A method for preparing a porous fiber membrane strip and its application in rapid immunoassay

Abstract

The application discloses a preparation method of a porous fiber membrane strip and application of the porous fiber membrane strip in rapid immune detection. The membrane preparation slurry in the preparation method has high viscosity, can fill the grooves on a rough base to increase the adhesion area, and forms a stable pattern strip; the forming agent used is green and environment-friendly, and can quickly replace the organic solvent in the membrane preparation slurry. Therefore, the preparation method can prepare a sheet porous fiber membrane strip in several minutes, or realize the patterning of the porous fiber membrane strip on demand, and significantly improves the production efficiency and expands the application. The application also provides the application of the porous fiber membrane strip in rapid immune detection. The porous fiber membrane strip has a micropore diameter, and can significantly shorten the diffusion distance of the molecules to be detected in a sample solution to the fiber surface.

Description

Technical Field

[0001] This invention belongs to the field of medical diagnostic technology, and more specifically, relates to a method for preparing porous fiber membrane strips and their application in rapid immunoassay. Background Technology

[0002] Porous fiber membranes and strips play a crucial role in biomedical detection, particularly in protein immunoassay and nucleic acid molecular detection. Within porous fiber membrane strips, micron-sized fibers intersect to form a 3D network structure and create pores, providing capillary channels for the flow of sample solutions. In immunoassay applications, the 3D network structure of porous fiber membranes also provides numerous binding sites for antibodies or antigens used to capture target molecules. More importantly, the pore size within porous fiber membranes is only a few micrometers; compared to the millimeter-sized pores in common microplates, the micron-sized fiber pores significantly shorten the distance required for analyte molecules in the sample solution to diffuse to the fiber surface and bind with antibodies or antigens on the fiber surface. Currently, porous fiber membranes and strips, as membrane materials capable of driving sample solution flow and performing immunocapture, are widely used in lateral flow immunoassay techniques.

[0003] Currently, in industrial applications, porous fiber membranes and strips used in lateral flow immunoassay are typically produced using a dry phase-inversion method (also known as a phase separation method), as illustrated in patents CN114130373B and CN116731399B. Specifically, this process involves first dissolving the fiber material in an organic solvent to prepare a membrane slurry; then, this slurry is spread evenly on a roller surface and dried to allow the organic solvent to evaporate. During drying, the porous fiber material precipitates at the edges of the organic solvent droplets, ultimately forming a fiber structure. This method requires strict control of the drying rate of the membrane slurry at various points on the roller surface, including drying temperature, airflow intensity, and vacuum level. During membrane formation, to reduce the drying rate at the surface and interior of the slurry, and to minimize the loss of effective components, the membrane-forming temperature is typically limited to 10–35°C, and the formation time usually requires 1–4 hours or more. This method limits the production efficiency of porous fiber membranes and strips.

[0004] Furthermore, to achieve uniform spreading on a large-area roller surface, the membrane-forming slurry must possess good fluidity to facilitate its spread over a large area, forming a large-area thin layer, thereby producing sheet-like porous fiber membranes. However, while good fluidity improves the spreading ability of the membrane-forming slurry on the substrate surface, it also significantly reduces the adhesion of the slurry to the substrate surface, making it difficult to maintain a specific shape. This production process and its characteristics are unsuitable for the fabrication of patterned porous fiber membrane strips, limiting their practical application. Therefore, there is an urgent need for a method to rapidly fabricate porous fiber membrane strips that can simultaneously produce sheet-like and patterned porous fiber membrane strips, and to solve the problem of low efficiency in the existing dry phase inversion process for porous fiber membrane strip fabrication. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a method for preparing porous fiber membrane strips.

[0006] A second objective of this invention is to provide the application of the method for preparing the porous fiber membrane strip in rapid immunoassay.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] This invention first provides a method for preparing porous fiber membrane strips, comprising the following steps:

[0009] S1. Dissolve cellulose derivative powder in an organic solvent to prepare a film-forming slurry;

[0010] S2. Scrape the film-forming slurry onto a smooth plate or extrude it into a pattern onto a rough substrate;

[0011] S3. Impregnate the film-forming slurry with a molding agent; the molding agent is a solution that is miscible with the organic solvent in step S1, but cannot dissolve the cellulose derivative powder in step S1;

[0012] S4. Remove the molding agent to obtain a formed sheet of porous fiber membrane strips or a patterned porous fiber membrane strip.

