Anti-interference trace immuno-chromatographic test paper and preparation method thereof

Abstract

The present application relates to a kind of anti-interference trace immunochromatography test paper and its preparation method, belong to immunochromatography test strip technical field.The present application is with TEMPO oxidized nanocellulose as base material, by zinc salt loading, in-situ growth ZIF-8 / Zn (OH) 2 heterojunction is prepared composite aerogel film, then load antibody and be made detection line and quality control line strip, assembly obtains test strip.The aerogel has high specific surface area and excellent stability, can greatly improve antibody loading capacity and binding strength, with the structural characteristics of ZIF-8 porous metal organic framework, effectively protect antibody activity under high temperature and high humidity environment, significantly improve detection sensitivity, signal stability and anti-aging ability, realize trace target super-sensitive, high-stable rapid detection.

Description

Technical Field

[0001] This invention belongs to the field of immunochromatographic test strip technology, and particularly relates to an anti-interference trace immunochromatographic test strip and its preparation method. Background Technology

[0002] In recent years, immunochromatography (ICA), as a rapid detection technology based on antigen-antibody specific binding reaction, has become one of the key means of point-of-care testing (POCT) for food safety due to its advantages such as simple operation, rapid detection, and no need for complex instruments. Its basic principle is to use the capillary action of the chromatographic membrane to drive the lateral flow of the sample liquid, so that the target analyte can specifically bind to the probe pre-fixed on the test strip, and the detection is achieved through visual signals. Traditional test strips are usually composed of a sample pad, a conjugation pad, a chromatographic membrane, an absorbent pad, and a backing, which are stacked in sequence.

[0003] Although traditional test strips are widely used in food safety screening due to their convenience and low cost, their reliable and quantitative detection of trace toxins, pesticide residues, or pathogens still faces significant challenges in complex environments such as agricultural product fields, processing workshops, or warehousing and logistics, characterized by high temperatures and humidity. Existing technologies mainly improve sensitivity and quantification by modifying markers or optimizing buffer formulations to improve the reaction environment; however, these improvements still have significant shortcomings. In extreme environments such as high temperature and humidity, biological probes such as antibodies are prone to rapid inactivation and detachment, leading to signal attenuation. Furthermore, the detection process of existing test strips typically requires manual, step-by-step addition of samples and multiple reagents, which is cumbersome, increases the risk of operational errors, and limits their widespread adoption in non-professional field environments.

[0004] To address these issues, researchers have attempted to improve detection performance and the automation level of immunochromatographic assays by modifying label performance, optimizing chromatographic membrane materials, and designing microfluidic structures. In recent years, aerogels have been widely used in biochips, sensors, and other fields due to their high specific surface area, large porosity, good biocompatibility, and excellent chemical stability, aiming to improve probe loading and molecular recognition efficiency. However, their ability to protect the activity of antibodies and other biological probes under extreme environments such as high temperature and humidity remains somewhat lacking. Even though current technologies have effectively integrated aerogels into immunochromatographic test strips, they still cannot effectively maintain antibody activity under extreme thermal and chemical conditions.

[0005] Therefore, there is an urgent need for an immunochromatographic test strip that can overcome the above-mentioned technical deficiencies. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an anti-interference trace immunochromatographic test strip and its preparation method. Using TEMPO-oxidized cellulose nanoparticles as a substrate, a composite aerogel membrane is prepared by zinc salt loading and in-situ growth of ZIF-8 / Zn(OH)2 heterostructures. Antibodies are then loaded to form detection and control lines, which are assembled into the test strip. This aerogel possesses both high specific surface area and excellent stability, significantly increasing antibody loading and binding strength. Utilizing the structural characteristics of the porous metal-organic framework of ZIF-8, it effectively protects antibody activity under high temperature and humidity conditions, significantly improving detection sensitivity, signal stability, and anti-aging ability, achieving ultrasensitive, highly stable, and rapid detection of trace targets.

