorbent pad, 240-adhesive support base). Detailed Implementation

[0057] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0058] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0059] Example 1: Design of a Chromatographic Membrane with a Fluid Diode Structure

[0060] This invention is based on the characteristics of a fluid diode: it can flow in one direction and is blocked in the opposite direction. By driving and locking the water through an arc-shaped curved wall, the structure has a high rectification coefficient, resulting in a chromatographic membrane with a fluid diode structure that enables rapid directional flow of liquid. This invention solves the problem of random liquid orientation in chromatographic membranes and avoids the problem of inaccurate detection signals and detection failure caused by differences in the residence time of the detected substance on different strips due to slowed flow rate.

[0061] The surface of the chromatography membrane designed in this invention is composed of a series of parallel, continuously distributed, barbed, arrow-shaped grooves, as shown in the diagram below. Figure 1 As shown in Figure a; the continuous barbed arrow-shaped groove 110 is formed by continuous barbed arrow-shaped groove units 120 connected end to end; the barbed arrow-shaped groove unit 120 is composed of an axisymmetric arc-shaped curved wall surface, the arc-shaped curved wall surface includes a first side wall surface 121 and a second side wall surface 122, the width L6 of the first side wall surface 121 and the second side wall surface 122 from the axis is the same; the tangent of the outer arc surface of the tail end of the first side wall surface 121 and the second side wall surface 122 intersects to form a barbed angle α123; the first end of the second side wall surface 122 intersects to form a sharp angle with the tangent of the outer arc surface of the first side wall surface 121 of the adjacent barbed arrow-shaped groove unit 120. The tangent of the outer arc of the first side wall 121 and the line parallel to the axis form an angle β125 between the wall axis and the axis.

[0062] The curved wall within the groove is a hydrophilic sidewall. This curved wall enhances the pressure difference between the liquid inside the groove and near the liquid surface, allowing the liquid to flow rapidly; sharp angles... The angle β between the wall axis and the continuous barbed arrow-shaped groove enables unidirectional flow. The design is based on the Laplace pressure theory of curved liquids, surface wetting theory, and surface tension analysis to achieve rapid directional flow of liquids.

[0063] Specifically: Where γGS is the surface energy of the gas-solid interface, γSL is the surface energy of the solid-liquid interface, γGL is the surface energy of the gas-liquid interface, and θc is the limiting contact angle at which the liquid remains stable at a sharp angle boundary; sharp angle The angle is defined as ranging from 0° to 90°, preferably ranging from 10° to 20°.

[0064] Wherein, θ is the inherent contact angle of the wall (reflecting the degree of wetting of the solid surface by the liquid, which is only related to the inherent properties of the liquid and the solid), L3 is the distance between the top and bottom of the first side wall 121 of the axisymmetric side wall, and H is the height of the groove; the included angle β of the wall axis is defined in the range of 5° to 40°, and more preferably β = 30°.

[0065] The setting of the barb angle α is mainly to provide a stronger liquid traction capillary effect on the liquid in the barb-shaped arrow-shaped groove unit, so that the liquid in the barb-shaped arrow-shaped groove unit structure flows towards the tail of the arrow faster; the angle of the barb angle α is defined as ranging from 0° to 90°, preferably 5° to 40°, and more preferably α = 26°.

[0066] The structural dimensions of the barbed arrow-shaped groove unit 120 are marked as follows: Figure 1 As shown in Figure b, the axial length L1 from the first end of the first sidewall 121 to the first end of the second sidewall 122 of the barbed arrow-shaped groove unit 120 is 100–2000 μm; the axial length L2 of the second sidewall 122 is 1–2 times L1; the distance L3 between the upper and lower ends of the axisymmetric first sidewall 121 is 10–200 μm; the distance L4 between the upper and lower ends of the axisymmetric second sidewall 122 is 1–10 times L3; and the height H of the groove unit 120 is 5–500 μm. The minimum distance L5 between adjacent parallel fluid diode grooves 120 is 5–500 μm. A physical diagram of the chromatographic membrane with a fluid diode structure is shown below. Figure 1 As shown in c.