[0013] This invention utilizes the difference in solubility between cellulose derivatives and organic solvents in a forming agent to rapidly separate the organic solvent from the slurry. During this process, due to its poor solubility in the forming agent, the cellulose derivatives quickly precipitate from the slurry and solidify, forming porous fiber membrane strips. Furthermore, a rough substrate can be selected; the grooves on the rough substrate increase the surface area of ​​contact between the slurry and the substrate, thereby increasing the adhesion of the slurry to the substrate surface and making it adhere more stably to the substrate surface, forming stable patterned strips. This manufacturing method can be used to produce large-area, sheet-like porous fiber membrane strips, or to produce patterned porous fiber membrane strips according to actual application requirements.

[0014] Furthermore, the film-forming slurry in this invention has a high viscosity, preferably with a contact angle of 80° to 90° on the rough substrate surface. This results in strong adhesion to the substrate surface, further enabling the film-forming slurry to adhere more stably to the substrate surface, thereby forming more stable patterned strips. This better meets the requirements for manufacturing patterned porous fiber membrane strips.

[0015] Further, the cellulose derivative powder in step S1 is selected from one or more of cellulose acetate (CA), cellulose nitrate (NC), and carboxymethyl cellulose nitrate (CMCN).

[0016] Furthermore, the content of the cellulose derivative powder in the film-forming slurry in step S1 can be flexibly varied according to actual production needs.

[0017] Preferably, the solid content of the cellulose derivative powder in the film-forming slurry is 5.5% to 7%.

[0018] Furthermore, the organic solvent in step S1 contains one or more of N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAC), dichloromethane (DCM), and ethyl acetate in any weight ratio as the main solvent.

[0019] Furthermore, the organic solvent in step S1 also contains one or two of a co-solvent and a modifier; the co-solvent is selected from one or more of acetone, butanone, and tetrabutylammonium fluoride (TBAF) in any weight ratio as a co-solvent; the modifier is selected from one of Tween-20, Tween-80, Span-20, perfluorodecyltrichlorosilane (FDTS), dodecyltrimethylammonium chloride, and hexadecyltrimethylammonium chloride.

[0020] Preferably, a certain amount of cellulose derivative powder is dissolved in the organic solvent described above to form a film-forming slurry with high viscosity.

[0021] Furthermore, the rough substrate mentioned in step S2 is a substrate with a rough depression depth of 30 to 300 μm.

[0022] Furthermore, the pattern described in step S2 is any pattern that can be constructed on a plane, such as a bar, a circle, or a wave, and its length, width, height, and other dimensions can be flexibly varied according to actual production needs.

[0023] Furthermore, the molding agent mentioned in step S3 is a green and environmentally friendly molding agent.

[0024] Preferably, the molding agent in step S3 is selected from water, ethanol, or a mixture thereof with a non-toxic plasticizer.

[0025] Preferably, the non-toxic plasticizer can be one of glycerol or citrate.

[0026] Preferably, the amount of the molding agent used can be flexibly adjusted according to the pore size requirements of the porous fiber membrane strip.

[0027] Furthermore, the method for removing the molding agent in step S4 can be self-drying, drying, or freeze-drying.

[0028] The present invention also provides porous fiber membrane strips prepared by any of the above-described preparation methods.

[0029] The present invention also provides the application of any of the above-described preparation methods or porous fiber membrane strips in immunoassay, nucleic acid molecular detection, and large particle biomolecule detection.

[0030] This invention also provides a rapid immunoassay chip based on the aforementioned porous fiber membrane strips and its applications. In immunoassays, antibodies or antigens used to capture analyte molecules are typically coated onto the surface of a solid substrate. The faster the analyte molecules in the sample solution are transported to the surface of these solid substrates, the faster they are captured by the antibodies or antigens on the solid substrate surface, thus shortening the time required for immunoassay. The porous fiber membrane strips provided by this invention have micron-sized pores. Compared to millimeter-sized micropores, the porous fiber membrane strips significantly shorten the diffusion distance of analyte molecules in the sample solution to the fiber surface, enabling analyte molecules to contact and be captured by the antibodies or antigens coated on the fiber surface more quickly, thereby achieving rapid immunoassay.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The method of this invention utilizes the difference in solubility between cellulose derivative materials and organic solvents in the forming agent to rapidly separate the organic solvents in the film-forming slurry. During this process, due to its poor solubility in the forming agent, the cellulose derivative materials will quickly precipitate from the film-forming slurry and form porous fiber membrane strips. The method for preparing porous fiber membrane strips provided by this invention not only simplifies the process of porous fiber membrane strip production, but more importantly, it accelerates the production efficiency of porous fiber membrane strips, enabling the production of porous fiber membrane strips within minutes; it also reduces the control equipment required in the film-forming process and lowers the film-forming cost.

