Nitrocellulose membrane as well as preparation method and application thereof

Abstract

The invention relates to the technical field of immunodetection, and discloses a nitrocellulose membrane treatment method, which comprises the following steps: carrying out plasma treatment on a nitrocellulose membrane; and coating the nitrocellulose membrane with polyvinylpyrrolidone, wherein the nitrocellulose membrane subjected to plasma treatment is coated with polyvinylpyrrolidone. The present invention achieves improvements in hydrophilicity, antibody binding rate, rate of change in chromatography rate, polyvinylpyrrolidone layer firmness, and / or lineation diffusivity in a low humidity environment.

Description

Technical Field

[0001] This invention relates to the field of immunoassay, specifically to a nitrocellulose membrane, its preparation method, and its uses. Background Technology

[0002] The natural hydrophobicity of nitrocellulose membranes results in an initial contact angle as high as 85°, which directly causes the chromatography speed to fluctuate by 25%-30%. Furthermore, in low humidity (≤30%RH) environments, the streaking is prone to a diffusion rate and breakage rate of 35%-40%, which cannot meet the basic requirements of immunochromatographic test strips for "chromatographic uniformity" and "streaking stability".

[0003] Limitations of traditional surfactant treatments (using Tween-20 as an example) Poor timeliness of effect: The nitrocellulose membrane coated with 0.5% Tween-20 initially had a contact angle of about 50°, but after being sealed and stored at 4°C for 1 month, the contact angle rose back to more than 75° due to the migration and loss of surfactant, and the hydrophilicity was basically lost. Insufficient biocompatibility: Tween-20 can non-specifically adsorb with gold-labeled antibodies, leading to a 15%-20% increase in false positive signals during the detection process. At the same time, the antibody binding rate drops from the initial 80% to below 65%, affecting the accuracy of the detection. Poor environmental adaptability: In scenarios with fluctuating humidity (40-80%RH), the chromatography rate still changes by 20%-25%, making it unsuitable for use in different regions and seasons.

[0004] The core bottleneck of individual plasma processing Rapid decline in hydrophilicity: The nitrocellulose membrane treated with a single argon gas (80W power, 60 seconds processing time) can initially reduce the contact angle to 35°, but due to the surface energy relaxation effect, the contact angle will rise back to more than 60° after 7 days of storage, which cannot meet the shelf life requirement of ≥6 months for the test strip. No long-term protection mechanism: It can only temporarily activate the surface and does not form a stable protective layer. The scratch diffusion rate is still 20%-25% in low humidity (25%RH) environment.

[0005] Inherent defects of polyvinylpyrrolidone (PVP) coating alone Poor adhesion: Nitrocellulose membranes that have not undergone plasma pretreatment lack active binding sites on their surface, and PVP can only adhere physically. After washing or storage for one month, the PVP layer peeling rate reaches 30%. Insufficient performance stability: After the PVP layer peels off, the contact angle rises back to over 70°, and the moisture resistance and hydrophilicity are completely lost (the tape peeling test shows that the PVP residue rate is only 50%). Summary of the Invention This invention provides a nitrocellulose (NC) membrane, its preparation method, and its uses to solve the problems of hydrophilic instability, low antibody binding rate, excessive rate of change in chromatography speed, easy shedding of the polyvinylpyrrolidone layer, and / or high streak diffusion rate under low humidity (≤30%RH) conditions.

[0006] In a first aspect, the present invention provides a method for treating nitrocellulose membranes, comprising: Plasma treatment, wherein a nitrocellulose membrane is subjected to plasma treatment; and, Polyvinylpyrrolidone coating, wherein a plasma-treated nitrocellulose membrane is coated with polyvinylpyrrolidone.

[0007] In one alternative embodiment, the plasma treatment employs argon and oxygen in a volume ratio of 3:1 to 5:1, optionally 4:1, and / or a power of 50-100W, and / or a treatment time of 30-150 seconds, and / or a vacuum degree of 10-50Pa.