[0007] The first objective of this invention is to provide a method for preparing an interference-resistant trace immunochromatographic test strip, comprising the following steps: S1. Add zinc salt to the TEMPO oxidized nanocellulose suspension and stir to dissolve. Adjust the pH of the system to 7.8-8.2, and then stir and let it stand to obtain a zinc-based nanocellulose suspension. S2. The zinc-based nanocellulose suspension described in S1 is vacuum filtered to form a membrane. After washing, it is vacuum filtered again. The vacuum filtration-washing operation is repeated to obtain a zinc-based nanocellulose membrane. The zinc-based nanocellulose membranes described in S3 and S2 were reacted in a 1,2-dimethylimidazolium methanol solution, and after washing and drying, a ZIF-8 / Zn(OH)2 modified nanocellulose aerogel membrane was obtained. S4. The ZIF-8 / Zn(OH)2 modified nanocellulose aerogel membrane described in S3 is immersed in an antibody solution, and after standing, drying, and shearing, antibody-loaded ZIF-8 / Zn(OH)2 modified nanocellulose aerogel strips are obtained; the antibody solution is an anti-AFP detection antibody solution or a goat anti-mouse IgG antibody solution. S5. The antibody-loaded ZIF-8 / Zn(OH)2 modified nanocellulose aerogel strip described in S4 is pasted onto the chromatography test strip to obtain the anti-interference trace immunochromatographic test strip; the chromatography test strip includes a base plate, a reaction pad, an absorbent pad, a conjugate pad, and a sample pad.

[0008] In one embodiment of the present invention, in S1, the solid content of the TEMPO-oxidized cellulose nanoparticle suspension is 0.08wt%-0.12wt%; And / or, the zinc salt is zinc nitrate; And / or, the mass ratio of the TEMPO oxidized nanocellulose suspension to the zinc salt is (38-42):3.

[0009] In one embodiment of the present invention, in S1, the stirring speed is 950 r / min-1050 r / min, and the time is 110-130 min; And / or, the settling time is 55 min-65 min.

[0010] In one embodiment of the present invention, in S3, the concentration of the 1,2-dimethylimidazolium methanol solution is 48 mg / mL to 52 mg / mL.

[0011] In one embodiment of the present invention, in S3, the temperature of the reaction is 5°C-35°C and the time is 11h-13h; And / or, the drying temperature is 55℃-65℃, and the time is 5.5h-6.5h.

[0012] In one embodiment of the present invention, in S4, the concentration of the anti-AFP detection antibody solution is 8 mg / mL-9 mg / mL; And / or, the concentration of the goat anti-mouse IgG antibody solution is 1.8 mg / mL to 2.2 mg / mL.

[0013] In one embodiment of the present invention, in S4, the impregnation time is 2.5h-3.5h; And / or, the settling time is 20 min to 30 min.

[0014] In one embodiment of the present invention, in S5, the binding pad is loaded with a gold-labeled anti-AFP capture antibody solution and a colloidal particle tracer label; the concentration of the gold-labeled anti-AFP capture antibody solution is 0.40 mg / mL-0.45 mg / mL.

[0015] In one embodiment of the present invention, in S5, the anti-interference trace immunochromatographic test strip uses a ZIF-8 / Zn(OH)2 modified nanocellulose aerogel strip loaded with anti-AFP detection antibody as a detection line; and a ZIF-8 / Zn(OH)2 modified nanocellulose aerogel strip loaded with goat anti-mouse IgG antibody as a control line.

[0016] A second objective of this invention is to provide an interference-resistant trace immunochromatographic test strip prepared by the method described above.

[0017] The technical solution of the present invention has the following advantages compared with the prior art: (1) The preparation method of the present invention uses TEMPO oxidized nanocellulose aerogel as the signal enhancement substrate, and grows ZIF-8 in situ in its three-dimensional porous structure to construct a ZIF-8 / Zn(OH)2 modified nanocellulose aerogel composite stabilization structure. This structure has large pore volume, high specific surface area and excellent mechanical properties, which can greatly improve the antibody loading and binding stability, significantly improve the detection signal intensity and sensitivity. At the same time, with the rigid shielding and hydrophobic moisture barrier effect of ZIF-8 and the coordination anchoring effect of Zn(OH)2, the antibody biological activity can be effectively protected in a wide temperature range and in harsh environments of high temperature and high humidity, giving the immunochromatographic test strip outstanding extreme environmental tolerance, thereby realizing ultrasensitive, highly stable, convenient and reliable instant detection of harmful substances in food. It has important technical value and market significance for improving the environmental adaptability, detection stability and long-term reliability of rapid on-site detection of food safety.