[0033] (2) The method of the present invention can be used to select the extrusion of patterns on a rough substrate or to further combine the use of a film-forming slurry with higher viscosity, so that the film-forming slurry is more stably attached to the substrate surface, thereby forming a stable pattern strip to meet the production requirements of patterned porous fiber membrane strips.

[0034] (3) The method for preparing porous fiber membrane strips provided by the present invention expands the application of porous fiber membrane manufacturing methods. It can produce large-area sheet porous fiber membrane strips and patterned porous fiber membrane strips according to actual use requirements.

[0035] (4) The preparation method provided by the present invention uses green and environmentally friendly molding agents (such as water and ethanol) in the film-making process, which can quickly replace the organic solvents in the film-making slurry. At the same time, it replaces the toxic and harmful organic molding agents (such as polysulfone and polyethersulfone) commonly used in existing film-making methods, reducing the proportion of organic reagents required by the manufacturing method and reducing pollution.

[0036] (5) Based on the porous fiber membrane strip prepared by the above preparation method, the present invention also provides its application in rapid immunoassay. The porous fiber membrane strip has a micron-sized pore size, which can shorten the distance required for the analyte molecules in the solution to be captured by the surface antibody or antibody on the fiber. Attached Figure Description

[0037] Figure 1 The image shows a porous fiber membrane prepared in Example 1 of this invention; the left image shows a sheet of porous cellulose acetate (CA) membrane, and the right image shows the fiber structure of the sheet of porous cellulose acetate (CA) membrane.

[0038] Figure 2 The image shows a porous fiber membrane prepared in Example 2 of this invention; the left image shows a patterned porous cellulose acetate (CA) membrane, and the right image shows the fiber structure of the patterned porous cellulose acetate (CA) membrane.

[0039] Figure 3The image shows the porous fiber membrane prepared in Example 3 of this invention; the left image shows a patterned porous nitrocellulose (NC) membrane, and the right image shows the fiber structure of the patterned porous nitrocellulose (NC) membrane.

[0040] Figure 4 A schematic diagram (1) of a porous CA membrane used for immunoassay of a lateral flow test strip and the results (2) of its qualitative detection of nucleocapsid protein (N protein) of the novel coronavirus; wherein, 1-sample pad, 2-labeled antibody binding pad, 3-lateral flow membrane, 31-porous cellulose acetate (CA) membrane embedded in the flow membrane (as the immunobinding area), 4-absorbent pad, 5-adhesive support base plate.

[0041] Figure 5 A schematic diagram (1) of porous nitrocellulose (NC) membrane used for microfluidic chip immunoassay and the results (2) of its use for qualitative detection of human immunoglobulin G (IgG). Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments 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 this technical field.

[0043] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0044] Example 1: Preparation of sheet-like porous cellulose acetate (CA) membranes

[0045] S1. Dissolve CA powder in a mixed solution of acetone and DMF to prepare CA film-forming slurry. The solid content of the CA powder is 5.5%; the mass ratio of acetone to DMF is 3:4.

[0046] S2. Spread 10 mL of CA film-forming slurry onto a smooth, flat glass surface to maintain a uniform thickness of 0.5 mm.

[0047] S3. Use 50 mL of water containing 20% ​​glycerol as a forming agent to fully impregnate the CA membrane-forming slurry. At this point, the CA membrane-forming slurry rapidly completes the wet phase inversion process, forming a porous CA membrane.

[0048] S4. Use a paper towel to absorb the residual forming agent on the porous CA membrane to obtain a sheet of porous CA membrane.

[0049] The sheet-like porous CA membrane, such as Figure 1 As shown in the left figure. Subsequently, the fibrous structure of the sheet-like porous CA membrane was observed using an inverted fluorescence microscope at a magnification of 40x, as shown... Figure 1As shown in the right figure, the results indicate that the sheet-like porous CA membrane has clearly visible fibers and pores, with the diameter of the pores being approximately 5–15 μm.

