CN111638370B - Gastric function and gastric cancer occurrence risk detection device and preparation method thereof - Google Patents
Gastric function and gastric cancer occurrence risk detection device and preparation method thereof Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/595—Gastrins; Cholecystokinins [CCK]
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- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96472—Aspartic endopeptidases (3.4.23)
- G01N2333/96475—Aspartic endopeptidases (3.4.23) with definite EC number
- G01N2333/96477—Pepsin (3.4.23.1; 3.4.23.2; 3.4.23.3)
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- G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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- G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis
Abstract
The invention relates to a gastric function and gastric cancer occurrence risk detection device and a preparation method thereof, belonging to the field of medical detection equipment. Is prepared by sticking solid phases of a nitrocellulose membrane with high specificity G-17, PGI, PGII antibody, anti-human IgG antibody, anti-human IgM antibody and goat anti-mouse IgG polyclonal antibody, a glass fiber membrane absorbed with colloidal gold labeled G-17, PGI, PGII antibody and HP urease antigen, a sample pad, absorbent paper and other auxiliary materials. The method effectively improves the reaction sensitivity on the basis of ensuring complete release of the immune colloidal gold, can reduce the using amount of the immune colloidal gold under the same threshold value, saves the cost, can simultaneously detect five gastric function assessment and gastric cancer risk markers of G-17, PGI, PGII, HP urease IgG antibody and IgM antibody in a specimen, and does not increase the complexity of production operation. The test paper has high sensitivity, strong specificity, simple and convenient operation, time saving and strong practicability.
Description
Technical Field
The invention relates to the field of medical detection equipment, in particular to a G-17, PGI, PGII, HP urease IgG and IgM antibody combined detection device and a preparation method thereof, and the detection device and the preparation method thereof can realize gastric function assessment and gastric cancer risk assessment and realize sensitive, specific and rapid detection of each marker by utilizing colloidal gold immunochromatography and a double-antibody sandwich method principle to quantitatively detect human gastrin 17 (G-17), pepsinogen I (PGI), pepsinogen II (PGII), helicobacter Pylori (HP) urease IgG and helicobacter pylori urease IgM in whole blood, serum and plasma samples.
Background
The pathological changes of the gastric mucosa are caused by various factors, including drugs, alcohol, abnormal gastric acid secretion, helicobacter pylori infection and the like, wherein the helicobacter pylori infection is a main factor; after the gastric mucosa is damaged, the function of the gastric mucosa can be changed; gastric cancer is a group of progressive epithelial cell malignant lesions with multifactorial participation, and is mostly developed gradually from chronic atrophic gastritis, and about 7 percent of chronic atrophic gastritis finally progresses to carcinoma in situ. Both abnormal cell proliferation and HP infection accelerate the rate of atrophic gastritis. China belongs to a high-incidence area of gastric cancer, and more than or equal to 20 ten thousand residents dying from gastric cancer every year. Although the five-year survival rate of the gastric cancer patient can be improved by radical operation combined with radiotherapy and chemotherapy, the effect of reducing the death rate is not obvious; early screening has been shown to be the key to reducing the incidence and mortality of gastric cancer.
Gastrin (GAS) is a class of gastrointestinal hormones secreted by G cells of the mucosa of the antrum and the upper duodenum and jejunum. Edkins was first found in the dog's antrum in 1905 and was later known. Gastrin in human serum mainly comprises two isoforms: gastrin 17 (gastrin-17, G-17) and gastrin-34 (gastrin-34, G-34), wherein gastrin 17 accounts for 85% -90%. One will generally select gastrin 17 to represent serum gastrin levels. Under physiological conditions, gastrin mainly plays a role by influencing the secretion of gastric acid, on one hand, gastrin can directly stimulate parietal cells to secrete gastric acid, the gastric acid activates pepsinogen into active pepsin so as to decompose protein, and meanwhile, the decomposition products can in turn stimulate the secretion of gastrin; on the other hand, gastrin can also act on cholecystokinin receptors on enterochromaffin-like cells to promote histamine secretion by the enterochromaffin-like cells, and further stimulate parietal cells to secrete gastric acid by histamine. Gastrin, while affecting gastric acid secretion, also reduces the tone of the lower esophageal sphincter. Some recent studies indicate that gastrin can act as a marker of gastric mucosal function: the level of gastrin-17 in chronic atrophic gastritis is significantly higher than in normal mucosa, and it is also believed that serum levels of gastrin-17 may be more accurately indicative of the severity of antral atrophy than serum total gastrin.