[0018] (2) The anti-interference trace immunochromatographic test strip of the present invention uses ZIF-8 / Zn(OH)2 modified nanocellulose aerogel. The aerogel uniformly and continuously coats ZIF-8 crystals onto the surface of nanofibers through in-situ reaction, improving the uniformity and structural continuity of ZIF-8 loading. At the same time, the high specific surface area of ​​the nanocellulose aerogel enhances the antibody loading capacity. Moreover, Zn(OH)2 is structurally stable in a wide temperature range, which can not only ensure the overall structural stability under high temperature conditions, but also provide sufficient metal coordination sites for the in-situ growth of ZIF-8. The protective mechanism of the aerogel for antibodies is mainly reflected in three aspects: First, the antibody is impregnated and adsorbed onto the pre-formed ZIF-8 / Zn(OH)2 heterostructure. Within the porous membrane, the rigid framework and microporous structure of ZIF-8 restrict antibody molecule movement, maintain its spatial conformational stability, and prevent harsh environmental factors such as high temperature and humidity from directly damaging the antibody structure. Secondly, Zn(OH)2 serves as an intermediate anchoring layer, providing a zinc source and nucleation sites for the in-situ growth of ZIF-8. It forms an interfacial coordination bridge between the nanocellulose fibers and ZIF-8 crystals, firmly anchoring the ZIF-8 crystals onto the cellulose framework. This prevents ZIF-8 particles from detaching during impregnation, chromatography, and long-term storage, ensuring the integrity of the carrier structure and improving the repeatability and stability of the test strip. Thirdly, the hydrophobic microporous environment of the ZIF-8 framework can selectively enrich target analytes, reduce interference from hydrophilic impurities, and improve the signal-to-noise ratio.

[0019] (3) The anti-interference trace immunochromatographic test strip of the present invention can significantly improve the effective antibody loading density per unit area of ​​aerogel strip through the synergistic effect of ZIF-8 and Zn(OH)2 bifunctional layers, increase the target capture probability in the detection area, stabilize the antibody conformation and maintain the spatial orientation of the active site, and maximize the retention of antibody-antigen specific binding efficiency. The Zn(OH)2 intermediate layer firmly anchors the ZIF-8 crystal to the surface of nanocellulose fiber through coordination bonds, improves the stability of the carrier structure, and enables the test strip to have a higher effective antibody adsorption capacity, better antibody stability in extreme environments, lower detection limit, wider quantitative linear range and better detection repeatability, and finally achieve highly sensitive and highly stable immunochromatographic detection. Attached Figure Description

[0020] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein: Figure 1 This is a schematic diagram of the structure of the anti-interference trace immunochromatographic test strip of the present invention. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0022] In this invention, unless otherwise stated, the preparation process of the anti-interference trace immunochromatographic test strip in the embodiments is carried out under full control of the operating environment. The antibody adsorption process is carried out at a temperature of 25±2℃ and a relative humidity of 50%. All antibody-related operations are carried out in a biosafety cabinet or a clean bench.

[0023] In this invention, unless otherwise stated, the solvent for the anti-AFP detection antibody solution, goat anti-mouse IgG antibody solution and gold-labeled anti-AFP capture antibody solution in the examples is PBS buffer.

