Integrated tumor marker detection membrane based on mesoporous silica

Abstract

The invention provides an integrated tumor marker detection membrane based on mesoporous silica, the detection membrane comprises a mesoporous silica membrane and a substrate, and the mesoporous silica membrane is arranged on the surface of the substrate as a chromatography membrane. The mesoporous silica film is adopted as a detection film, the specific surface area is large, the porosity is high, more colloidal gold particles can be borne, and the detection sensitivity is improved. The mesoporous silica film adopted by the invention is simple in preparation process, low in cost, safe and environment-friendly, required equipment is simple, and expansion and popularization of production and manufacturing are facilitated. According to the invention, a commercial PVDF film or nylon film is used as a substrate, which is low in price, good in biocompatibility, low in biotoxicity, not easy to deform and beneficial to storage and transfer.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor marker detection, and in particular relates to an integrated tumor marker detection membrane based on mesoporous silica. Background Art

[0002] Tumor markers refer to a class of substances that reflect the existence and growth of tumors, which are produced by tumor cells themselves or by the body's response to tumor cells during the occurrence and proliferation of tumors, including proteins, hormones, enzymes, cytosomes, and oncogene products. Some of these substances do not exist in the normal human body and are only found in embryos, while the content of some in tumor patients exceeds that in normal people. In clinical practice, the detection of tumor markers generally uses enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography. Since ELISA requires multiple incubations and a long detection time, it is not suitable for rapid detection. Colloidal gold immunochromatography technology has the advantages of simplicity, rapidity, sensitivity, and on-site operation, which meets the needs of rapid detection such as tumor screening and personal tumor prognosis monitoring.

[0003] Carcinoembryonic antigen (CEA) is synthesized in the small intestine, liver and pancreas during the embryonic period. The serum content of CEA in adults is extremely low (below 2.5 ng / mL). It is mainly used for clinical auxiliary diagnosis of patients with colorectal cancer, gastric cancer, pancreatic cancer, liver cancer, lung cancer, breast cancer and medullary thyroid cancer. Quantitative detection of CEA can significantly improve the sensitivity and accuracy of diagnosing colorectal cancer, and can also be used for the evaluation of the efficacy of colorectal cancer surgery, disease staging and recurrence monitoring.

[0004] Tumor markers commonly used in clinical practice also include cancer antigen 125 (CA125), which is a specific marker for ovarian cancer. In clinical practice, the level of CA125 in the blood is often tested as a criterion for early diagnosis of ovarian cancer. Since other types of tumors such as endometrial cancer, peritoneal cancer, and fallopian tube cancer can also cause an increase in the level of CA125 in the blood, it is often necessary to measure the levels of multiple markers to confirm different tumor types.

[0005] Medical workers have reached a consensus: the best way to improve survival rate is early diagnosis, early detection and early treatment, among which early diagnosis is the most critical. Liver cancer usually progresses slowly and is asymptomatic in the early stages. At present, combining serum alpha-fetoprotein (AFP) detection and liver ultrasound examination is the standard method for monitoring and screening liver cancer. The serum AFP content of normal adults is about 10ng / mL, while the serum AFP value of liver cancer patients can be as high as 500ng / mL. Similarly, pancreatic cancer treatment is highly dependent on the accuracy of early diagnosis. To this end, detecting the content of its preferred tumor marker CA19-9 and its secondary marker CA125 can provide effective judgment for pancreatic cancer, and gastric cancer can be effectively diagnosed early through the preferred tumor marker CA72-4 and the secondary marker CEA. In addition, uterine cancer, which poses a great threat to women's health, can also be effectively judged by comprehensive detection of the content of tumor markers CA125 and CA19-9. Ovarian cancer is detected by detecting CA125 and CA72-4.

[0006] Bladder cancer is a common urogenital tumor in clinical practice. The tumor markers used for the diagnosis of bladder cancer are mainly bladder tumor antigen (BTA) and nuclear matrix protein 22 (NMP22) in urine. Prostate-specific antigen (PSA) is a 237-amino acid protease secreted by prostate epithelial cells and periurethral tissues. It exists in free and bound forms and is a clinically recognized tumor marker for prostate cancer. Testicular choriocarcinoma is a common malignant tumor in urology. Its main feature is trophoblast differentiation. The detection of human chorionic gonadotropin (β-hCG) levels is helpful for the diagnosis and prognosis of testicular choriocarcinoma. Human calcitonin (hCT) is a peptide hormone secreted by parathyroid follicular C cells. It is a specific and sensitive tumor marker for the diagnosis and monitoring of medullary thyroid carcinoma (C cell carcinoma).

[0007] Chinese patent document CN108918894A discloses an integrated multi-tumor early diagnosis detection membrane, which is a gold-labeled test paper that can simultaneously detect tumor markers of alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA125, CA19-9 and CA72-4. Specifically, the detection membrane integrates the left-handed polylactic acid / polyethylene oxide polymer membrane on an aluminum foil support substrate by electrostatic spinning technology, including a sample area, a filtration area, a colloidal gold area, a detection area and a water absorption area that are sequentially connected on the support substrate. A plurality of detection strips and a quality control line are provided in the detection area, and a colloidal gold antibody conjugate is contained in the colloidal gold area. The colloidal gold antibody is a color-developing antibody, and the detection strip is composed of a plurality of line segments immobilized with different capture antibodies. The color-developing antibody and the capture antibody are antibodies that can form a double antibody sandwich pairing. Through the detection of a variety of tumor markers in human body fluids, the detection of liver cancer, pancreatic cancer, gastric cancer, colorectal cancer, uterine cancer, and ovarian cancer is achieved. However, due to the high price of L-polylactic acid, the cost of the prepared L-polylactic acid / polyethylene oxide polymer film is high. Moreover, since the aluminum foil supporting substrate is easily deformed, it is not conducive to the preservation and transfer of the detection membrane. At the same time, it is difficult to ensure that the substrate deformation causes uneven diffusion of the liquid to be tested, which in turn brings about detection deviation, which also affects the accuracy of the detection membrane. In addition, organic solvents need to be used in the electrospinning process, which is easy to cause pollution, and the production process is complicated and time-consuming. Summary of the invention

[0008] In order to improve the deficiencies of the prior art, the present invention provides an integrated tumor marker detection membrane based on mesoporous silica. The present invention uses a mesoporous silica membrane as a detection membrane, which has the characteristics of large specific surface area, high porosity, and can carry more colloidal gold particles. The use of the detection membrane can greatly improve the detection sensitivity of the existing tumor marker detection membrane. The detection membrane has low preparation cost, simple manufacturing process, high stability, safety, fastness, not easy to deform, and easy storage and transfer.

[0009] The present invention is achieved through the following technical solutions:

[0010] The invention discloses a use of a mesoporous silica membrane as a chromatography membrane.

[0011] According to an embodiment of the present invention, the chromatographic membrane is a chromatographic membrane in a detection membrane.

[0012] According to an embodiment of the present invention, the chromatographic membrane is a chromatographic membrane used in a tumor marker detection membrane.

[0013] An integrated tumor marker detection membrane comprises a mesoporous silica membrane and a substrate, wherein the mesoporous silica membrane is arranged on the surface of the substrate as a chromatographic membrane.

[0014] According to an embodiment of the present invention, the mesoporous silica membrane is provided with a sample region to be tested, a filtration region, a colloidal gold region, a detection region and a terminal region which are connected in sequence.

[0015] According to an embodiment of the present invention, the thickness of the mesoporous silica film is 50-500 nm.

[0016] According to an embodiment of the present invention, the porosity of the mesoporous silica film is 0.990-0.995.

[0017] According to an embodiment of the present invention, the specific surface area of ​​the mesoporous silica film is 600-900m 2 / g.

[0018] According to an embodiment of the present invention, the pore volume of the mesoporous silica membrane is 0.4-0.55 cm 3 / g.

[0019] According to an embodiment of the present invention, the pore size of the mesoporous silica membrane is 2-50 nm.

[0020] According to an embodiment of the present invention, the contact angle between the mesoporous silica film and water is 0°.

[0021] According to an embodiment of the present invention, bovine serum albumin (BSA) is loaded on the surface of the mesoporous silica membrane.

[0022] According to an embodiment of the present invention, the substrate is selected from a polyvinylidene fluoride substrate or a nylon substrate.

[0023] According to an embodiment of the present invention, a glue layer is further included between the mesoporous silica film and the substrate.

[0024] According to an embodiment of the present invention, the adhesive layer includes at least one of adhesives such as polyurethane, epoxy resin, and acrylate.

[0025] In one embodiment, the sample region to be tested includes a mesoporous silica film. Exemplarily, the sample region to be tested is a mesoporous silica film.