[0067] A schematic diagram of directional flow of liquid is shown below. Figure 2 As shown, after the liquid enters the barbed arrow-shaped groove unit 120, the hydrophilic arc-shaped curved wall of the groove increases the pressure difference between the liquid inside the groove and near the liquid surface, thereby driving the liquid to spread and flow. Simultaneously, the groove prevents the liquid from spreading and flowing in directions outside the axial direction, creating a sharp angle. This makes it more difficult for the liquid to wet the inner wall of the barbed arrow-shaped groove unit 120 structure, while the barb angle α provides a stronger capillary effect on the liquid within the barbed arrow-shaped groove unit 120, allowing the liquid within the structure to flow faster towards the arrow's tail. The specific flow principle is as follows:

[0068] (1) When the liquid flows towards the arrow's tail and just enters a barbed arrow-shaped groove unit 120, the hydrophilic arc-shaped wall within the barbed arrow-shaped groove unit 120 causes the liquid to form a meniscus-shaped concave liquid surface. At this time, the internal pressure of the liquid near the arrowhead is greater than the pressure near the arrow's tail, and the liquid will spread and flow towards the arrow's tail under the action of the pressure difference. When the liquid's leading surface reaches the sharp angle at the connection between adjacent barbed arrow-shaped groove units 120... When positioned, the liquid will split, flowing to the two wings of the barb and the next barb-shaped arrow-shaped groove unit 120 structure respectively; then repeating in this way, the liquid achieves continuous rapid flow within the structure based on the pressure difference.

[0069] (2) When the liquid flows in the direction of the arrow, the liquid will flow along the gradually narrowing channel to fill and reach the sharp angle at the connection of the adjacent barbed arrow-shaped groove unit 120. At this location, due to the surface tension of the liquid, it will gradually form a crescent-shaped convex surface. Only when the liquid continuously accumulates at this point, and the curvature of the convex surface continuously increases until the liquid surface contacts the second side wall of the next adjacent barbed arrow-shaped groove unit 120, can it break through the restriction and flow into the next adjacent barbed arrow-shaped groove unit 120, wetting the inner wall of the structure. Because the barb angle α provides a stronger capillary effect on the liquid in the barbed arrow-shaped groove unit 120, and the liquid will spread and flow more rapidly towards the arrow tail direction according to the pressure gradient, it is difficult for the liquid at this location to accumulate in large quantities and thus break through the restriction to flow into the next barbed arrow-shaped groove unit 120. Therefore, the liquid surface flowing in the arrow direction is pinned, which is a control of the directional flow of the liquid.

[0070] Simultaneously, this invention also calculates the rectification coefficient of the structure. The rectification coefficient is a dimensionless parameter that measures the unidirectional flow capability of the surface structure, denoted by the letter k, and the calculation formula is: k = Ls / Lp; where Ls represents the forward spreading length of the liquid, defined as the distance between the terminating liquid surface on the forward spreading side and the liquid surface at the starting position when the liquid stops flowing on the surface film; Lp represents the reverse breakthrough length of the liquid, defined as the distance between the terminating liquid surface on the reverse breakthrough side and the liquid surface at the starting position when the liquid stops flowing on the surface film.

[0071] The rectification coefficient of the chromatography membrane of this invention is k = 14.5 mm / 1 mm = 14.5, indicating a rectification coefficient of 14.5. This demonstrates that the chromatography membrane possesses a high rectification coefficient, resulting in a longer flow distance, higher mass transfer efficiency during detection, and lower reagent consumption. For example, with conventionally used NC membranes, the rectification coefficient after droplet release from the surface can be calculated as 1, meaning the distribution of matter on the membrane is random and non-directional. In contrast, the flow of this invention achieves a forward flow that is 14.5 times faster than the reverse flow, resulting in higher mass transfer efficiency and a longer transport distance (with less loss).

[0072] Example 2: Preparation of Chromatographic Membrane with Fluid Diode Structure

[0073] S1. Place a flat PMMA sheet into a CO2 laser marking machine and adjust the laser focus on the PMMA sheet surface. Load the surface fluid diode structure drawing into the computer control system of the marking machine, and set suitable laser intensity parameters so that the marking thickness on the PMMA sheet surface is exactly the height H of the fluid diode groove. The program will control the laser marking machine to move continuously within the fluid diode groove area in the drawing until the fluid diode groove is constructed, thus obtaining the chromatographic film with the fluid diode structure.

[0074] S2. After thoroughly cleaning and drying the chromatographic membrane prepared in step S1, place it in a plasma cleaner for surface hydrophilic functionalization treatment to obtain a hydrophilic chromatographic membrane with a fluid diode structure.