[0024] In this invention, unless otherwise stated, each pad material in the embodiments needs to be pretreated with a treatment solution before use: the treatment solution for the sample pad is prepared by dissolving 1.5 wt% Tween-20 in water; the treatment solution for the conjugate pad is prepared by dissolving 2.0 w / v% BSA and 1.5 w / v% Tween-20 in a 2.0 mol / L phosphate buffer solution; the treatment solution for the reaction pad is prepared by dissolving 2.0 w / v% BSA and 1.5 w / v% Tween-20 in a 1.5 mol / L phosphate buffer solution.

[0025] In this invention, unless otherwise stated, the colloidal particle tracer used in the embodiments was purchased from Kerui Biotechnology Co., Ltd., catalog number bsm-33081M-Gold. Example 1

[0026] The interference-resistant trace immunochromatographic test strip and its preparation method in this embodiment specifically include the following steps: S1. Preparation of TEMPO oxidized cellulose nanoparticle suspension: At room temperature (25±2℃), 4.0g of TEMPO oxidized cellulose nanoparticle slurry with a solid content of 1.0wt%, a carboxylate content of 2.0mmol / g, and an average diameter of 10nm was added to a beaker. Then, deionized water was slowly added while stirring with a glass rod to adjust the solid content of the system to 0.1wt%. The beaker was then placed on a magnetic stirrer and stirred at 500r / min for 30min to ensure that the cellulose nanoparticles were completely and uniformly dispersed, thus obtaining the TEMPO oxidized cellulose nanoparticle suspension. S2. Preparation of zinc-based nanocellulose suspension: Add 3g of zinc nitrate hexahydrate to 40g of TEMPO oxidized nanocellulose suspension and stir at 800r / min for 20min until the zinc nitrate is completely dissolved. Then, add sodium hydroxide solution with a concentration of 4mg / mL dropwise at 1 drop / s at 1000r / min to adjust the pH value to 8.0±0.1. Then maintain stirring at 1000r / min for 2h to allow zinc ions to be fully adsorbed on the surface of nanocellulose to form a Zn(OH)2 layer. After stopping stirring, let stand for 1h to obtain zinc-based nanocellulose suspension. S3. Preparation of zinc-based nanocellulose membrane: The zinc-based nanocellulose suspension was transferred to a vacuum filtration device equipped with a 0.1 μm PVDF filter membrane. The vacuum pump was turned on for vacuum filtration to make the suspension form a uniform film on the filter membrane surface. After the initial gel membrane was formed, the vacuum pump was turned off and 50 mL of methanol was taken to wash the surface of the gel membrane evenly. Then the vacuum pump was turned on again for filtration. This vacuum filtration-methanol washing operation was repeated 3 times to obtain the zinc-based nanocellulose membrane. Preparation of ZIF-8 / Zn(OH)2 modified nanocellulose aerogel membrane: Zinc-based nanocellulose membrane was transferred to a clean petri dish, and 1,2-dimethylimidazole methanol solution with a concentration of 50 mg / mL was added to completely immerse the gel membrane. Then, the petri dish was covered and allowed to stand at room temperature for 12 h to allow Zn(OH)2 on the surface of the gel membrane to fully react with 1,2-dimethylimidazole and generate ZIF-8 crystals in situ. After the reaction, the gel membrane sample was removed with tweezers, washed in methanol for 30 min, and then the washed sample was transferred to a clean petri dish and dried in an oven at 60 ℃ for 6 h to obtain ZIF-8 / Zn(OH)2 modified nanocellulose aerogel membrane. S5. Preparation of antibody-loaded ZIF-8 / Zn(OH)2 modified cellulose nanoparticle aerogel strips: The ZIF-8 / Zn(OH)2 modified cellulose nanoparticle aerogel membrane was immersed in an 8.4 mg / mL anti-AFP detection antibody solution for 3 h, allowed to stand for 20 min, and then dried in a 37℃ constant temperature drying oven for 2 h; the ZIF-8 / Zn(OH)2 modified cellulose nanoparticle aerogel membrane was immersed in a 2 mg / mL goat anti-mouse IgG antibody solution for 3 h, allowed to stand for 30 min, and then dried in a 37℃ constant temperature drying oven for 2 h; then, ZIF-8 / Zn(OH)2 modified cellulose nanoparticle aerogel strips loaded with anti-AFP detection antibody (detection line) and ZIF-8 / Zn(OH)2 modified cellulose nanoparticle aerogel strips loaded with goat anti-mouse IgG antibody (control line) were cut into strips with a length of 2 mm, a width of 8 mm, and a thickness of 1 mm. S6. Preparation of anti-interference trace immunochromatographic test strips: according to... Figure 1 The structure shown uses a PVC board as the base. On the corresponding positions of the reaction pad, ZIF-8 / Zn(OH)2 modified nanocellulose aerogel strips loaded with anti-AFP detection antibody and goat anti-mouse IgG antibody are sequentially pasted in the order of detection line, blank area, and control line. Then, the reaction pad, absorbent pad, conjugate pad loaded with 0.42 mg / mL gold-labeled anti-AFP capture antibody solution and colloidal particle tracer, and sample pad are sequentially pasted onto the PVC board from bottom to top. The reaction pad overlaps with the conjugate pad, the absorbent pad, and the sample pad by 1.5 mm, resulting in an interference-resistant trace immunochromatographic test strip. The sample pad, conjugate pad, and reaction pad are all made of glass fiber membrane, and the absorbent pad is made of highly absorbent cotton fiber membrane. Comparative Example 1