[0008] In one optional embodiment, in the polyvinylpyrrolidone coating, a polyvinylpyrrolidone solution with a concentration of 0.3 g / 100 ml to 2.5 g / 100 ml is used. Optionally, the polyvinylpyrrolidone solution with a concentration of 0.3 g / 100 ml to 2.5 g / 100 ml contains 0.025 g / 100 ml to 0.15 g / 100 ml of crosslinking agent. Optionally, the solvent in the polyvinylpyrrolidone solution with a concentration of 0.3 g / 100 ml to 2.5 g / 100 ml is deionized water and / or ethanol. Further, optionally, the solvent in the polyvinylpyrrolidone solution with a concentration of 0.3 g / 100 ml to 2.5 g / 100 ml is deionized water and ethanol in a volume ratio of 9:1.

[0009] In one optional embodiment, the molecular weight of polyvinylpyrrolidone is greater than 15,000 and less than 130,000; optionally, the molecular weight of polyvinylpyrrolidone is greater than 30,000 and less than 130,000; and more preferably, the molecular weight of polyvinylpyrrolidone is 38,000. Alternatively, the crosslinking agent may be glutaraldehyde or genipin.

[0010] In an alternative embodiment, the step of drying the nitrocellulose film coated with polyvinylpyrrolidone is further included, optionally at 60°C.

[0011] In one alternative embodiment, polyvinylpyrrolidone coating is performed by placing a plasma-treated nitrocellulose membrane in a polyvinylpyrrolidone solution for 10-30 seconds.

[0012] In one alternative implementation, at least one of the following is satisfied: In plasma processing, the power is 50 W, 80 W, or 100 W; In plasma treatment, the treatment time is 30, 90, 120, or 150 seconds; In plasma processing, the vacuum level is 10 Pa, 30 Pa, or 50 Pa; In polyvinylpyrrolidone coating, polyvinylpyrrolidone solutions with concentrations of 0.3 g / 100 ml, 0.5 g / 100 ml, 1 g / 100 ml, 1.5 g / 100 ml, 2 g / 100 ml, or 2.5 g / 100 ml are used. In polyvinylpyrrolidone coating, a polyvinylpyrrolidone solution with a concentration of 0.3 g / 100 ml to 2.5 g / 100 ml contains 0.025 g / 100 ml, 0.1 g / 100 ml, or 0.15 g / 100 ml of crosslinking agent.

[0013] Secondly, the present invention provides a nitrocellulose membrane, characterized in that it is prepared by the above-described processing method.

[0014] Thirdly, the present invention provides a kit comprising the nitrocellulose membrane described above.

[0015] Fourthly, the present invention provides an application of the above-mentioned nitrocellulose membrane in immunoassay.

[0016] The technical solution of this invention has the following advantages: 1. The nitrocellulose membrane treatment method provided by the present invention improves hydrophilicity, antibody binding rate, chromatography speed change rate, polyvinylpyrrolidone layer firmness and / or streak diffusion rate under low humidity environment by using a specific concentration of polyvinylpyrrolidone and a crosslinking agent (glutaraldehyde / genipin).

[0017] 2. The nitrocellulose membrane treatment method provided by this invention achieves better technical results through a specific volume ratio of argon and oxygen. If the oxygen ratio is too high (e.g., argon / oxygen = 1:1), it will cause the nitrocellulose membrane to become brittle (the tensile strength will decrease by 15%); if the oxygen ratio is too low (e.g., argon / oxygen = 8:1), the introduction of active groups (-COOH / -OH) will be insufficient, only 60% of the optimal ratio (4:1).

[0018] 3. The nitrocellulose membrane treatment method provided by this invention achieves better coating uniformity through polyvinylpyrrolidone of a specific molecular weight. If PVPK90 (molecular weight 130,000) is used, uneven coating is easily caused by molecular aggregation; if PVPK15 (molecular weight 15,000) is used, it is easy to penetrate into the membrane, resulting in a slower chromatography rate (30% lower than normal).