[0050] Example 2: Preparation of Patterned Porous CA Membranes

[0051] S1. Dissolve CA powder in a mixed solution of acetone, DMF, and Tween-20 to prepare CA film-forming slurry. The solid content of the CA powder is 6.5%; the mass ratio of acetone, DMF, and Tween-20 is 3:4:1.

[0052] S2. Use a pipette to take 50 μL of CA film-forming slurry and extrude it onto a rough polymethyl methacrylate (PMMA) sheet to form two parallel strip patterns.

[0053] S3. Use 100μL of water as a forming agent to fully impregnate the CA membrane-forming slurry. At this point, the CA membrane-forming slurry quickly completes the wet phase inversion process, forming a porous CA membrane.

[0054] S4. Treat the porous CA membrane on a heating plate at 37°C for 10 minutes to evaporate any residual moisture inside, thus obtaining a patterned porous CA membrane.

[0055] The patterned porous CA membrane, such as Figure 2 As shown in the left figure. Subsequently, the fibrous structure of the patterned porous CA membrane was observed using an inverted fluorescence microscope at 40x magnification, as shown... Figure 2 As shown in the right figure, the results indicate that the patterned porous CA membrane has clearly visible fibers and pores, with the diameter of the pores being approximately 4–7 μm.

[0056] Example 3: Preparation of Patterned Porous Nitrocellulose (NC) Membranes

[0057] S1. Dissolve NC powder in a mixed solution of butanone, DMAC, and Tween-20 to prepare NC film-forming slurry. The solid content of the NC powder is 7%; the mass ratio of butanone, DMAC, and Tween-20 is 1:1:0.25.

[0058] S2. Use a pipette to take 30 μL of NC film-forming slurry and extrude it onto a rough PMMA sheet to form two parallel strip patterns.

[0059] S3. Use 60μL of water as a forming agent to fully impregnate the NC membrane slurry. At this point, the NC membrane slurry quickly completes the wet phase inversion process to form a porous NC membrane.

[0060] S4. The patterned porous NC membrane can be obtained by vacuum drying the water in the porous NC membrane at room temperature.

[0061] The patterned porous NC membrane, such as Figure 3 As shown in the left figure. Subsequently, the fiber structure of the patterned porous NC membrane was observed using an inverted fluorescence microscope at 40x magnification, as shown... Figure 3 As shown in the right figure, the results indicate that the patterned porous NC membrane has clearly visible fibers and pores, with the diameter of the pores being approximately 3–5 μm.

[0062] Example 4: Qualitative detection of the N protein of the novel coronavirus using a porous CA membrane.

[0063] 1) Fabrication of the porous CA membrane

[0064] The porous CA membrane preparation method in this embodiment is the same as that in Example 2. When the porous CA membrane is formed on the rough surface of the PMMA sheet, it is embedded in the groove of the rough surface of the PMMA sheet, thereby fixing a patterned porous CA membrane 31 on the surface of the PMMA sheet. The PMMA sheet with the patterned porous CA membrane fixed on its surface is cut into 2.5cm × 0.4cm membranes as lateral flow membranes 3. Each lateral flow membrane 3 has two patterned porous CA membranes 31 on its surface for later use.

[0065] 2) Preparation of lateral flow immunoassay strip with the porous CA membrane

[0066] S1. Immobilization of novel coronavirus N protein antibody on porous CA membrane. 1–2 μL of crosslinking agent was used to wet the porous CA membrane 31 on the lateral flow membrane 3. Immediately, 1–2 μL of novel coronavirus N protein antibody solution (1 mg / mL) was added to the left porous CA membrane as the detection line antibody, and goat anti-mouse IgG solution (1 mg / mL) was added to the right porous CA membrane as the quality control line antibody. The reaction was carried out overnight at 4°C to complete the crosslinking of novel coronavirus N protein antibody onto the porous CA membrane 31.

[0067] S2. Place absorbent pads 4 and conjugate pads 2 loaded with colloidal gold-labeled antibodies (40 μg / mL) at both ends of the lateral flow membrane 3, respectively; wherein the length of overlap between the absorbent pads 4 and conjugate pads 2 and the lateral flow membrane 3 is 2-3 mm, and the remaining parts are bonded and fixed to the adhesive support base plate 5.