[0027] The process is basically the same as in Example 1, except that the ZIF-8 / Zn(OH)2 modified nanocellulose aerogel membrane is replaced with a SiO2 aerogel membrane. The SiO2 aerogel membrane was purchased from Shenzhen Ruijite Biotechnology Co., Ltd., with the product number RGT00650. Comparative Example 2

[0028] The basic structure is the same as in Example 1, except that the ZIF-8 / Zn(OH)2 modified nanocellulose aerogel membrane is replaced with a nanocellulose aerogel membrane. The nanocellulose aerogel membrane was purchased from Xi'an Qiyue Biotechnology Co., Ltd., with the product number Q-0071101. Comparative Example 3

[0029] The procedure is basically the same as in Example 1, except that: the nanocellulose aerogel membrane modified with ZIF-8 / Zn(OH)2 was not used, and the anti-AFP detection antibody solution and goat anti-mouse IgG antibody solution were directly added to the detection line and control line. Test Example 1

[0030] Based on Example 1 and Comparative Examples 1-2, the aerogel membranes before and after antibody loading were tested: (1) BET specific surface area, pore volume and average pore size: 20 mg of aerogel membrane sample before antibody loading was taken and degassed under vacuum at 250℃ for 4 h. The N2 adsorption-desorption method (BET method) was used. The surface area and pore size analyzer of Micromeritics ASAP 2460 was used to perform isothermal adsorption-desorption test under liquid nitrogen atmosphere at -196℃. The data were recorded and the BET specific surface area, pore volume and average pore size of the sample were calculated. (2) Maximum BSA loading: Take 20 mg of aerogel membrane sample before antibody loading and immerse it in a series of concentration gradients (0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 8 mg / mL, 16 mg / mL) of bovine serum albumin (BSA) PBS solution (pH 7.4). After equilibration at room temperature, transfer it to a 4℃ environment and let it stand for 5 h to allow BSA to be fully adsorbed onto the aerogel membrane. After adsorption, take the supernatant and use the Thermo Fisher BCA protein quantification kit to determine the concentration of unbound BSA in the supernatant. Calculate the maximum saturated adsorption capacity of the aerogel membrane for BSA based on the concentration difference. (3) Effective adsorption capacity of anti-AFP antibody: Take 20 mg of the aerogel membrane sample loaded with antibody, and use 0.1 mol / L NaOH solution to fully elute the anti-AFP antibody fixed on the membrane surface. Collect the eluent and determine the concentration using the BCA protein quantification method. Calculate the effective adsorption capacity of anti-AFP antibody per unit area of ​​aerogel membrane based on the volume of eluent, antibody concentration and effective contact area of ​​aerogel membrane. Table 1 shows the final measured parameters: Table 1

[0031] As can be seen from Table 1, the ZIF-8 / Zn(OH)2 modified nanocellulose aerogel membrane prepared in Example 1 showed the best performance in terms of specific surface area, pore structure parameters, protein loading capacity and effective antibody adsorption.