[0019] 4. The nitrocellulose membrane treatment method provided by the present invention forms hydrogen bonds between -COOH / -OH introduced by plasma and the pyrrolidone groups of PVP molecules, and at the same time undergoes a covalent reaction through a crosslinking agent (glutaraldehyde / genipin) (the formation of chemical bonds can be verified by Fourier transform infrared spectroscopy), so that the PVP layer changes from "physical adhesion" to "chemical bonding + physical intercalation", thus solving the problem of easy peeling off of PVP coating alone.

[0020] 5. The nitrocellulose membrane treatment method provided by this invention has wide environmental adaptability: it can be used stably in high humidity (>80%RH) or low humidity (<20%RH) scenarios without the need for additional moisture-proof packaging, reducing production costs by 20%; it has high detection sensitivity: the improved antibody binding rate reduces the minimum detection concentration of human IgG to 0.2mg / mL, which is twice the sensitivity of the untreated membrane (0.4mg / mL), and can be used for the detection of low-concentration targets such as early disease diagnostic markers; it is safe and compliant: the selected glutaraldehyde and genipin (natural cross-linking agent) have no toxic residues and can be adapted to the production requirements of medical-grade and food-grade immunochromatographic detection products. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 This is a graph showing the effect of immunochromatographic detection of the NC membrane manufactured in Example 2 under the conditions of Experiment 2.

[0023] Figure 2 This is a graph showing the effect of the NC membrane manufactured in Example 1 at different human IgG (H+L) concentrations in Experiment 2. Detailed Implementation

[0024] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0025] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0026] Example 1 The method for treating the nitrocellulose membrane provided in this embodiment is as follows: 1. Plasma treatment: Fix the cut NC membrane onto the treatment tray, place it in the plasma reaction chamber, set the low-pressure radio frequency plasma generator power to 50W, maintain a vacuum of 30Pa, and a plasma argon / oxygen volume ratio of 4:1 for 30s. Exhaust the gas and break the vacuum, then remove the treated NC membrane.

[0027] 2. Prepare 100ml PVP solution: Weigh 0.5g of PVP K30 (molecular weight 38,000) powder and dissolve it in a 9:1 ratio of deionized water and anhydrous ethanol. Add 0.25 g / ml glutaraldehyde solution to obtain 100ml PVP solution containing 0.1g glutaraldehyde.

[0028] 3. Immersion coating: Immerse the plasma-pretreated NC membrane in the above PVP solution for 10 seconds, then remove and drain excess liquid. 4. Curing: Dry the NC film from step 3 in hot air at 60°C for 5 minutes, and obtain the modified NC film after cooling.

[0029] Example 2-12 The method described in Example 1 differs from that in Example 1 only in the method parameters shown in the table below: Table 1 Method Parameters

[0030] Example 1 of process parameters The process parameters for plasma treatment were screened using the method described in Example 1.

[0031] Table 2. Process parameters and effects of plasma treatment

[0032] Micropore etching effect: After gold sputtering, the surface morphology and pore structure of the NC film sample were directly observed under SEM.

[0033] Membrane mechanical strength: The NC membrane was cut into standard dumbbell-shaped specimens, and its breaking strength and elongation at break were measured using a universal testing machine.

[0034] Carboxyl (-COOH) content: The relative percentage of OC=O functional groups was calculated by high-resolution scanning and peak fitting of the C1s spectrum.

[0035] Reactive group density: XPS analysis was performed on samples treated for different times, and curves were plotted showing the change in the content of oxygen-containing functional groups (-COOH / -OH) over time.

[0036] Antibody permeation and loss: The antibody solution labeled with FITC fluorescent dye was subjected to chromatography on the treated NC membrane. The membrane was washed with buffer and the eluent was collected. The fluorescence intensity of the eluent was measured using a fluorescence spectrophotometer. The higher the intensity, the weaker the antibody adsorption and the more serious the loss.