[0026] In one embodiment, the filtration region comprises a mesoporous silica membrane. Exemplarily, the filtration region is a mesoporous silica membrane.

[0027] In one embodiment, the colloidal gold region comprises a gold-labeled antibody and a mesoporous silica membrane, wherein the gold-labeled antibody is bound to the surface of the mesoporous silica membrane, preferably to the surface and pores of the mesoporous silica.

[0028] In one embodiment, the gold-labeled antibody is a colloidal gold-first antibody conjugate, and the colloidal gold-first antibody conjugate is a conjugate in which colloidal gold and the first antibody are connected by a covalent bond.

[0029] In one embodiment, the colloidal gold surface has a hydrophilic functionalized layer, and the first antibody is covalently linked to the colloidal gold through the hydrophilic functionalized layer. The hydrophilic functionalized layer on the colloidal gold surface contains a carboxylic acid group, which can be coupled to the amino group, alcohol group and other reactive groups on the surface of the first antibody through the carboxylic acid group. Exemplarily, the hydrophilic functionalized layer on the colloidal gold surface is a citric acid layer.

[0030] In one embodiment, the first antibody is an anti-human tumor marker antibody, such as at least one of a mouse anti-human tumor marker antibody, a guinea pig anti-human tumor marker antibody, a rat anti-human tumor marker antibody, and a rabbit anti-human tumor marker antibody.

[0031] In one embodiment, the loading amount of the gold-labeled antibody in the colloidal gold region is 8-20 pL / cm 2 .

[0032] In one embodiment, the detection area is provided with a plurality of detection strips and a quality control line; the plurality of detection strips include a second antibody and a mesoporous silica membrane, the second antibody is bound to the surface of the mesoporous silica membrane, preferably to the surface and pores of the mesoporous silica; the quality control line includes a third antibody and a mesoporous silica membrane, the third antibody is bound to the surface of the mesoporous silica membrane, preferably to the surface and pores of the mesoporous silica.

[0033] In one embodiment, the second antibody is an anti-human tumor marker antibody, such as at least one of a mouse anti-human tumor marker antibody, a guinea pig anti-human tumor marker antibody, a rat anti-human tumor marker antibody, and a rabbit anti-human tumor marker antibody.

[0034] In one embodiment, the third antibody is a specific antibody of the first antibody and the second antibody, such as at least one of anti-mouse IgG antibody, anti-rat IgG antibody, anti-guinea pig IgG antibody and anti-rabbit IgG antibody.

[0035] In one embodiment, the loading amount of the second antibody in the detection area is 0.1-10 μL / mm 2 .

[0036] In one embodiment, the loading amount of the third antibody in the detection area is 0.1-10 μL / mm 2 .

[0037] In one embodiment, the detection strip is close to the colloidal gold area, and the quality control line is close to the end area.

[0038] In one embodiment, the plurality of detection strips are sequentially spaced apart along the length direction of the mesoporous silica membrane.

[0039] In one embodiment, the detection strip includes at least two detection strip segments.

[0040] In one embodiment, the detection area is provided with a first detection strip, a second detection strip and a quality control line; the two detection strip segments arranged on the first detection strip are detection strip segment A and detection strip segment B from left to right; the two detection strip segments arranged on the second detection strip are detection strip segment C and detection strip segment D from left to right; at least three of the four detection strip segments include a second antibody.

[0041] In one embodiment, the detection area is provided with a first detection strip, a second detection strip, a third detection strip and a quality control line; the two detection strip segments arranged on the first detection strip are detection strip segment E and detection strip segment F from left to right; the two detection strip segments arranged on the second detection strip are detection strip segment G and detection strip segment H from left to right; the two detection strip segments arranged on the third detection strip are detection strip segment I and detection strip segment J from left to right; at least five of the six detection strip segments include a second antibody.

[0042] In one embodiment, the terminal region comprises a mesoporous silica film. Exemplarily, the terminal region is a mesoporous silica film.

[0043] Beneficial effects of the present invention:

[0044] The present invention adopts a mesoporous silica membrane as a detection membrane, which has a large specific surface area and a high porosity, can carry more colloidal gold particles, and improves the detection sensitivity. The mesoporous silica membrane used in the present invention has a simple preparation process, low cost, safety, environmental friendliness, simple equipment, and is conducive to the expansion and popularization of production and manufacturing. The present invention adopts a commercial PVDF membrane or a nylon membrane as a substrate, which is cheap, has good biocompatibility, low biotoxicity, is not easy to deform, and is conducive to storage and transfer. The detection membrane of the present invention does not contain a multilayer structure, which overcomes the influence of inconsistent interlayer contact quality control on the detection stability. The change of the multilayer structure on the color reaction efficiency in the prior art is reduced, and accurate quantitative detection data is obtained. The mesoporous silica membrane of the present invention arranges multiple color development detection strips in parallel, which overcomes the error caused by the concentration change of the detection liquid during the diffusion process. The present invention is designed using colloidal gold technology and chromatography principles, and uses the principle of double antibody sandwich method to design detection strips to detect the presence of tumor markers. The detection membrane of the present invention has a filtering area, which can remove body fluid samples, including other components of serum, saliva, urine, etc., improve the selectivity of the combination of antigen and colloidal gold antibody conjugate, and further improve the detection efficiency. The colloidal gold and the first antibody conjugate of the present invention is formed by covalently connecting the carboxylic acid group on the surface of the colloidal gold nanoparticles with the first antibody. The formed colloidal gold-first antibody conjugate cannot be reversibly separated, has excellent stability, and can maintain the ability to specifically bind to the antigen for a long time. The conjugates of various colloidal gold and different antibodies provided by the present invention (i.e., multi-gold-labeled antibodies) are used in combination with the multi-gold-labeled antibodies and their corresponding monoclonal antibody strips, and the content of various tumor markers is reflected through a specific color development reaction, thereby comprehensively detecting the content of two or more specific types of tumor markers, and significantly improving the accuracy of tumor diagnosis. The present invention can simultaneously detect multiple tumor markers according to age, industry, and region (including living habits and eating habits), focusing on the detection of liver cancer, pancreatic cancer, colorectal cancer, gastric cancer, uterine cancer, and ovarian cancer. Moreover, targeted detection chromatographic membranes can be prepared according to different tumor types to expand the scope of early tumor detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a schematic diagram of the structure of a detection membrane according to a preferred embodiment of the present invention.

[0046] Figure 2 This is a schematic diagram of the structure of a detection membrane according to a preferred embodiment of the present invention.

[0047] Figure 3 This is a schematic diagram of the structure of a detection membrane according to a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0048] <Application of mesoporous silica membrane as chromatography membrane>

[0049] As mentioned above, the present invention provides a use of a mesoporous silica membrane as a chromatographic membrane.

[0050] According to an embodiment of the present invention, the chromatographic membrane is a chromatographic membrane in a detection membrane.

[0051] According to an embodiment of the present invention, the chromatographic membrane is a chromatographic membrane used in a tumor marker detection membrane.

[0052] <Integrated Tumor Marker Detection Membrane>

[0053] As mentioned above, the present invention provides an integrated tumor marker detection membrane, which comprises a mesoporous silica membrane and a substrate, wherein the mesoporous silica membrane is disposed on the surface of the substrate as a chromatographic membrane.

[0054] According to an embodiment of the present invention, the mesoporous silica membrane is provided with a sample region to be tested, a filtration region, a colloidal gold region, a detection region and a terminal region which are connected in sequence.

[0055] <Mesoporous Silica Film>

[0056] According to an embodiment of the present invention, the thickness of the mesoporous silica film is 50-500nm, for example, 100-300nm, such as 180-220nm, and illustratively, it is 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm or 300nm.

[0057] According to an embodiment of the present invention, the porosity of the mesoporous silica membrane is 0.990-0.995, for example, 0.990, 0.991, 0.992, 0.993, 0.994 or 0.995.

[0058] According to an embodiment of the present invention, the specific surface area of ​​the mesoporous silica film is 600-900m 2 / g, for example, 600m 2 / g, 620m 2 / g, 650m 2 / g, 680m 2 / g、700m 2 / g, 720m 2 / g, 750m 2 / g, 780m 2 / g、800m 2 / g, 820m2 / g, 834m 2 / g, 850m 2 / g, 880m 2 / g or 900m 2 / g.

[0059] According to an embodiment of the present invention, the pore volume of the mesoporous silica membrane is 0.4-0.55 cm 3 / g, for example 0.4cm 3 / g, 0.42cm 3 / g, 0.45cm 3 / g, 0.48cm 3 / g, 0.49cm 3 / g, 0.5cm 3 / g, 0.52cm 3 / g or 0.55cm 3 / g.

[0060] According to an embodiment of the present invention, the pore size of the mesoporous silica membrane is 2-50 nm, for example, 2-25 nm, such as 2-10 nm, for example, 2 nm, 3 nm, 4 nm, 5 nm or 8 nm.