[0075] Example 3: Replication of Chromatographic Membranes with Fluid Diode Structure

[0076] S1. First, place the flat aluminum sheet on the marking table of the UV laser marking machine, load the fluid diode structure drawing into the laser marking machine control software, and set parameters such as a marking speed of 400mm / s for continuous marking. The computer control system will process the fluid diode groove on the surface of the aluminum sheet according to the drawing;

[0077] S2. Take polydimethylsiloxane and curing agent, mix them thoroughly at a mass ratio of 10:1, pour the mixture into the surface of the aluminum plate obtained in step S1, place it in a vacuum pump to remove air bubbles, and then place it in an 80℃ oven for 30 minutes to fully cure. Gently peel off the cured PDMS layer to obtain a mold for processing fluid diode structures;

[0078] S3. With the side of the PDMS mold with the fluid diode structure facing up, take a flat HDPE film and stack it on the surface of the PDMS mold to fully cover the part with the fluid diode structure. Then send the mold and the film to be processed into a hot press and press them together, raising the temperature to 150°C.

[0079] S4. After maintaining the pressed film state for 5 minutes, adjust the parameters and allow the hot press to cool until room temperature.

[0080] S5. Remove the PDMS mold and HDPE film from the platform of the hot press and separate them to obtain the chromatographic film with the fluid diode structure on the surface.

[0081] S6. The chromatography membrane is subjected to hydrophilic functionalization treatment. First, the surface of the chromatography membrane is subjected to plasma treatment using oxygen as a gas source. Then, the chip is added to a cysteine ​​hydrochloric acid solution and kept heated for several hours. After removal, the surface is rinsed with pure water 3 to 5 times to obtain a hydrophilic chromatography membrane with a fluid diode structure.

[0082] Comparative Example 1

[0083] This comparative example uses a fiber-based chromatographic membrane from a commercial test strip, and compares it with the chromatographic membrane of Example 3, which has a fluid diode structure after hydrophilization treatment, to assess its liquid directional flow performance. 20 μL of pure water was added dropwise between the two membranes, and the flow was timed and observed.

[0084] Comparison results of directional liquid flow are as follows: Figure 3As shown in the figure, in the chromatographic membrane with a fluid diode structure of the present invention, the liquid rapidly spreads and flows along the surface fluid diode structure towards the fishtail structure, while the liquid surface is pinned in the direction of the arrow, indicating that the chromatographic membrane of the present invention has a directional flow effect on the liquid. In contrast, liquid added in the middle of a fiber-based chromatographic membrane spreads outwards on the membrane with the droplet position as the center. Clearly, the chromatographic membrane provided by the present invention possesses a directional liquid flow capability that fiber-based chromatographic membranes do not have.

[0085] Comparative Example 2

[0086] This comparative example uses a fiber-based chromatographic membrane from a commercial test strip, and compares it with the chromatographic membrane of Example 3, which has a fluid diode structure after hydrophilization treatment, in terms of rapid liquid spreading and flow performance.

[0087] Cut the chromatographic membrane and the fiber filament chromatographic membrane to a length of 25 mm. Add 10 μL of red ink to the starting point of both chromatographic membranes and observe the liquid flow and spreading performance of the liquid before the liquid flow to compare the liquid flow and spreading performance of the membrane.

[0088] The comparison results are as follows Figure 4 As shown in the figure, red ink droplets can spread rapidly on the chromatographic membrane of this invention, flowing quickly to the endpoint after 2 seconds, and the length of liquid spreading on the membrane is the same for adjacent 1-second intervals. In contrast, the filament-based chromatographic membrane requires a total of 40 seconds to flow to the endpoint, and the length of liquid spreading on the membrane differs between adjacent 10-second intervals, showing a gradually decreasing trend, that is, the flow speed slows down as the flow distance increases. It can be seen that the liquid flow on the filament-based chromatographic membrane becomes slower and slower, while the chromatographic membrane of this invention can maintain a consistent and stable flow rate, allowing for faster liquid spreading.

[0089] Comparative Example 3

[0090] This comparative example uses a fiber-based chromatographic membrane from a commercial test strip, and compares it with the chromatographic membrane of Example 3, which has a fluid diode structure after hydrophilization treatment, in terms of non-specificity of material components on the membrane or retention performance of sample solution.

[0091] Chromatographic test strips with a chromatographic length of 1 cm were prepared using the aforementioned chromatographic membrane and the fiber optic chromatographic membrane, respectively. The results are as follows: Figure 5 As shown in the figure, the chromatography membrane of the present invention can quickly complete the chromatography process. Comparing the state of the chromatography membrane surface without liquid after chromatography with that before chromatography, it can be seen that the residual amount of components on the chromatography membrane is significantly less than that of the fiber-based chromatography membrane.