[0037] Membrane structure integrity: After sputtering gold onto the NC membrane sample, its surface morphology and pore structure were directly observed under SEM.

[0038] Processing efficiency: Except for the vacuum level, all other parameters remain consistent. NC membranes are processed under different vacuum levels, and their water contact angles are measured.

[0039] Processing uniformity: On the same treated membrane, select the center and the perimeter, measure the contact angle, and calculate the standard deviation.

[0040] Example 2 of process parameter screening The process parameters for polyvinylpyrrolidone coating were screened using the method in Example 2.

[0041] The PVP formulation used is: PVPK301%, 0.1% glutaraldehyde, and deionized water / ethanol (9:1).

[0042] The Tween-20 formula used is: Tween-20 0.5%, deionized water / ethanol (9:1).

[0043] Table 3. Process parameters and effects of polyvinylpyrrolidone coating.

[0044] Further research revealed that in polyvinylpyrrolidone (PVP) coating: immersing the plasma-pretreated NC membrane in the above PVP solution for 10-30 seconds (time < 10 seconds: incomplete coating; time > 30 seconds: excessive liquid absorption by the membrane, resulting in uneven coating after drying). Curing: Dry with hot air at 60℃ for 5 minutes (temperature < 60℃: drying is incomplete and crosslinking reaction is incomplete; temperature > 60℃: PVP undergoes thermal degradation), so that PVP and the active groups on the membrane surface crosslink to form a film through "hydrogen bonds + covalent bonds", forming a stable protective layer.

[0045] Experimental Example 1 The effectiveness of the implementation examples was tested, and the specific performance tests are as follows: Contact angle test: Take the NC membrane prepared in the example, cut it into a 2*4cm rectangle, use tweezers to pick up the cut NC membrane and stick it flat on the glass slide of the sample stage, turn on the contact angle measuring instrument, calibrate the horizontal direction, focus, calibrate the pixels, slowly drop a drop of 2μL ultrapure water onto the surface of the NC membrane, capture the image at the moment the droplet contacts the membrane surface, calculate the contact angle with the software, and take the average value.

[0046] Contact angle test after 6 months of accelerated aging: The membrane was placed in an environment of 40℃ and 75%RH for 6 months of accelerated aging, and the contact angle was tested according to the above method.

[0047] Antibody binding rate: Coating: The NC membrane prepared in the example and the untreated NC membrane were cut into circular pieces with a diameter of 8 mm and placed in a 96-well microplate. 100 μL of 1 mg / mL human IgG solution was added to each well and incubated at 4 °C for 12 hours. Blocking: Discard the liquid in the wells, add 200 μL of 5% BSA blocking solution to each well, incubate at 37°C for 1 hour, and wash the plate 3 times with PBS (30 seconds each time). Incubation with primary antibody: Add 100 μL of 0.1 mg / mL goat anti-human IgG-HRP solution to each well, incubate at 37°C for 1 hour, and wash the plate 3 times with PBS; Color development and reading: Add 100 μL of TMB color development solution to each well, incubate in the dark for 15 minutes, then add 50 μL of 2 mol / L H2SO4 to terminate the reaction, and read the absorbance (OD value) at 450 nm using a microplate reader. Binding rate calculation: Antibody binding rate = 100% × OD value of untreated NC membrane (control) / OD value of modified NC membrane Chromatography rate change rate during humidity fluctuations (40-80% RH): Sample preparation: Cut the NC membrane prepared in the example into strips of 25mm×300mm, and use a gold spraying device to draw 1mg / mL human IgG lines (1mm wide) on the membrane. After air drying at room temperature, it is ready for use. Chromatography speed test at 40%RH: Fix the membrane strip in the chromatography card, add 100μL of gold-labeled goat anti-human IgG solution, and place it in a constant temperature and humidity chamber at 40%RH and 25℃. Use a stopwatch to record the time it takes for the gold-labeled solution to travel from the sample application area to the IgG line (denoted as t1). The chromatography distance is 20mm, so the speed v1 = 20 / t1 (unit: mm / min). Chromatography rate test at 80%RH: Take the same batch of membrane strips, repeat step (2), and test at 80%RH and 25℃ to obtain the rate v2; Calculation of rate of change:

[0048] Low humidity (25% RH) streak diffusion rate test method: Operating steps: (1) Preparation for scribing: Place the NC membrane prepared in the example in a constant temperature and humidity chamber at 25%RH and 25℃ for equilibration for 30 minutes; (2) Scibing operation: Use a scribing sputtering instrument (parameters: line width 1mm, speed 5mm / s) to scribing a line of 1mg / mL human IgG on the membrane, and immediately let it stand in the chamber for 10 minutes after scribing (to avoid interference from ambient humidity); (3) Measurement of line width: Observe the line at 5 different positions with a microscope and measure the actual line width (referred to as Wi); (4) Calculation of diffusion rate: Line diffusion rate = 100% × (average actual line width) 1mm) / 1mm (Note: The average actual line width is the average of 5 Wi.) Low-humidity scribing uniformity test: Under 25%RH environment, 10 lines (1mm wide) were continuously scribed using a scribing and gold spraying machine, and the smoothness of the line edges was observed under a microscope; the experimental results are shown below. Figure 2 Table (Example 2) The detection results of the example are as follows: Table 4

[0049] Experiment Example 2 The membranes prepared in Examples 1 and 2 were tested. The specific methods are as follows: (1) Basic marking and closure treatment Human IgG (H+L) (50 mg / mL) was diluted to 1 mg / mL with PBS and streaked onto NC membranes treated in the examples, untreated NC membranes, Tween-treated NC membranes, NC membranes coated with PVP alone, or NC membranes treated with plasma alone using a streaking gold sputtering apparatus (parameters: line width 1 mm, speed 5 mm / s). The membranes were then air-dried at room temperature (air velocity 0.5 m / s to avoid membrane deformation). Immerse the NC membrane after marking in 5% BSA blocking solution for 30 minutes (shake intermittently every 5 minutes to ensure uniform sealing), then remove and air dry at room temperature.

[0050] (2) Determination of standard concentration of gold-labeled goat anti-human IgG Gold-labeled goat anti-human IgG (0.4 mg / mL) was diluted 10, 20, 30, 40, and 60 times with PBS, respectively. Chromatography (chromatographic speed 10 mm / min) was used to sequentially pass gold-labeled antibodies of different dilutions through the NC membrane treated in step (1); Judgment criteria: The minimum concentration at which the appearance of the lines tends to be stable (without serious single-sidedness, diffusion, non-denseness, non-straightness, blurriness, or incomplete color development) is taken as the standard concentration of gold-labeled goat anti-human IgG (the standard concentration of the treated membrane of this invention can be as low as 40 times dilution, while the untreated membrane needs to be diluted more than 20 times to achieve a stable appearance).

[0051] (3) Determination of the minimum detection concentration of human IgG (H+L) Preliminary screening: Human IgG (H+L) was serially diluted 10-fold (10-1000 times) with PBS, and the color development was observed by chromatography to determine the color development range; Precise screening: Based on the preliminary screening results, human IgG (H+L) was further diluted to 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, and 0.8 mg / mL, and the streaking and blocking operations of step (1) were repeated; Detection and judgment: Chromatography was performed using the gold-labeled antibody standard concentration determined in step (2), and the color development on the membrane was observed. The lowest concentration with "stable line appearance" was taken as the lowest detection concentration of human IgG (H+L) (the lowest detection concentration of the treated membrane in this invention is 0.2 mg / mL, and that of the untreated membrane is 0.4 mg / mL).