[0061] According to an embodiment of the present invention, the mesoporous silica film includes mesoporous silica particles; preferably, the mesoporous silica film is a film formed by mesoporous silica particles; for example, the mesoporous silica film is a film formed by spontaneous assembly of mesoporous silica particles.

[0062] According to an embodiment of the present invention, the particle size of the mesoporous silica particles is 50-100 nm, for example, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm or 100 nm.

[0063] According to an embodiment of the present invention, the contact angle between the mesoporous silica film and water is 0°, which indicates that the mesoporous silica film has good hydrophilicity and can achieve the effect of being completely wetted by water.

[0064] According to an embodiment of the present invention, the surface of the mesoporous silica membrane is loaded with bovine serum albumin (BSA), which indicates that the mesoporous silica membrane has good biocompatibility.

[0065] <Base>

[0066] According to an embodiment of the present invention, the substrate is selected from a polyvinylidene fluoride substrate or a nylon substrate.

[0067] According to an embodiment of the present invention, the thickness of the substrate is 0.5-3 mm, for example, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm or 3 mm.

[0068] According to an embodiment of the present invention, the substrate can provide mechanical support for the mesoporous silica membrane, ensuring that the obtained detection membrane is not easily deformed, so as to facilitate storage and transfer.

[0069] <Glue layer>

[0070] According to an embodiment of the present invention, a glue layer is further included between the mesoporous silica film and the substrate.

[0071] According to an embodiment of the present invention, the thickness of the glue layer is 2-20 nm, for example, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 12 nm, 15 nm, 18 nm or 20 nm.

[0072] According to an embodiment of the present invention, the adhesive layer includes at least one of adhesives such as polyurethane, epoxy resin, and acrylate.

[0073] <Method for preparing mesoporous silica film>

[0074] According to an embodiment of the present invention, the mesoporous silica film can be prepared by the following preparation methods: chemical vapor deposition, physical vapor deposition, sol-gel method, liquid phase deposition method or thermal oxidation method.

[0075] According to an embodiment of the present invention, exemplarily, the mesoporous silica film is formed by The solution was prepared.

[0076] According to an embodiment of the present invention, the mesoporous silica film is prepared by the following method:

[0077] 1) mixing alkyl orthosilicate, a template, a solvent and an alkaline substance to form a reaction system;

[0078] 2) immersing the substrate in the reaction system of step 1) and allowing it to stand for self-assembly reaction to form a templated silicon dioxide film on the surface of the substrate;

[0079] 3) cleaning, drying and aging the templated silicon dioxide film of step 2);

[0080] 4) placing the dried and aged membrane in an acidic solution to prepare the mesoporous silica membrane.

[0081] According to an embodiment of the present invention, in step 1), the alkyl orthosilicate is selected from at least one of tetraethyl orthosilicate (TEOS), methyl orthosilicate and propyl orthosilicate.

[0082] According to an embodiment of the present invention, in step 1), the template is selected from cetyltrimethylammonium bromide (CTAB).

[0083] According to an embodiment of the present invention, in step 1), the solvent is selected from a combination of water and an alcohol solvent, and the alcohol solvent is selected from ethanol, methanol or propanol, preferably a combination of water and ethanol.

[0084] According to an embodiment of the present invention, in step 1), the alkaline substance is selected from any one or more of ammonia water, sodium hydroxide, potassium hydroxide, ammonium fluoride, ammonium bicarbonate, sodium carbonate, sodium bicarbonate, ethanolamine, diethanolamine, methylamine, dimethylamine, ethylamine, diethylamine, propylamine, dipropylamine, isopropanolamine, aniline, o-phenylenediamine, m-phenylenediamine and p-phenylenediamine.

[0085] According to an embodiment of the present invention, in step 1), the mass ratio of the alkyl orthosilicate to the template is 1-3:5, for example, 1:5, 1.5:5, 2:5 or 3:5.

[0086] According to an embodiment of the present invention, in step 1), the mass ratio of the alkyl orthosilicate to the solvent is 1:1000-2000, for example, 1:1000, 1:1100, 1:1200, 1:1260, 1:1300, 1:1340, 1:1400, 1:1450, 1:1500, 1:1600, 1:1700, 1:1800, 1:1900 or 1:2000.

[0087] According to an embodiment of the present invention, in step 1), the mass ratio of the alkyl orthosilicate to the alkaline substance is 6-12:1, for example, 6:1, 7:1, 8:1, 8.5:1, 9:1, 9.4:1, 9.5:1, 9.8:1, 10:1, 11:1, or 12:1.

[0088] According to an embodiment of the present invention, step 1) may specifically include the following steps: mixing a template, a solvent and an alkaline substance; and then adding an alkyl orthosilicate to the mixed system under stirring to form a reaction system.

[0089] According to an embodiment of the present invention, in step 2), the standing temperature is 50-80°C, for example, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C or 80°C; the standing time is 24-96 hours, for example, 24 hours, 36 hours, 48 ​​hours, 72 hours or 96 hours.

[0090] According to an embodiment of the present invention, in step 2), during the standing process, the alkyl orthosilicate undergoes a hydrolysis reaction to form mesoporous silica particles.

[0091] According to an embodiment of the present invention, in step 2), the substrate is preferably cleaned before use, for example by soaking it in a sodium hydroxide solution at room temperature for 10 hours to clean organic residues, and then rinsed with deionized water before use.

[0092] According to an embodiment of the present invention, in step 3), the cleaning is, for example, gently washing the templated silicon dioxide film formed on the substrate with running water. The purpose of the cleaning is to remove other unreacted substances on the surface and the template agent in the silicon dioxide film.

[0093] According to an embodiment of the present invention, in step 3), the drying and aging is, for example, drying in an oven at 80-120° C. for 8-24 hours.

[0094] According to an embodiment of the present invention, in step 4), the acidic solution is, for example, an alcohol solution containing an acid, preferably an ethanol solution containing hydrochloric acid. Treatment with an acidic solution is beneficial for peeling the mesoporous silica film off the substrate.

[0095] According to an embodiment of the present invention, a mesoporous silica membrane loaded with bovine serum albumin (BSA) on the surface is prepared by the following method:

[0096] The mesoporous silica membrane is placed in a prefabricated treatment solution and soaked for a period of time, and then taken out and dried; wherein the prefabricated treatment solution includes bovine serum albumin (BSA) and phosphate buffer.

[0097] According to an embodiment of the present invention, the concentration of bovine serum albumin (BSA) in the prefabricated treatment solution is 0.2-1 wt %; and the pH of the phosphate buffer in the prefabricated treatment solution is 7.4.

[0098] <Sample area to be tested>

[0099] In one embodiment, the width of the sample area to be tested is 0.5-1.5 cm, preferably 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm or 1.5 cm.

[0100] In one embodiment, the length of the sample area to be tested is 0.5-1.5 cm, preferably 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm or 1.5 cm.

[0101] In one embodiment, the sample region to be tested includes a mesoporous silica film. Exemplarily, the sample region to be tested is a mesoporous silica film.

[0102] In one embodiment, the mesoporous silica membrane has the characteristics of integration and uniformity. The mesoporous silica membrane can be used as a chromatographic membrane to transport the sample to be tested dropped into the sample area through chromatography (capillary action) and pass through the filtration area, colloidal gold area, detection area and terminal area to achieve detection of the sample to be tested.

[0103] <Filter Area>

[0104] In one embodiment, the width of the filtration area is 0.5-1.5 cm, preferably 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm or 1.5 cm.

[0105] In one embodiment, the length of the filtration area is 0.2-0.8 cm, preferably 0.2 cm, 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm or 0.8 cm.

[0106] In one embodiment, the filtration region comprises a mesoporous silica membrane. Exemplarily, the filtration region is a mesoporous silica membrane.

[0107] Since the sample to be tested is a body fluid, which usually includes serum, saliva, urine, etc., there are other components in addition to antigen proteins, such as blood cells, epithelial cell residues in saliva, and cytosomes in urine. These components are easy to bind to the gold-labeled antibody, interfering with the detection of the antigen, and must be removed. The mesoporous silica membrane in the filter area between the colloidal gold area and the sample area to be tested can play a role in filtering large-volume substances such as cells in the sample; and the sample to be tested added to the sample area to be tested has different paths through the mesoporous silica membrane, especially the part of the sample to be tested close to the filter area has not been fully filtered. Therefore, the setting of the filter area can ensure the filtering effect and improve the detection specificity.

[0108] <Colloidal Gold Area>

[0109] In one embodiment, the width of the colloidal gold area is 0.5-1.5 cm, preferably 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm or 1.5 cm.

[0110] In one embodiment, the length of the colloidal gold region is 0.3-1.5 cm, preferably 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm or 1.5 cm.