[0068] S3. Place sample pad 1 at the other end of the conjugate pad 2. The overlap between sample pad 1 and conjugate pad 2 should be 2-3 mm. The remaining portion should be adhered and fixed to the surface of the adhesive support base plate. This yields a lateral flow immunoassay test strip for detecting the N protein of the novel coronavirus, with the structure shown below. Figure 4 As shown in (1).

[0069] 3) Detection of the N protein of the novel coronavirus

[0070] An immunoassay was performed using a 50 μL standard solution of the novel coronavirus N protein as a simulated sample. The lateral flow immunoassay strip can complete the test within 5 minutes and provides accurate negative and positive results. The test results are as follows: Figure 4 As shown in (2).

[0071] Example 5: Qualitative detection of human IgG using porous NC membrane.

[0072] 1) Fabrication of the porous NC membrane

[0073] The porous NC membrane preparation method in this embodiment is the same as that in Example 3. When the porous NC membrane is formed on the rough surface of the PMMA sheet, it is embedded in the grooves of the rough surface of the PMMA sheet, thereby fixing a patterned porous NC membrane on the surface of the PMMA sheet.

[0074] 2) Fabrication of the microfluidic chip with the porous NC membrane

[0075] S1. Immobilization of human IgG antibody on porous NC membrane. Soak the porous NC membrane with 1–2 μL of goat anti-human IgG solution (1 mg / mL) and incubate overnight at 4°C.

[0076] S2. Remove the porous NC membrane from the PMMA sheet and fill it into the channel of the microfluidic chip (for example, fill it into the middle of the microchannel or into the corners at both ends of the microchannel) to seal the flow cross section of the microfluidic chip channel. Figure 5 (1) A schematic diagram of a microfluidic chip in which the porous NC membrane is filled in the middle of a microchannel is shown.

[0077] 3) Detection of human IgG

[0078] S1. Using the standard solution of human IgG as a simulated sample, mix it in advance with a 50 μg / mL colloidal gold-labeled mouse anti-human IgG antibody solution at an equal volume ratio to prepare a sample solution for immunoassay.

[0079] S2. Add 50 μL of sample solution into the channel of the microfluidic chip, and push it through the porous NC membrane in the channel of the microfluidic chip by air pressure.

[0080] S3. Add 50 μL of phosphate buffer solution as a cleaning solution to the channel of the microfluidic chip, and use air pressure to push it through the porous NC membrane in the microfluidic chip channel. Repeat three times to complete the cleaning step.

[0081] The microfluidic chip can complete the detection within 5 minutes and provide accurate negative and positive results. Detection results are as follows: Figure 5 As shown in (2).

[0082] The above comparative examples and embodiments are preferred embodiments of the present invention, but the embodiments and effects of the present invention are not limited to the above comparative examples and embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a porous fiber membrane strip, characterized in that, Includes the following steps: S1. Dissolve cellulose derivative powder in an organic solvent to prepare a film-forming slurry; S2. Extruding the film-forming slurry onto a rough substrate to form a pattern; S3. Impregnate the film-forming slurry with a molding agent; the molding agent is a solution that is miscible with the organic solvent in step S1, but cannot dissolve the cellulose derivative powder in step S1; S4. Remove the molding agent to obtain a formed sheet of porous fiber membrane strips or a patterned porous fiber membrane strip; The organic solvent in step S1 contains one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dichloromethane, and ethyl acetate in any weight ratio as the main solvent; the organic solvent also contains one or two of a co-solvent and a modifier; the co-solvent is selected from acetone, butanone, and tetrabutylammonium fluoride in any weight ratio as the co-solvent; the modifier is selected from Tween-20, Tween-80, Span-20, perfluorodecyltrichlorosilane, dodecyltrimethylammonium chloride, and hexadecyltrimethylammonium chloride; The contact angle of the film-forming slurry on the rough substrate surface is 80° to 90°; The molding agent in step S3 is selected from water, ethanol, or a mixture thereof with a non-toxic plasticizer; the non-toxic plasticizer is glycerol or citrate.

2. The preparation method according to claim 1, characterized in that, The rough substrate mentioned in step S2 is a substrate with a rough depression depth of 30 to 300 μm.

3. The preparation method according to claim 1, characterized in that, The pattern described in step S2 is a bar, a circle, or a wave.

4. The preparation method according to claim 1, characterized in that, The method for removing the molding agent in step S4 is selected from suction drying, drying or freeze drying.

5. The porous fiber membrane strip prepared by any one of the preparation methods described in claims 1 to 4.

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