[0032] Comparing Example 1 and Comparative Example 1, it can be seen that the SiO2 aerogel membrane used in Comparative Example 1 has a significantly lower specific surface area and pore volume, resulting in a limited number of adsorption sites and weaker protein loading and antibody immobilization effects, making it difficult to meet the requirements of immunochromatography for a high-efficiency antibody carrier.

[0033] Comparing Example 1 and Comparative Example 2, it can be seen that the nanocellulose aerogel membrane used in Comparative Example 2 has a significantly lower degree of pore structure development and adsorption capacity. It has not been modified with ZIF-8 crystals, and its antibody loading capacity and molecular recognition efficiency are much lower than those of the aerogel membrane modified with ZIF-8 / Zn(OH)2. Test Example 2

[0034] The immunochromatographic test strips of Example 1 and Comparative Examples 1-3 were set as the initial group and the aging group, respectively. The initial group was stored in a normal environment of 25°C and 45% RH for 7 days, and the aging group was stored continuously in a constant temperature and humidity chamber of 60°C and 80% RH for 7 days. After the aging treatment, performance tests were performed. (1) T-line signal intensity and signal retention rate: Take the test strips of the initial group and the aged group respectively, add 10 μL of AFP positive sample with a concentration of 100 ng / mL prepared with PBS buffer to the sample pad, let it stand for 15 min at room temperature for chromatography, use Biomaxima Reader BM-300 colloidal gold immunochromatographic reader to quantitatively scan the gray value of the detection line (T line), record the initial T-line signal intensity and the T-line signal intensity after aging, and calculate the signal retention rate by the ratio of the T-line signal value after aging to the initial T-line signal value; (2) Limit of detection (LOD): AFP series concentration gradient standards of 0 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL and 100 ng / mL were prepared using PBS buffer. The initial group and the high temperature and humidity aging group of test strips were tested for gradient detection. The lowest sample concentration corresponding to the ratio of T line signal intensity to C line signal intensity ≥ 1.0 was determined as the limit of detection. The LOD values ​​of each group of test strips in the initial state and after high temperature and humidity aging were recorded. The LOD drift factor was calculated by the ratio of the LOD after aging to the initial LOD. (3) Antibody desorption rate: Take the immunochromatographic test strips after high temperature and high humidity aging treatment, remove the aerogel strips on them, and use 0.1mol / L NaOH solution to fully elute the strips. Collect all the eluent and use the BCA protein quantification method to determine the concentration of residual antibody in the eluent. Calculate the antibody desorption rate according to the formula: antibody desorption rate (%) = (initial loading - residual amount after aging) / initial loading × 100%. Table 2 shows the final measured parameters: Table 2

[0035] As can be seen from Table 2, the immunochromatographic test strips of the embodiments can still maintain excellent detection stability after high temperature and high humidity aging treatment. The signal intensity, detection sensitivity and antibody fixation effect are significantly better than those of the respective comparative examples, which fully demonstrates the highly efficient protective effect of the aerogel carrier of this patent on antibodies under extreme environments.

[0036] Comparing Example 1 and Comparative Example 1, it can be seen that the signal retention rate of the immunochromatographic test strip in Comparative Example 1 decreased significantly after aging, the detection limit drifted significantly, and antibody desorption was more severe. This is because the silica aerogel relies only on weak physical interaction to load the antibody. Its surface is hydrophilic and easily invaded by water molecules. It lacks a rigid protective framework and stable anchoring points, and cannot maintain the antibody structure and binding activity under high temperature and high humidity.