[0111] In one embodiment, the colloidal gold region comprises a gold-labeled antibody and a mesoporous silica membrane, wherein the gold-labeled antibody is bound to the surface of the mesoporous silica membrane, preferably to the surface and pores of the mesoporous silica.

[0112] In one embodiment, the gold-labeled antibody is a colloidal gold-first antibody conjugate, and the colloidal gold-first antibody conjugate is a conjugate in which colloidal gold and the first antibody are connected by a covalent bond.

[0113] In one embodiment, the colloidal gold solution is mixed with the first antibody solution, and the first antibody is covalently immobilized on the surface of the colloidal gold under the action of a coupling agent to form the colloidal gold-first antibody conjugate.

[0114] In one embodiment, the colloidal gold surface has a hydrophilic functionalized layer, and the first antibody is covalently linked to the colloidal gold through the hydrophilic functionalized layer. The hydrophilic functionalized layer on the colloidal gold surface contains a carboxylic acid group, which can be coupled to the amino group, alcohol group and other reactive groups on the surface of the first antibody through the carboxylic acid group. Exemplarily, the hydrophilic functionalized layer on the colloidal gold surface is a citric acid layer.

[0115] In one embodiment, the diameter of the colloidal gold particles is 20-80 nm, preferably 20-60 nm, and more preferably 30-50 nm, for example, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm or 80 nm.

[0116] In one embodiment, the first antibody is an anti-human tumor marker antibody, such as at least one of a mouse anti-human tumor marker antibody, a guinea pig anti-human tumor marker antibody, a rat anti-human tumor marker antibody, and a rabbit anti-human tumor marker antibody.

[0117] Exemplarily, the first antibody is a mouse anti-human carcinoembryonic antigen monoclonal antibody, a mouse anti-human alpha-fetoprotein monoclonal antibody, a mouse anti-human CA125 monoclonal antibody, a mouse anti-human CA19-9 monoclonal antibody, a mouse anti-human CA72-4 monoclonal antibody, a mouse anti-human CA15-3 monoclonal antibody, a mouse anti-human bladder tumor antigen monoclonal antibody, a mouse anti-human nuclear matrix protein 22 monoclonal antibody, a mouse anti-human prostate specific antigen monoclonal antibody, a mouse anti-human chorionic gonadotropin monoclonal antibody and a mouse Anti-human calcitonin monoclonal antibody, rat anti-human carcinoembryonic antigen monoclonal antibody, rat anti-human alpha-fetoprotein monoclonal antibody, rat anti-human CA125 monoclonal antibody, rat anti-human CA19-9 monoclonal antibody, rat anti-human CA72-4 monoclonal antibody, rat anti-human CA15-3 monoclonal antibody, rat anti-human bladder tumor antigen monoclonal antibody, rat anti-human nuclear matrix protein 22 monoclonal antibody, rat anti-human prostate specific antigen monoclonal antibody, rat anti-human chorionic gonadotropin monoclonal antibody, rat anti-human Calcitonin monoclonal antibody, guinea pig anti-human carcinoembryonic antigen monoclonal antibody, guinea pig anti-human alpha-fetoprotein monoclonal antibody, guinea pig anti-human CA125 monoclonal antibody, guinea pig anti-human CA19-9 monoclonal antibody, guinea pig anti-human CA72-4 monoclonal antibody, guinea pig anti-human CA15-3 monoclonal antibody, guinea pig anti-human bladder tumor antigen monoclonal antibody, guinea pig anti-human nuclear matrix protein 22 monoclonal antibody, guinea pig anti-human prostate specific antigen monoclonal antibody, guinea pig anti-human chorionic gonadotropin monoclonal antibody, guinea pig anti-human calcitonin At least one of rabbit anti-human calcitonin monoclonal antibody, rabbit anti-human carcinoembryonic antigen monoclonal antibody, rabbit anti-human alpha-fetoprotein monoclonal antibody, rabbit anti-human CA125 monoclonal antibody, rabbit anti-human CA19-9 monoclonal antibody, rabbit anti-human CA72-4 monoclonal antibody, rabbit anti-human CA15-3 monoclonal antibody, rabbit anti-human bladder tumor antigen monoclonal antibody, rabbit anti-human nuclear matrix protein 22 monoclonal antibody, rabbit anti-human prostate specific antigen monoclonal antibody, rabbit anti-human chorionic gonadotropin monoclonal antibody and rabbit anti-human calcitonin monoclonal antibody.

[0118] In one embodiment, the loading amount of the gold-labeled antibody in the colloidal gold region is 8-20 pL / cm 2 , for example 8pL / cm 2 、10pL / cm 2 , 12pL / cm 2 、15pL / cm 2 、18pL / cm 2 or 20pL / cm 2 .

[0119] In one embodiment, the gold-labeled antibody is coated on the surface area of ​​the mesoporous silica membrane to form a colloidal gold area. The coating is, for example, sprayed, illustratively, sprayed using a film sprayer. The sprayed gold-labeled antibody is uniformly bound to the surface of the mesoporous silica membrane, preferably to the surface and pores of the mesoporous silica.

[0120] In one embodiment, the gold-labeled antibody is dissolved in a boric acid buffer at pH 8 and then sprayed onto the surface area of ​​the mesoporous silica membrane to form a colloidal gold area.

[0121] In one embodiment, a gold-labeled antibody is sprayed on the colloidal gold region using a film sprayer and embedded on the surface and pores of the mesoporous silica to form a colloidal gold region. Due to the superior high specific surface area and porosity of mesoporous silica, more colloidal gold particles can be stably loaded, thereby improving detection sensitivity and reducing detection limits.

[0122] <Detection Area>

[0123] In one embodiment, the width of the detection area is 0.5-1.5 cm, preferably 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm or 1.5 cm.

[0124] In one embodiment, the length of the detection area is 0.8-3.2 cm, preferably 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm, 1.5 cm, 1.6 cm, 1.7 cm, 1.8 cm, 1.9 cm, 2 cm, 2.1 cm, 2.2 cm, 2.3 cm, 2.4 cm, 2.5 cm, 2.6 cm, 2.7 cm, 2.8 cm, 2.9 cm, 3.0 cm or 3.2 cm.

[0125] In one embodiment, the detection area is provided with a plurality of detection strips and a quality control line; the plurality of detection strips include a second antibody and a mesoporous silica membrane, the second antibody is bound to the surface of the mesoporous silica membrane, preferably to the surface and pores of the mesoporous silica; the quality control line includes a third antibody and a mesoporous silica membrane, the third antibody is bound to the surface of the mesoporous silica membrane, preferably to the surface and pores of the mesoporous silica.

[0126] In one embodiment, the second antibodies on different detection strips are the same or different, preferably different.

[0127] In one embodiment, the detection strip is close to the colloidal gold area, and the quality control line is close to the end area.

[0128] In one embodiment, the loading amount of the second antibody in the detection area is 0.1-10 μL / mm 2 , for example 0.1 μL / mm 2 , 0.2μL / mm 2 , 0.5μL / mm 2 , 1μL / mm 2 , 2μL / mm 2 , 5μL / mm 2 or 10μL / mm 2 .

[0129] In one embodiment, the second antibody is coated on the surface area of ​​the mesoporous silica membrane to form a plurality of detection strips. The coating is, for example, sprayed, illustratively, sprayed using a film sprayer. The sprayed second antibody is uniformly bound to the surface of the mesoporous silica membrane, preferably to the surface and pores of the mesoporous silica.

[0130] In one embodiment, the loading amount of the third antibody in the detection area is 0.1-10 μL / mm 2 , for example 0.1 μL / mm 2 , 0.2μL / mm 2 , 0.5μL / mm 2 , 1μL / mm 2 , 2μL / mm 2 , 5μL / mm 2 or 10μL / mm 2 .

[0131] In one embodiment, the third antibody is coated on the surface area of ​​the mesoporous silica membrane to form a quality control line. The coating is, for example, sprayed, illustratively, sprayed using a film sprayer. The sprayed third antibody is uniformly bound to the surface of the mesoporous silica membrane, preferably to the surface and pores of the mesoporous silica.

[0132] In one embodiment, the second antibody is an anti-human tumor marker antibody, such as at least one of a mouse anti-human tumor marker antibody, a guinea pig anti-human tumor marker antibody, a rat anti-human tumor marker antibody, and a rabbit anti-human tumor marker antibody.