[0092] Comparative Example 4

[0093] This comparative example uses a fiber-based chromatographic membrane from a commercial test strip, and compares its performance in micro-liquid transport with that of the chromatographic membrane in Example 3, which has a fluid diode structure after hydrophilization treatment.

[0094] Cut a 25mm length of the chromatography membrane from Example 3 and a fiber chromatography membrane. The membrane surface was marked with the positions corresponding to the C and T lines during the detection process. Add 5μL and 10μL of red ink to the starting point of the two types of chromatography membranes, respectively. By observing how well the liquid spreads on the membrane to the C and T lines, the differences in the micro-sample transfer performance between the membranes were compared.

[0095] The comparison results are as follows Figure 6 As shown, when 5 μL of liquid is added, the liquid droplet on the surface of the fiber chromatography membrane can only be transported to line C, and cannot complete the full liquid mass transfer; while the chromatography membrane of the present invention can transport the surface liquid to the endpoint. When the liquid volume is increased to 10 μL, the fiber chromatography membrane can transport the liquid to the endpoint.

[0096] Example 4: Multi-index immunochromatographic test strip with a fluid diode structure

[0097] The multi-index immunochromatographic test strip with a fluid diode structure provided in this embodiment includes a chromatographic membrane 100 with a fluid diode structure, an adhesive support base plate 240, a sample pad 210, a conjugation pad 220, and an absorbent pad 230. The chromatographic membrane with the fluid diode structure can be obtained by the methods in embodiments 2 and 3 of this invention, and the other immunochromatographic components and membrane materials can be purchased commercially.

[0098] The chromatography membrane with a fluid diode structure contains bands / regions for various immunoassay indicators, and the bands / regions contain protein molecules that specifically capture antigens / antibodies.

[0099] The width of each membrane component in the multi-index immunochromatographic test strip is approximately 2.0–10.0 mm, the length of the sample pad 210 is approximately 1.5–2.0 cm, the length of the conjugation pad 220 is approximately 1.0–1.5 cm, the length of the chromatography membrane 100 is approximately 2.0–5.0 cm, and the length of the absorbent pad 230 is approximately 1.5–2.5 cm. The assembly method of the above immunochromatographic test strip is as follows:

[0100] S1. A chromatography membrane 100 with a variety of specific antigen / antibody protein molecules on its surface is bonded and fixed to the surface of an adhesive support base plate 240.

[0101] S2. At the same horizontal position at both ends of the chromatography membrane 100, attach the binding pad 220 and the absorbent pad 230 to the adhesive support base plate 240. The binding pad 220 and the absorbent pad 230 need to cover the length of the chromatography membrane by 2 to 4 mm.

[0102] S3. Place the sample pad 210 at the other end of the conjugate pad 220. The overlap length between the sample pad 210 and the conjugate pad 220 is 2-4 mm. The remaining part is bonded and fixed to the surface of the adhesive support base plate 240 to complete the assembly of the multi-index immunochromatographic test strip.

[0103] When using this multi-index immunochromatographic test strip, the sample solution needs to be added to the sample pad. After capillary flow, the sample solution fully penetrates into the conjugation pad 220, where labeled antibodies are released and bind to the test sample in the sample solution. Simultaneously, the sample solution is immersed in the overlap between the conjugation pad 220 and the chromatography membrane 100. At this point, the fluid diode structure on the surface of the chromatography membrane 100 drags the sample solution, causing it to be released from the conjugation pad 220 and continuously move forward along the chromatography membrane 100. During the liquid flow, the sample solution makes full contact with each immunobinding region between the chromatography membrane 100, completing the immunoreaction. Finally, the sample solution reaches the flow endpoint, i.e., the absorbent pad 230. The absorbent pad 230 absorbs water, allowing (or most) of the sample solution carrying the analyte to pass through the immunobinding region. This completes the immunochromatographic reaction process. By interpreting the detection signal on the surface of the chromatography membrane using various methods, real-time detection results can be obtained.