[0037] Comparing Example 1 and Comparative Example 2, it can be seen that the immunochromatographic test strip of Comparative Example 2 showed more significant signal attenuation, a larger detection limit drift, and a higher antibody detachment rate after aging. This is because ordinary nanocellulose aerogel has not been modified with crystals, has limited pore structure and adsorption capacity, and relies solely on physical adsorption to fix antibodies. It lacks hydrophobic and moisture-proof and rigid protection mechanisms, making it difficult to resist the damage of antibodies caused by high temperature and high humidity.

[0038] Comparing Example 1 and Comparative Example 3, it can be seen that the immunochromatographic test strip of Comparative Example 3 has the lowest signal retention rate, the most severe detection limit drift, and the highest antibody desorption rate. This is because traditional membrane materials rely on electrostatic adsorption to fix antibodies, which has the weakest binding force and no protective structure. Under high temperature and high humidity, antibodies are easily inactivated and detached, leading to a sharp deterioration in detection performance. Test Example 3

[0039] The immunochromatographic test strips of Example 1 and Comparative Examples 1-3 were tested for performance under normal conditions of 25°C and 45% RH: (1) Limit of detection (LOD): AFP series concentration gradient standards of 0 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL and 100 ng / mL were prepared using PBS buffer. The lowest sample concentration corresponding to the signal-to-noise ratio (SNR) ≥ 3 was determined as the limit of detection (LOD). The test was performed using colloidal gold labeling method. Quantitative scanning and data reading were completed using Biomaxima Reader BM-300 colloidal gold immunochromatographic reader. (2) AFP quantitative linear range: Based on the above gradient standard test results, a log-log coordinate graph was constructed with the ratio of the signal intensity of the detection line to the control line (T / C) as the vertical axis and the standard concentration as the horizontal axis. The linear correlation coefficient R was then calculated. 2 The concentration range corresponding to ≥0.99 was determined as the quantitative linear range of the test strip, and the linear fitting R-value was recorded. 2 ; (3) Intra-batch and inter-batch precision (CV): For intra-batch CV, the same batch of test strips was used, and the test was repeated 10 times with 10 ng / mL AFP standard. The relative standard deviation (RSD) of the detection signal value was calculated. For inter-batch precision CV, three different batches of test strips were used, and each batch was repeatedly tested 5 times with 10 ng / mL AFP standard. A total of 15 sets of valid data were accumulated, and the relative standard deviation (RSD) of the detection signal value between different batches was calculated. Table 3 shows the final measured parameters: Table 3

[0040] As can be seen from Table 3, the immunochromatographic test strips of the examples exhibit the best overall performance under conventional testing conditions, with detection sensitivity, quantitative linear range, linear fit and detection repeatability all significantly better than the respective comparative examples.

[0041] Comparing Example 1 and Comparative Example 1, it can be seen that Comparative Example 1 has lower detection sensitivity, narrower quantitative linear range, and worse linear fitting effect. The intra-batch and inter-batch detection precision is also significantly lower. This is because the specific surface area and pore volume of silica aerogel are limited, the antibody loading and immobilization stability are weak, and it cannot provide sufficient and stable reaction sites for antigen-antibody binding, resulting in lower detection sensitivity and repeatability than the Example.

[0042] Comparing Example 1 and Comparative Example 2, it can be seen that Comparative Example 2 has significantly lower detection sensitivity, narrower quantitative linear range, and worse linear correlation. The fluctuation range of the detection results is larger and the precision is the worst. This is because ordinary nanocellulose aerogel has not been modified by crystals, and its pore structure and adsorption capacity are insufficient. It relies solely on physical adsorption to fix the antibody, resulting in poor antibody loading efficiency and binding stability, making it difficult to achieve highly sensitive, wide linear, and highly stable quantitative detection.

[0043] Comparing Example 1 and Comparative Example 3, it can be seen that the detection sensitivity and linear range of Comparative Example 3 are at a medium level, but the detection repeatability is poor, and the intra-batch and inter-batch precision is not as good as that of Example 1. This is because traditional membrane materials rely on weak electrostatic effects to fix antibodies, which are easy to detach and have difficulty in maintaining stable activity, resulting in large fluctuations in detection signals and poor precision, which cannot achieve the stable detection level of the present invention.