[0133] Exemplarily, the second antibody is a mouse anti-human carcinoembryonic antigen monoclonal antibody, a mouse anti-human alpha-fetoprotein monoclonal antibody, a mouse anti-human CA125 monoclonal antibody, a mouse anti-human CA19-9 monoclonal antibody, a mouse anti-human CA72-4 monoclonal antibody, a mouse anti-human CA15-3 monoclonal antibody, a mouse anti-human bladder tumor antigen monoclonal antibody, a mouse anti-human nuclear matrix protein 22 monoclonal antibody, a mouse anti-human prostate specific antigen monoclonal antibody, a mouse anti-human chorionic gonadotropin monoclonal antibody and a mouse Anti-human calcitonin monoclonal antibody, rat anti-human carcinoembryonic antigen monoclonal antibody, rat anti-human alpha-fetoprotein monoclonal antibody, rat anti-human CA125 monoclonal antibody, rat anti-human CA19-9 monoclonal antibody, rat anti-human CA72-4 monoclonal antibody, rat anti-human CA15-3 monoclonal antibody, rat anti-human bladder tumor antigen monoclonal antibody, rat anti-human nuclear matrix protein 22 monoclonal antibody, rat anti-human prostate specific antigen monoclonal antibody, rat anti-human chorionic gonadotropin monoclonal antibody, rat anti-human Calcitonin monoclonal antibody, guinea pig anti-human carcinoembryonic antigen monoclonal antibody, guinea pig anti-human alpha-fetoprotein monoclonal antibody, guinea pig anti-human CA125 monoclonal antibody, guinea pig anti-human CA19-9 monoclonal antibody, guinea pig anti-human CA72-4 monoclonal antibody, guinea pig anti-human CA15-3 monoclonal antibody, guinea pig anti-human bladder tumor antigen monoclonal antibody, guinea pig anti-human nuclear matrix protein 22 monoclonal antibody, guinea pig anti-human prostate specific antigen monoclonal antibody, guinea pig anti-human chorionic gonadotropin monoclonal antibody, guinea pig anti-human calcitonin At least one of rabbit anti-human calcitonin monoclonal antibody, rabbit anti-human carcinoembryonic antigen monoclonal antibody, rabbit anti-human alpha-fetoprotein monoclonal antibody, rabbit anti-human CA125 monoclonal antibody, rabbit anti-human CA19-9 monoclonal antibody, rabbit anti-human CA72-4 monoclonal antibody, rabbit anti-human CA15-3 monoclonal antibody, rabbit anti-human bladder tumor antigen monoclonal antibody, rabbit anti-human nuclear matrix protein 22 monoclonal antibody, rabbit anti-human prostate specific antigen monoclonal antibody, rabbit anti-human chorionic gonadotropin monoclonal antibody and rabbit anti-human calcitonin monoclonal antibody.

[0134] In one embodiment, the first antibody and the second antibody are preferably different antibodies against the same tumor marker, such as antibodies against different antigenic determinants of the same tumor marker.

[0135] In one embodiment, the third antibody is a specific antibody of the first antibody and the second antibody, such as at least one of anti-mouse IgG antibody, anti-rat IgG antibody, anti-guinea pig IgG antibody and anti-rabbit IgG antibody; for example, at least one of goat anti-mouse IgG monoclonal antibody, rat anti-mouse IgG monoclonal antibody, guinea pig anti-mouse IgG monoclonal antibody, rabbit anti-mouse IgG monoclonal antibody, goat anti-rat IgG monoclonal antibody, mouse anti-rat IgG monoclonal antibody, guinea pig anti-rat IgG monoclonal antibody, rabbit anti-rat IgG monoclonal antibody, goat anti-guinea pig IgG monoclonal antibody, mouse anti-guinea pig IgG monoclonal antibody, rat anti-guinea pig IgG monoclonal antibody, rabbit anti-guinea pig IgG monoclonal antibody, goat anti-rabbit IgG monoclonal antibody, rat anti-rabbit IgG monoclonal antibody, guinea pig anti-rabbit IgG monoclonal antibody and mouse anti-rabbit IgG monoclonal antibody.

[0136] Exemplarily, when the first antibody and the second antibody are selected from mouse anti-human tumor marker antibodies, the third antibody is selected from anti-mouse IgG antibodies; when the first antibody and the second antibody are selected from guinea pig anti-human tumor marker antibodies, the third antibody is selected from anti-guinea pig IgG antibodies; when the first antibody and the second antibody are selected from rat anti-human tumor marker antibodies, the third antibody is selected from anti-rat IgG antibodies; when the first antibody and the second antibody are selected from rabbit anti-human tumor marker antibodies, the third antibody is selected from anti-rat IgG antibodies.

[0137] In one embodiment, the plurality of detection strips are sequentially spaced apart along the length direction of the mesoporous silica membrane, with a spacing of 0.5-5 mm, preferably 1-3 mm, for example, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 4 mm or 5 mm.

[0138] In one embodiment, the width of the detection strip along the length direction of the mesoporous silica membrane is 0.5-5 mm, preferably 1-3 mm, for example, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 4 mm or 5 mm.

[0139] In one embodiment, the width of the quality control line along the length direction of the mesoporous silica membrane is 0.5-5 mm, preferably 1-3 mm, for example, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 4 mm or 5 mm.

[0140] In one embodiment, the distance between the quality control line and the detection strip is 0.5-5 mm, preferably 1-3 mm, for example, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 4 mm or 5 mm.

[0141] In one embodiment, the detection strip includes at least two detection strip segments.

[0142] In one embodiment, the second antibodies on different test strip segments are the same or different, preferably different.

[0143] In one embodiment, the distance between adjacent detection strip segments is 0.5-5 mm, preferably 1-3 mm, for example 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 4 mm or 5 mm.

[0144] In one embodiment, the length of the detection strip segment along the width direction of the mesoporous silica membrane is 1-5 mm, preferably 1-4 mm, for example, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 4 mm or 5 mm.

[0145] In one embodiment, the detection area is provided with a first detection strip, a second detection strip and a quality control line; the two detection strip segments arranged on the first detection strip are detection strip segment A and detection strip segment B from left to right; the two detection strip segments arranged on the second detection strip are detection strip segment C and detection strip segment D from left to right; at least three of the four detection strip segments include a second antibody. Exemplarily, the four detection strip segments include four different second antibodies. Exemplarily, one of the four detection strip segments is a blank detection strip segment, and the remaining three detection strip segments include three different second antibodies.

[0146] In one embodiment, the detection area is provided with a first detection strip, a second detection strip, a third detection strip and a quality control line; the two detection strip segments arranged on the first detection strip are detection strip segment E and detection strip segment F from left to right; the two detection strip segments arranged on the second detection strip are detection strip segment G and detection strip segment H from left to right; the two detection strip segments arranged on the third detection strip are detection strip segment I and detection strip segment J from left to right; at least five of the six detection strip segments include a second antibody. Exemplarily, the six detection strip segments include six different second antibodies. Exemplarily, one of the six detection strip segments is a blank detection strip segment, and the remaining five detection strip segments include five different second antibodies.

[0147] In one embodiment, the detection area is marked with the second antibody at the designated position using a film spraying instrument to form a plurality of detection strips; and the third antibody is marked with the third antibody at the designated position using a film spraying instrument to form a quality control line.

[0148] <End Area>

[0149] In one embodiment, the width of the end region is 0.5-1.5 cm, preferably 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm or 1.5 cm.

[0150] In one embodiment, the length of the end region is 0.5-1.5 cm, preferably 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.1 cm, 1.2 cm, 1.3 cm, 1.4 cm or 1.5 cm.

[0151] In one embodiment, the terminal region comprises a mesoporous silica film. Exemplarily, the terminal region is a mesoporous silica film.

[0152] In one embodiment, the configuration of the end region can enhance the capillary action and promote the diffusion of the liquid to be tested.

[0153] <Size of the detection membrane>

[0154] In one embodiment, the widths of the sample area, the filtration area, the colloidal gold area, the detection area and the terminal area are the same.

[0155] In one embodiment, the length of the sample area to be tested is 0.7 cm, the length of the filter area is 0.3 cm, the length of the colloidal gold area is 0.5 cm, the length between the first detection strip and the edge of the colloidal gold area on the detection area is 0.3 cm, the length between the first detection strip and the second detection strip is 0.3 cm, the length between the second detection strip and the quality control line is 0.3 cm, the length between the quality control line and the edge of the terminal area is 0.3 cm, and the length of the terminal area is preferably 0.8 cm.

[0156] <Tumor markers>

[0157] According to an embodiment of the present invention, the tumor markers include but are not limited to carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA125 (CA125), CA19-9 (CA19-9), CA72-4 (CA72-4), CA15-3 (CA15-3), bladder tumor antigen (BTA), nuclear matrix protein 22 (NMP22), prostate specific antigen (PSA), human chorionic gonadotropin (β-hCG) and calcitonin (hCT).

[0158] <Definition of terms>

[0159] In the present invention, unless otherwise specified, the direction in which the width lies refers to a direction perpendicular to the chromatography direction of the mesoporous silica membrane. The direction in which the length lies refers to a direction parallel to the chromatography direction of the mesoporous silica membrane.