[0052] For relevant experimental results, please refer to Figure 1 (The corresponding detection conditions include: gold-labeled goat anti-human IgG concentration of 0.02 mg / mL and human IgG (H+L) concentration of 0.4 mg / mL) and Figure 2 . Figure 1 This demonstrates that: untreated NC films have uneven edges; NC films coated with PVP alone have a lighter color; NC films treated with plasma alone show significantly lighter color and blurred lines after aging; the NC films of this invention (plasma + PVP coating) exhibit uniform and clear color development; and Tween treatment results in diffusion. In other words, Figure 1 This demonstrates that the present invention achieves a very significant synergistic effect of 1+1>2.

[0053] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for treating nitrocellulose membranes, characterized in that, include: Plasma treatment, in which nitrocellulose membranes are subjected to plasma treatment; and, Polyvinylpyrrolidone coating, wherein a plasma-treated nitrocellulose membrane is coated with polyvinylpyrrolidone.

2. The processing method according to claim 1, characterized in that, In plasma treatment, argon and oxygen are used in a volume ratio of 3:1-5:1, optionally 4:1, and / or, the power is 50-100W, and / or, the treatment time is 30-150 seconds, and / or, the vacuum degree is 10-50Pa.

3. The processing method according to claim 1 or 2, characterized in that, In polyvinylpyrrolidone coating, a polyvinylpyrrolidone solution with a concentration of 0.3 g / 100 ml to 2.5 g / 100 ml is used. Optionally, the polyvinylpyrrolidone solution with a concentration of 0.3 g / 100 ml to 2.5 g / 100 ml contains 0.025 g / 100 ml to 0.15 g / 100 ml of crosslinking agent. Optionally, the solvent in the polyvinylpyrrolidone solution with a concentration of 0.3 g / 100 ml to 2.5 g / 100 ml is deionized water and / or ethanol. Further, optionally, the solvent in the polyvinylpyrrolidone solution with a concentration of 0.3 g / 100 ml to 2.5 g / 100 ml is deionized water and ethanol in a volume ratio of 9:

1.

4. The processing method according to any one of claims 1-3, characterized in that, The molecular weight of polyvinylpyrrolidone is greater than 15,000 and less than 130,000. Optionally, the molecular weight of polyvinylpyrrolidone is greater than 30,000 and less than 130,000. More preferably, the molecular weight of polyvinylpyrrolidone is 38,000. Alternatively, the crosslinking agent may be glutaraldehyde or genipin.

5. The processing method according to any one of claims 1-4, characterized in that, It also includes the step of drying the nitrocellulose film coated with polyvinylpyrrolidone, optionally at 60°C.

6. The processing method according to any one of claims 1-5, characterized in that, Polyvinylpyrrolidone coating is performed by placing a plasma-treated nitrocellulose membrane in a polyvinylpyrrolidone solution for 10-30 seconds.

7. The processing method according to any one of claims 1-5, characterized in that, Meet at least one of the following: In plasma processing, the power is 50 W, 80 W, or 100 W; In plasma treatment, the treatment time is 30, 90, 120, or 150 seconds; In plasma processing, the vacuum level is 10 Pa, 30 Pa, or 50 Pa; In polyvinylpyrrolidone coating, polyvinylpyrrolidone solutions with concentrations of 0.3 g / 100 ml, 0.5 g / 100 ml, 1 g / 100 ml, 1.5 g / 100 ml, 2 g / 100 ml, or 2.5 g / 100 ml are used. In polyvinylpyrrolidone coating, a polyvinylpyrrolidone solution with a concentration of 0.3 g / 100 ml to 2.5 g / 100 ml contains 0.025 g / 100 ml, 0.1 g / 100 ml, or 0.15 g / 100 ml of crosslinking agent.

8. A nitrocellulose membrane, characterized in that, Prepared by the processing method according to any one of claims 1-7.

9. A reagent kit, characterized in that, Includes the nitrocellulose membrane as described in claim 8.

10. The application of the nitrocellulose membrane according to claim 8 in immunoassay.

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