[0104] Example 5: Multi-index immunochromatographic test strip with a fluid diode structure

[0105] The chromatographic membrane prepared in Example 3 was used to prepare multi-index immunochromatographic test strips for multi-index immunoassay. The specific method is as follows:

[0106] 1. Immobilization of multi-index immunoassay antigens / antibodies on chromatography membrane 100:

[0107] To meet the requirements of antigen-antibody binding, a hydrophilic three-dimensional substrate strip was added to the surface of the chromatographic membrane with a fluid diode structure prepared in Example 3 to form an immune binding zone, thereby increasing the antibody capture capacity. First, cellulose acetate powder was dissolved in dimethyl sulfoxide-acetone solution, and Tween 20 was added as a hydrophilic additive to prepare a membrane-forming solution. This membrane-forming solution was then layered from top to bottom onto the hydrophilized surface of the chromatographic membrane with the fluid diode structure, and immediately immersed in water. At this time, the membrane-forming solution containing cellulose acetate rapidly formed on the hydrophilized fluid diode structure surface and embedded itself into the fluid diode structure. Thus, a hydrophilic chromatographic membrane with a hydrophilic fiber layer fixed on the surface fluid diode structure was obtained.

[0108] Take 1-2 μL of crosslinking agent and wet the hydrophilic fiber layer of the above hydrophilic chromatography membrane. Immediately add 1-2 μL of various immunoassay antigen / antibody reagents. The band near the tail of the arrow with a length the same as the width of the multi-index immunochromatographic test strip is designated as the binding C line 320, and the band therewith is designated as the T line 310. React overnight at 37°C to complete the crosslinking of antigen / antibody in the hydrophilic fiber layer.

[0109] 2. Assembly of multi-index immunochromatographic test strips:

[0110] The assembly structure of the multi-index immunochromatographic test strip is as follows: Figure 7 As shown, the cured chromatography membrane 100 from step 2 is bonded and fixed to the surface of the adhesive support plate 240. On the support plate 240 surface where the chromatography membrane 100 is fixed, absorbent pads 230 and binding pads 220 immobilized with labeled antibodies are placed at both ends of the chromatography membrane 100, respectively. The overlap length between the absorbent pads 230 and 220 and the chromatography membrane 100 is 2-3 mm, and the remaining portions are bonded and fixed to the surface of the adhesive support plate 240. A sample pad 210 is placed at the other end of the binding pad 220, with an overlap length of 2-3 mm, and the remaining portions are bonded and fixed to the surface of the adhesive support plate 240.

[0111] 3. Detection of multiple proteins using multi-index immunochromatographic test strips:

[0112] Take the antibody / antigen of the protein to be tested at the working concentration, add the sample solution to the sample pad of the chromatography test strip, wait for the solution to complete the chromatographic flow on the chromatography test strip, and read the test result after 5 minutes.

[0113] Example 6: Multi-index immunochromatographic test strip with a fluid diode structure

[0114] Multi-index immunochromatographic test strips were prepared using the chromatographic membrane with a fluid diode structure obtained in Example 2 for multi-index immunoassay. The specific method is as follows:

[0115] 1. Immobilization of multi-index immunoassay antigens / antibodies on chromatographic membranes:

[0116] First, a PDMS film with micropores is fabricated as a mold for coating antigen / antibody regions on the surface of a chromatography membrane with a fluid diode structure. The membrane has multiple square pores, each approximately 1-2 mm wide, penetrating the PMMS membrane and arranged neatly in a straight line from front to back. The length of these square pores is shorter than the length of the surface structure membrane. Each square pore corresponds to the position of the multi-index T-line 310 and C-line 320 on the surface structure membrane, with the square pore closest to the arrow tail of the fluid diode chromatography membrane corresponding to the detected C-line 320; the other square pores are used for the multi-index detection T-line 310. The PDMS film is placed on the surface of the chromatography membrane with the fluid diode structure and pressed firmly onto the PMMA chromatography membrane using external clamps or other methods. The chromatography membrane is then placed in a plasma treatment device and treated with oxygen or air for 300 seconds. An activating crosslinking agent is added to the square groove regions formed by the PDMS membrane and the PMMA chromatography membrane, respectively. The membrane is then incubated at 37°C to hydrophilize it and provide sites for covalent binding of proteins.

[0117] After removing the residual activating crosslinking agent solution from the square wells, the antispecific antigen / antibody reagents used to capture the analyte were added to the square wells, and the mixtures were incubated at 37°C. After incubation, the liquid in each well was removed, and the wells were washed with PBS solution containing 0.5% Tween-20, repeated three times. After drying at 37°C, the PDMS membrane was peeled off to obtain a surface structure membrane with specific antibodies covalently immobilized in multiple localized areas.