[0160] The present invention will be described in further detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0161] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, materials, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0162] Example 1

[0163] A. Preparation of mesoporous silica membrane: on a glass substrate, a CTAB-containing Solution preparation of mesoporous silica film: The substrate is first soaked in 10M sodium hydroxide solution at room temperature for 10 hours to clean organic residues, and rinsed with deionized water before use. 0.095g CTAB, 15mL ethanol, 35mL water and 5μL concentrated ammonia solution (25wt.%) are mixed to form a reaction system, and 0.04mL tetraethyl orthosilicate TEOS is added thereto under continuous stirring. The substrate is then immersed in the solution, the water bath is maintained at a constant temperature of 60°C, and it is allowed to stand to spontaneously assemble into a film. After 72 hours, the templated silica film formed on the substrate is gently rinsed with running water, and then the substrate and the silica film are placed in an oven at 100°C for drying and aging overnight. After cooling to room temperature, the mesoporous silica film on the substrate was placed in an ethanol solution containing 0.1M HCl, stirred moderately for 10 minutes, and repeated three times to obtain a mesoporous silica film with a thickness of 200nm. According to the commonly used nanofilm transfer method, the mesoporous silica film was carefully lifted out of the solution with a commercial PVDF membrane and transferred to deionized water for rinsing 3 times. The polyurethane adhesive was evenly spin-coated on the surface of a commercial PVDF membrane with a thickness of 1mm using a glue spreader, and then the prepared mesoporous silica film was laminated with a commercial PVDF membrane using a dry laminator to form a composite membrane. The water contact angle of the mesoporous silica film on the PVDF membrane was measured to be 0°, showing good hydrophilicity. The mesoporous silica membrane and its PVDF substrate were cut into 30mm×12mm strips with a slicer for use.

[0164] B. Pretreatment of biocompatibility of mesoporous silica membrane: Place the above-mentioned strip-shaped mesoporous silica membrane in the pre-treatment solution, soak for 1 hour, take it out, and dry it at 37°C for 2 hours. The pre-treatment solution is: 0.5% bovine serum albumin (BSA), 20mM phosphate buffer with pH 7.4. After preparation, filter and sterilize it with a 0.22μm microporous filter membrane and store it at 4°C for use. The validity period is one week.

[0165] C. Antibody preparation and loading: 1. Prepare loading buffer: 20mM phosphate buffer with a pH of 7.4. After preparation, filter with a 0.22μm microporous filter to sterilize and dialyze overnight for use. The remaining should be stored at 4°C for use. The shelf life is one week. 2. Select commercial mouse anti-human alpha-fetoprotein monoclonal antibody I (αAFP-I), mouse anti-human carcinoembryonic antigen monoclonal antibody I (αCEA-I), mouse anti-human CA19-9 monoclonal antibody I (αCA19-9-I), mouse anti-human CA72-4 monoclonal antibody I (αCA72-4-I) and goat anti-mouse IgG antibody. Use the dialyzed loading buffer to dilute αAFP-I, αCEA-I, αCA19-9-I and αCA72-4-I to a concentration of 1 mg / mL, respectively, and use the quantitative film sprayer BioDotRR120 at 1μL / mm 2 The antibody solution is uniformly sprayed on the mesoporous silica membrane. Figure 1 The detection strips are arranged in a structure, that is, detection strip segment a is mouse anti-human alpha-fetoprotein monoclonal antibody I (αAFP-I), detection strip segment b is mouse anti-human carcinoembryonic antigen monoclonal antibody I (αCEA-I), detection strip segment c is mouse anti-human CA19-9 monoclonal antibody I (αCA19-9-I), and detection strip segment d is mouse anti-human CA72-4 monoclonal antibody I (αCA72-4-I). The contact speed and time of the BioDotRR120 spray point and the mesoporous silica membrane are controlled so that the length of each detection strip is 5 mm and the width is 2 mm. The parallel detection strip segments a and b are separated by 2 mm, the parallel detection strip segments c and d are separated by 2 mm, the center points of the front-to-back detection strip segments a and c are separated by 3 mm, and the center points of the front-to-back detection strip segments b and d are separated by 3 mm. Dilute goat anti-mouse IgG monoclonal antibody to a concentration of 1 mg / mL with loading buffer, and continuously spray goat anti-mouse IgG monoclonal antibody on the parallel detection strip c and the detection strip d 3 mm behind the center point of the quality control line through BioDotRR120, and the quality control line width is 2 mm. After drying at room temperature for 30 minutes, dry at 37°C for 1 hour, add desiccant, seal and store at 4°C for use.

[0166] D. Gold-labeled antibody: Use a commercially available sample (Leiming Bio Au010040) - a colloidal gold solution containing colloidal gold particles with a diameter of 40nm. Concentrate the colloidal gold solution through a centrifuge tube to a final OD value of 3 for later use. Take 7mL of colloidal gold with an OD value of 3 and a diameter of 40nm and 200pg of paired mouse anti-human alpha-fetoprotein monoclonal antibody II (αAFP-II), and combine them by magnetic stirring and oscillation at a pH of 8.5. Add bovine serum albumin (BSA) as a stabilizer, and make the final concentration of BSA 1%. Use high-speed centrifugation (10000r / min, 30min, 4°C) to remove unbound monoclonal antibodies. The deep red precipitate at the bottom of the centrifuge tube is the colloidal gold-antibody complex. The colloidal gold-antibody complex was collected and redissolved in 1 mL of 20 mM, pH 7.4 salt-free phosphate buffer, 1-ethyl-3,3-dimethylaminopropylcarbodiimide hydrochloride was added to 1 mg / mL, and the reaction was carried out at room temperature for 15 minutes. 1.4 pL 2-aminoethanol was added to quench EDC. Thus, a stable gold-labeled antibody covalently linked to gold nanoparticles, namely, αAFP-II gold-labeled antibody, was prepared. The gold-labeled antibody was slowly washed with 10 mL of 20 mM, pH 7.4 salt-free phosphate buffer containing 0.2% polyethylene glycol, and the supernatant was removed by centrifugation to obtain a deep red precipitate. The purified precipitate was dissolved with 2 mL of 20 mM, pH 8 boric acid buffer and stored at 4 ° C for use. Using the same process, covalently linked stable gold-labeled antibodies, αCEA-II gold-labeled antibody, αCA19-9-II gold-labeled antibody, and αCA72-4-II gold-labeled antibody, were prepared respectively.

[0167] E. Colloidal gold region: Mix the covalently linked αAFP-II gold-labeled antibody, αCEA-II gold-labeled antibody, αCA19-9-II gold-labeled antibody, and αCA72-4-II gold-labeled antibody stock solutions prepared above. Use BioDotRR120 film sprayer at 10 pL / cm 2 The mixed solution is evenly sprayed on the reserved gold-labeled antibody region of the mesoporous silica membrane. Figure 1 The colloidal gold region is a continuous region starting from 5 mm from the center of the parallel detection strip segment a and the detection strip segment b and ending at 5 mm from one end of the detection membrane. Dry under vacuum at 37°C for 1 hour. Thus, an integrated tumor marker detection membrane is obtained. Add a desiccant and seal at 4°C for later use.

[0168] Example 2

[0169] An integrated tumor marker detection membrane was prepared in the manner of Example 1, except that the antibodies in the detection membrane were different. Specifically, in step C, commercial mouse anti-human carcinoembryonic antigen monoclonal antibody I (αCEA-I), mouse anti-human CA125 monoclonal antibody I (αCA125-I), mouse anti-human CA19-9 monoclonal antibody I (αCA19-9-I), mouse anti-human CA72-4 monoclonal antibody I (αCA72-4-I) and goat anti-mouse IgG antibody were selected. Figure 1 The detection strips are arranged in a structure of, that is, detection strip segment a is mouse anti-human carcinoembryonic antigen monoclonal antibody I (αCEA-I), detection strip segment b is mouse anti-human CA125 monoclonal antibody I (αCA125-I), detection strip segment c is mouse anti-human CA19-9 monoclonal antibody I (αCA19-9-I), and detection strip segment d is mouse anti-human CA72-4 monoclonal antibody I (αCA72-4-I). Correspondingly, in step D "gold-labeled antibody", covalently linked αCEA-II gold-labeled antibody, αCA125-II gold-labeled antibody, αCA19-9-II gold-labeled antibody and αCA72-4-II gold-labeled antibody are prepared respectively. In step E, "colloidal gold region", the αCEA-II gold-labeled antibody, αCA125-II gold-labeled antibody, αCA19-9-II gold-labeled antibody, and αCA72-4-II gold-labeled antibody stock solutions were mixed using a BioDotRR120 film sprayer and evenly sprayed on the reserved gold-labeled antibody region of the above-mentioned mesoporous silica membrane. A desiccant was added and sealed at 4°C for later use.