[0044] 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 preparing an interference-resistant trace immunochromatographic test strip, characterized in that, Includes the following steps: S1. Add zinc salt to the TEMPO oxidized nanocellulose suspension and stir to dissolve. Adjust the pH of the system to 7.8-8.2, and then stir and let it stand to obtain a zinc-based nanocellulose suspension. S2. The zinc-based nanocellulose suspension described in S1 is vacuum filtered to form a membrane. After washing, it is vacuum filtered again. The vacuum filtration-washing operation is repeated to obtain a zinc-based nanocellulose membrane. The zinc-based nanocellulose membranes described in S3 and S2 were reacted in a 1,2-dimethylimidazolium methanol solution, and after washing and drying, a ZIF-8 / Zn(OH)2 modified nanocellulose aerogel membrane was obtained. S4. The ZIF-8 / Zn(OH)2 modified nanocellulose aerogel membrane described in S3 is immersed in an antibody solution, and after standing, drying, and shearing, antibody-loaded ZIF-8 / Zn(OH)2 modified nanocellulose aerogel strips are obtained; the antibody solution is an anti-AFP detection antibody solution or a goat anti-mouse IgG antibody solution. S5. The antibody-loaded ZIF-8 / Zn(OH)2 modified nanocellulose aerogel strip described in S4 is pasted onto the chromatography test strip to obtain the anti-interference trace immunochromatographic test strip; the chromatography test strip includes a base plate, a reaction pad, an absorbent pad, a conjugate pad, and a sample pad.

2. The method for preparing the anti-interference trace immunochromatographic test strip according to claim 1, characterized in that, In S1, the solid content of the TEMPO-oxidized cellulose nanoparticle suspension is 0.08wt%-0.12wt%. And / or, the zinc salt is zinc nitrate; And / or, the mass ratio of the TEMPO oxidized nanocellulose suspension to the zinc salt is (38-42):

3.

3. The method for preparing the anti-interference trace immunochromatographic test strip according to claim 1, characterized in that, In S1, the stirring speed is 950 r / min-1050 r / min, and the time is 110-130 min; And / or, the settling time is 55 min-65 min.

4. The method for preparing the anti-interference trace immunochromatographic test strip according to claim 1, characterized in that, In S3, the concentration of the 1,2-dimethylimidazolium methanol solution is 48 mg / mL to 52 mg / mL.

5. The method for preparing the anti-interference trace immunochromatographic test strip according to claim 1, characterized in that, In S3, the reaction temperature is 5℃-35℃ and the time is 11h-13h; And / or, the drying temperature is 55℃-65℃, and the time is 5.5h-6.5h.

6. The method for preparing the anti-interference trace immunochromatographic test strip according to claim 1, characterized in that, In S4, the concentration of the anti-AFP detection antibody solution is 8 mg / mL-9 mg / mL; And / or, the concentration of the goat anti-mouse IgG antibody solution is 1.8 mg / mL to 2.2 mg / mL.

7. The method for preparing the anti-interference trace immunochromatographic test strip according to claim 1, characterized in that, In S4, the impregnation time is 2.5h-3.5h; And / or, the settling time is 20 min to 30 min.

8. The method for preparing the anti-interference trace immunochromatographic test strip according to claim 1, characterized in that, In S5, the binding pad is loaded with a gold-labeled anti-AFP capture antibody solution and a colloidal particle tracer label; the concentration of the gold-labeled anti-AFP capture antibody solution is 0.40 mg / mL-0.45 mg / mL.

9. The method for preparing the anti-interference trace immunochromatographic test strip according to claim 1, characterized in that, In S5, in the anti-interference trace immunochromatographic test strip, the ZIF-8 / Zn(OH)2 modified nanocellulose aerogel strip loaded with anti-AFP detection antibody serves as the detection line; the ZIF-8 / Zn(OH)2 modified nanocellulose aerogel strip loaded with goat anti-mouse IgG antibody serves as the control line.

10. The interference-resistant trace immunochromatographic test strip prepared by the method of any one of claims 1-9.

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