[0118] 2. Assembly of multi-index immunochromatographic test strips:

[0119] The assembly structure of the multi-index immunochromatographic test strip is as follows: Figure 8 As shown, the cured chromatography membrane 100 from step 2 is bonded and fixed to the surface of the adhesive support plate 240. On the support plate 240 surface where the chromatography membrane 100 is fixed, absorbent pads 230 and binding pads 220 immobilized with labeled antibodies are placed at both ends of the chromatography membrane 100, respectively. The overlap length between the absorbent pads 230 and 220 and the chromatography membrane 100 is 2-3 mm, and the remaining portions are bonded and fixed to the surface of the adhesive support plate 240. At the other end of the binding pad 220, a sample pad 210 is placed, with an overlap length of 2-3 mm between the sample pad 210 and the binding pad 220, and the remaining portions are bonded and fixed to the surface of the adhesive support plate 240.

[0120] 3. Detection of multiple proteins using multi-index immunochromatographic test strips:

[0121] Take the antibody / antigen of the protein to be tested at the working concentration, add the sample solution to the sample pad of the chromatography test strip, wait for the solution to complete the chromatographic flow on the chromatography test strip, and read the test result after 5 minutes.

[0122] In summary, the chromatographic membrane with a fluid diode structure and the multi-index immunochromatographic test strip provided by this invention not only solve the problems of existing fiber filament chromatographic membranes and multi-index detection, but also offer advantages such as: faster flow rate, enabling faster and better / uniform (liquid flow rate decreases with increasing flow distance) water transport; directional flow and reverse blocking, resulting in a higher rectification coefficient, fast and efficient mass transfer, longer flow distance, and reduced reagent consumption and residue (enabling the transfer of trace samples). Using the chromatographic membrane for multi-index immunochromatographic detection also offers advantages such as: accelerated multi-index immunochromatographic detection speed, reduced differences in contact / reaction time with different detection bands, and reduced sample volume during detection. It can be well applied in immunoassay, nucleic acid detection, and biological particle detection.

[0123] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A chromatographic membrane having a fluid diode structure, characterized in that, The surface of the chromatography membrane is composed of a series of parallel, continuously barbed arrow-shaped grooves (110); The continuous barbed arrow-shaped groove (110) is formed by connecting the ends of continuous barbed arrow-shaped groove units (120); The barbed arrow-shaped groove unit (120) is composed of an axisymmetric arc-shaped curved wall surface, which includes a first side wall surface (121) and a second side wall surface (122). The width L6 of the first side wall surface (121) and the second side wall surface (122) from the axis is the same. The tangents of the outer arc of the tail end of the first side wall surface (121) and the second side wall surface (122) intersect to form a barbed angle α (123). The head end of the second side wall surface (122) is connected to the adjacent... The outer arc tangents of the first side wall (121) of the barbed arrow-shaped groove unit (120) intersect to form a sharp angle φ (124); the outer arc tangent of the first side wall (121) and the line parallel to the axis form a wall axis angle β (125); the barbed angle α (123) is 5° to 40°, the sharp angle φ (124) is 10° to 20°, and the wall axis angle β (125) is 5° to 40°; The axial length L1 from the first end of the first sidewall (121) to the first end of the second sidewall (122) of the barbed arrow-shaped groove unit (120) is 100 to 2000 μm; the axial length L2 of the second sidewall (122) is 1 to 2 times L1; the distance L3 between the upper and lower ends of the first sidewall (121) which is symmetrical is 10 to 200 μm; the distance L4 between the upper and lower ends of the second sidewall (122) which is symmetrical is 1 to 10 times L3; and the height H of the barbed arrow-shaped groove unit (120) is 5 to 500 μm.

2. The chromatography membrane according to claim 1, characterized in that, The minimum distance L5 between the parallel distributed adjacent continuous barbed arrow-shaped grooves (110) is 5 to 500 μm.

3. The application of the chromatography membrane according to claim 1 or 2 in immunoassay, nucleic acid assay, and biological particle assay.

4. The application of the chromatography membrane according to claim 1 or 2 in the preparation of immunochromatographic detection products.

5. The application according to claim 4, characterized in that, The product is a multi-index immunochromatographic test strip.

6. A multi-index immunochromatographic test strip with a fluid diode structure, characterized in that, The chromatographic membrane according to claim 1 or 2 has a variety of specifically captured antigens / antibodies on its surface, forming a specific immune binding region.

7. The multi-index immunochromatographic test strip according to claim 6, characterized in that, It also includes other immunochromatographic components: a support base plate, a conjugation pad, an absorbent pad, and a sample pad.

8. The application of the multi-index immunochromatographic test strip according to claim 6 or 7 in liquid flow, chromatography, immunoassay, nucleic acid detection, and biological particle detection.

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