[0170] Example 3

[0171] A. Preparation of mesoporous silica membrane: Other operations are the same as those in Example 1, except that the mesoporous silica membrane and its PVDF substrate are cut into 33 mm×12 mm strips by a slicer for later use.

[0172] B. Pretreatment of mesoporous silica membrane for biocompatibility: Same as Example 1.

[0173] C. Antibody preparation and loading: 1. Prepare loading buffer: 20 mM phosphate buffer with a pH of 7.4. After preparation, filter and sterilize with a 0.22 μm microporous filter membrane, then dialyze overnight for use. The remainder is stored at 4°C for use. The shelf life is one week. 2. Select commercial mouse anti-human alpha-fetoprotein monoclonal antibody I (αAFP-I), mouse anti-human carcinoembryonic antigen monoclonal antibody I (αCEA-I), mouse anti-human CA125 monoclonal antibody I (αCA125-I), mouse anti-human CA19-9 monoclonal antibody I (αCA19-9-I), mouse anti-human CA15-3 monoclonal antibody I (αCA15-3-I), mouse anti-human CA72-4 monoclonal antibody I (αCA72-4-I) and goat anti-mouse IgG antibody. αAFP-I, αCEA-I, αCA125-I, αCA19-9-I, αCA15-3-I and αCA72-4-I were diluted to a concentration of 1 mg / mL using the dialyzed loading buffer and then sprayed at 1 μL / mm using a quantitative film sprayer BioDotRR120. 2 The antibody solution is uniformly sprayed on the mesoporous silica membrane. Figure 2 The detection strips are arranged in a structure of, that is, detection strip segment e is mouse anti-human alpha-fetoprotein monoclonal antibody I (αAFP-I), detection strip segment f is mouse anti-human carcinoembryonic antigen monoclonal antibody I (αCEA-I), detection strip segment g is mouse anti-human CA125 monoclonal antibody I (αCA125-I), detection strip segment h is mouse anti-human CA19-9 monoclonal antibody I (αCA19-9-I), detection strip segment i is mouse anti-human CA15-3 monoclonal antibody I (αCA15-3-I), and detection strip segment j is mouse anti-human CA72-4 monoclonal antibody I (αCA72-4-I). The contact speed and time of the BioDotRR120 spray point and the mesoporous silica membrane are controlled so that the length of each detection strip is 5 mm and the width is 2 mm. The parallel detection strip segments e and f are separated by 2 mm, the parallel detection strip segments g and h are separated by 2 mm, the parallel detection strip segments i and j are separated by 2 mm, the center points of the front and back detection strip segments e and g are separated by 3 mm, the center points of the front and back detection strip segments g and i are separated by 3 mm, the center points of the front and back detection strip segments f and h are separated by 3 mm, and the center points of the front and back detection strip segments h and j are separated by 3 mm. The sheep anti-mouse IgG monoclonal antibody is diluted to a concentration of 1 mg / mL with the loading buffer, and the quality control line is continuously sprayed with the sheep anti-mouse IgG monoclonal antibody by BioDotRR120 at 3 mm behind the center points of the parallel detection strip segments i and j, and the quality control line width is 2 mm. After drying at room temperature for 30 minutes, it is dried at 37°C for 1 hour, and a desiccant is added to seal it at 4°C for standby use.

[0174] D. Gold-labeled antibodies: Prepare covalently linked stable gold-labeled antibodies respectively - αAFP-II gold-labeled antibody, αCEA-II gold-labeled antibody, αCA125-II gold-labeled antibody, αCA19-9-II gold-labeled antibody, αCA15-3-II gold-labeled antibody and αCA72-4-II gold-labeled antibody.

[0175] E. Colloidal gold region: Mix the covalently linked αAFP-II gold-labeled antibody, αCEA-II gold-labeled antibody, αCA125-II gold-labeled antibody, αCA19-9-II gold-labeled antibody, αCA15-3-II gold-labeled antibody, and αCA72-4-II gold-labeled antibody stock solutions prepared above. Use BioDotRR120 film sprayer at 10 pL / cm 2 The mixed solution is evenly sprayed on the reserved gold-labeled antibody region of the mesoporous silica membrane. Figure 2 The colloidal gold region is a continuous region starting from 5 mm from the center of the parallel detection strip segment e and the detection strip segment f and ending at 5 mm from one end of the detection membrane. Dry under vacuum at 37°C for 1 hour. Thus, an integrated tumor marker detection membrane is prepared. Add a desiccant and seal at 4°C for later use.

[0176] Example 4

[0177] The detection membrane prepared in Example 1 is used for the detection of liver cancer, colorectal cancer, pancreatic cancer, and gastric cancer. 25 μL of blood sample is dripped into the central area of ​​the sample area to be tested, and 40 μL of sample treatment solution is quickly dripped, the sample treatment solution includes a phosphate buffer with a pH of 7.4, 0.5wt% bovine serum albumin BSA, 1.0wt% polyvinyl alcohol, and 0.2wt% Tween 20. After filtration and sterilization with a 0.22μm microporous filter membrane, it is stored at 4°C for use, with a validity period of one week. The sample diffuses to the filtration area, colloidal gold area, detection area, and terminal area in sequence through the capillary action of the mesoporous silica layer. After the sample passes the quality control line, the color change of the quality control line and each detection strip is immediately checked. In the detection membrane prepared in Example 1, detection segment a is mouse anti-human alpha-fetoprotein monoclonal antibody I (αAFP-I), detection segment b is mouse anti-human carcinoembryonic antigen monoclonal antibody I (αCEA-I), detection segment c is mouse anti-human CA19-9 monoclonal antibody I (αCA19-9-I), and detection segment d is mouse anti-human CA72-4 monoclonal antibody I (αCA72-4-I). First, check the color reaction of the quality control line. If the quality control line has no color reaction, the test result has no substantive meaning. The reason may be that the sample is wrong or the detection membrane fails due to improper storage. If the quality control line is red, the detection membrane is normal, and the color reaction of the tumor marker is shown in Table 1, where color (+), no color (-), color or no color (- / +), indicating that the positivity or negativity of the tumor marker cannot be used as the main judgment basis, but only as an auxiliary judgment basis.

[0178] Table 1 Description of the detection of various tumors by the detection membrane prepared in Example 1

[0179]

[0180]

[0181] Example 5

[0182] The detection membrane prepared in Example 2 is used for the detection of uterine cancer, ovarian cancer, colorectal cancer, and gastric cancer. The specific method of using the detection membrane is the same as that in Example 4. In the detection membrane prepared in Example 2, detection segment a is mouse anti-human carcinoembryonic antigen monoclonal antibody I (αCEA-I), detection segment b is mouse anti-human CA125 monoclonal antibody I (αCA125-I), detection segment c is mouse anti-human CA19-9 monoclonal antibody I (αCA19-9-I), and detection segment d is mouse anti-human CA72-4 monoclonal antibody I (αCA72-4-I).

[0183] First, check the color reaction of the quality control line. If the quality control line has no color reaction, the test result is meaningless. The reason may be that the sample is wrong or the test membrane has failed due to improper storage. If the quality control line is red, the test membrane is normal, and the color reaction of the tumor marker is shown in Table 2, where color (+), no color (-), color or no color (- / +), indicating that the positive or negative of the tumor marker cannot be used as the main judgment basis, but only as an auxiliary judgment basis.

[0184] Table 2 Description of the detection of various tumors by the detection membrane prepared in Example 2

[0185] Tumor type a b c d Uterine cancer - / + + + - Ovarian cancer - / + + - + Colorectal cancer - - - - Cancer - / + - - +

[0186] Example 6

[0187] The detection membrane prepared in Example 3 is used for the detection of liver cancer, ovarian cancer, uterine cancer, gastric cancer, colorectal cancer, pancreatic cancer, and breast cancer. The specific method of using the detection membrane is the same as that of Example 4. In the detection membrane prepared in Example 3, the detection segment e is mouse anti-human alpha-fetoprotein monoclonal antibody I (αAFP-I), the detection segment f is mouse anti-human carcinoembryonic antigen monoclonal antibody I (αCEA-I), the detection segment g is mouse anti-human CA125 monoclonal antibody I (αCA125-I), the detection segment h is mouse anti-human CA19-9 monoclonal antibody I (αCA19-9-I), the detection segment i is mouse anti-human CA15-3 monoclonal antibody I (αCA15-3-I), and the detection segment j is mouse anti-human CA72-4 monoclonal antibody I (αCA72-4-I). First, check the color reaction of the quality control line. If the quality control line has no color reaction, the test result has no substantive meaning. The reason may be that the sample is wrong or the detection membrane fails due to improper storage. If the quality control line is red, the test membrane is normal, and the color reaction of the tumor marker is as shown in Table 3, where color development (+), no color development (-), color development or no color development (- / +) indicates that the positivity or negativity of the tumor marker cannot be used as the main basis for judgment, but only as an auxiliary basis for judgment.

[0188] Table 3 Description of the detection of various tumors by the detection membrane prepared in Example 3

[0189] Tumor type e f g h i j Liver cancer + + - - - - Ovarian cancer - - / + + - - + Uterine cancer - - / + + + - - Colorectal cancer - + - - - - Pancreatic cancer - - - + - - Gastric cancer - - / + - - - + Breast cancer - - / + - - / + + -

[0190] Example 7

[0191] A. Preparation of mesoporous silica film: Same as Example 3.

[0192] B. Biocompatibility pretreatment of mesoporous silica film: same as Example 3.

[0193] C. Antibody preparation and loading: Same as Example 3, except that commercial mouse anti-human bladder tumor antigen monoclonal antibody I (αBTA-I), mouse anti-human nuclear matrix protein 22 monoclonal antibody I (αNMP22-I), mouse anti-human prostate specific antigen monoclonal antibody I (αPSA-I), mouse anti-human chorionic gonadotropin monoclonal antibody I (αβ-hCG-I), mouse anti-human calcitonin monoclonal antibody I (αhCT-I) and goat anti-mouse IgG antibody were used. Figure 2 The detection strips are arranged in a structure of, that is, detection strip segment e is mouse anti-human bladder tumor antigen monoclonal antibody I (αBTA-I), detection strip segment f is mouse anti-human nuclear matrix protein 22 monoclonal antibody I (αNMP22-I), detection strip segment g is mouse anti-human prostate specific antigen monoclonal antibody I (αPSA-I), detection strip segment h is mouse anti-human chorionic gonadotropin monoclonal antibody I (αβ-hCG-I), detection strip segment i is sprayed with loading buffer as a blank, and detection strip segment j is mouse anti-human calcitonin monoclonal antibody I (αhCT-I).

[0194] D. Gold-labeled antibodies: Prepare covalently linked stable gold-labeled antibodies respectively - αhCT-II gold-labeled antibody, αBTA-II gold-labeled antibody, αNMP22-II gold-labeled antibody, αPSA-II gold-labeled antibody, and αβ-hCG-II gold-labeled antibody.

[0195] E. Colloidal gold region: Mix the covalently linked αBTA-II gold-labeled antibody, αNMP22-II gold-labeled antibody, αPSA-II gold-labeled antibody, αβ-hCG-II gold-labeled antibody, and αhCT-II gold-labeled antibody stock solutions prepared above. Use BioDotRR120 film sprayer at 10 pL / cm 2 The mixed solution is evenly sprayed on the reserved gold-labeled antibody region of the mesoporous silica membrane. Figure 2 The colloidal gold region is a continuous region starting from 5 mm from the center of the parallel detection strip segment e and the detection strip segment f and ending at 5 mm from one end of the detection membrane. Dry under vacuum at 37°C for 1 hour. Thus, an integrated tumor marker detection membrane is prepared. Add a desiccant and seal at 4°C for later use.

[0196] Example 8

[0197] The detection membrane prepared in Example 7 is used for the detection of bladder cancer, prostate cancer, testicular choriocarcinoma, and medullary thyroid cancer. The specific method of using the detection membrane is the same as in Example 4. In the detection membrane prepared in Example 7, the detection segment e is mouse anti-human bladder tumor antigen monoclonal antibody I (αBTA-I), the detection segment f is mouse anti-human nuclear matrix protein 22 monoclonal antibody I (αNMP22-I), the detection segment g is mouse anti-human prostate specific antigen monoclonal antibody I (αPSA-I), the detection segment h is mouse anti-human chorionic gonadotropin monoclonal antibody I (αβ-hCG-I), the detection segment i is sprayed with loading buffer as a blank, and the detection segment j is mouse anti-human calcitonin monoclonal antibody I (αhCT-I). First, check the color reaction of the quality control line. If the quality control line has no color reaction, the test result has no substantive meaning. The reason may be that the sample is wrong or the detection membrane fails due to improper storage. If the quality control line is red, the test membrane is normal, and the color reaction of the tumor marker is as shown in Table 4, where color is developed (+) and no color is developed (-).

[0198] Table 4 Description of the detection of various tumors by the detection membrane prepared in Example 7

[0199] Tumor type e f g h j Bladder cancer + + - - - Prostate cancer - - + - - Testicular choriocarcinoma - - - + - Medullary thyroid cancer - - - - +

[0200] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a mesoporous silica membrane as a chromatographic membrane.

2. The use according to claim 1, characterized in that The chromatographic membrane is a chromatographic membrane in a detection membrane; preferably, the chromatographic membrane is a chromatographic membrane used in a tumor marker detection membrane.

3. The use according to claim 1 or 2, characterized in that The mesoporous silica film satisfies at least one of the following conditions: (1) The thickness of the mesoporous silica film is 50-500 nm; (2) The porosity of the mesoporous silica membrane is 0.990-0.995; (3) The specific surface area of ​​the mesoporous silica membrane is 600-900m 2 / g; (4) The pore volume of the mesoporous silica membrane is 0.4-0.55 cm 3 / g; (5) The pore size of the mesoporous silica membrane is 2-50 nm; (6) The contact angle between the mesoporous silica film and water is 0°; (7) The surface of the mesoporous silica membrane is loaded with bovine serum albumin (BSA).

4. An integrated tumor marker detection membrane, characterized in that: The detection membrane comprises a mesoporous silica membrane and a substrate, wherein the mesoporous silica membrane is arranged on the surface of the substrate as a chromatographic membrane.

5. The integrated tumor marker detection membrane according to claim 4, characterized in that: The mesoporous silica film satisfies at least one of the following conditions: (1) The thickness of the mesoporous silica film is 50-500 nm; (2) The porosity of the mesoporous silica membrane is 0.990-0.995; (3) The specific surface area of ​​the mesoporous silica membrane is 600-900m 2 / g; (4) The pore volume of the mesoporous silica membrane is 0.4-0.55 cm 3 / g; (5) The pore size of the mesoporous silica membrane is 2-50 nm; (6) The contact angle between the mesoporous silica film and water is 0°; (7) The surface of the mesoporous silica membrane is loaded with bovine serum albumin (BSA).

6. The integrated tumor marker detection membrane according to claim 5, characterized in that: The substrate is selected from a polyvinylidene fluoride substrate or a nylon substrate. Preferably, a glue layer is further included between the mesoporous silica film and the substrate. Preferably, the adhesive layer comprises at least one of polyurethane, epoxy resin and acrylate.

7. The integrated tumor marker detection membrane according to any one of claims 4 to 6, characterized in that: The mesoporous silica membrane is provided with a sample area to be tested, a filtering area, a colloidal gold area, a detection area and a terminal area which are connected in sequence.

8. The integrated tumor marker detection membrane according to claim 7, characterized in that: The sample area to be tested includes a mesoporous silica membrane; and / or, the filtration area includes a mesoporous silica membrane; and / or, the terminal area includes a mesoporous silica membrane.

9. The integrated tumor marker detection membrane according to claim 7, characterized in that: The colloidal gold region comprises a gold-labeled antibody and a mesoporous silica membrane, and the gold-labeled antibody is bound to the surface of the mesoporous silica membrane. Preferably, the gold-labeled antibody is a colloidal gold-first antibody conjugate, and the colloidal gold-first antibody conjugate is a conjugate in which colloidal gold and the first antibody are connected by a covalent bond. Preferably, the first antibody is an anti-human tumor marker antibody, such as at least one of a mouse anti-human tumor marker antibody, a guinea pig anti-human tumor marker antibody, a rat anti-human tumor marker antibody and a rabbit anti-human tumor marker antibody. Preferably, the loading amount of the gold-labeled antibody in the colloidal gold region is 8-20 pL / cm 2 .

10. The integrated tumor marker detection membrane according to claim 7, characterized in that: The detection area is provided with a plurality of detection strips and a quality control line; the plurality of detection strips include a second antibody and a mesoporous silica membrane, and the second antibody is bound to the surface of the mesoporous silica membrane; the quality control line includes a third antibody and a mesoporous silica membrane, and the third antibody is bound to the surface of the mesoporous silica membrane. Preferably, the detection strip is close to the colloidal gold area, and the quality control line is close to the terminal area. Preferably, the second antibody is an anti-human tumor marker antibody, such as at least one of a mouse anti-human tumor marker antibody, a guinea pig anti-human tumor marker antibody, a rat anti-human tumor marker antibody and a rabbit anti-human tumor marker antibody.

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