Pepsinogen (PG) is a protein polypeptide chain secreted by the host cells in the gastric mucosa. Human pepsinogen is largely divided into two biochemically and immunologically distinct isoenzymes (PGI and PGII). Pepsinogen I is separated from the gastric gland main cell and the cervical cell, and pepsinogen II can also be secreted from the cardiac gland, the pyloric gland and the proximal duodenal gland. The pepsinogen itself has no biological activity, and can be activated into pepsin with a digestion function under the action of gastric acid, so that the protein taken in by the body is decomposed into small molecular peptides, amino acids and the like which are easy to absorb. Most of the pepsinogen is secreted into the gastric lumen, and only a small fraction can be absorbed into the blood. We generally examined the pepsin in serum that originally represented the pepsin levels in humans. Pepsinogen I and pepsinogen II levels in serum are reported to accurately reflect gastric mucosal function and histological state. Thus, serum pepsinogen is also referred to as a "serological biopsy". Detection of serum pepsinogen I and pepsinogen II in combination with sophisticated endoscopy allows for efficient identification of gastric diseases, and in particular allows for the discovery of early stage gastric mucosal lesions. Previous studies have shown that serum pepsinogen I and pepsinogen II levels are significantly abnormal in erosive gastritis, atrophic gastritis, gastric ulcers, gastric cancer and other gastric disorders and can provide valuable screening methods for these disorders.
Helicobacter Pylori (HP) is a gram-negative, microaerophilic bacterium that colonizes the human stomach. Commonly found in 1983 by Marshall and Warren. The Malnick et al study demonstrated that HP has existed in humans for at least 58000 years. Epidemiological studies have indicated that the prevalence of HP infection can reach 20% to 50% in industrialized countries and even up to over 80% in developing countries. Meanwhile, as the modern medicine has proved that HP infection is related to gastric and duodenal ulcer, gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma, the established consensus criteria suggest that the first-class relatives of patients with peptic ulcer, mucosa-associated lymphoid tissue lymphoma (MALT), gastric cancer and gastric cancer, unknown iron-deficiency anemia and immune thrombocytopenia should be treated by eradicating HP.
Currently, there is still a lack of a unified early gastric cancer screening method in the clinic. The tumor marker and the related protein can effectively reflect the abnormal hyperplasia of cells, and are suitable for early screening and postoperative follow-up of cancers. Gastrointestinal radiography and chest fluoroscopy are mainly used for observing gastric ulcer and middle and late gastric cancer, but the sensitivity and accuracy of in-situ cancer are low; live examination under gastroscopy belongs to traumatic operation, has more contraindications and has large development difficulty. Gastrin 17, pepsinogen I and pepsinogen II are digestive enzymes secreted by gastric mucosa tissues, and the level of the digestive enzymes can be changed due to pathological changes of gastric mucosa parts such as gastric fundus, gastric antrum and the like. The joint detection of gastrin 17, pepsinogen I, pepsinogen II and helicobacter pylori has obvious effect when being applied to screening early gastric cancer, can effectively improve the diagnosis rate of gastric cancer, and is worthy of clinical application and popularization.
The Gold Immunochromatography (GICA) technique is a solid-phase membrane immunoassay technique using microporous membrane as carrier, which combines the gold immunochromatography technique with the protein chromatography technique. The colloidal gold immunochromatographic assay is a commonly used immunochromatographic assay, is very suitable for field detection due to the characteristics of simple operation, time saving, low manufacturing cost, easy interpretation of results and the like, and is widely applied to the fields of biology, medicine, food and the like. Because the colloidal gold immunochromatographic assay is used for completing detection in one step, the interference factors in the detection process are more, the low sensitivity is a main factor for limiting the application range of the colloidal gold immunochromatographic assay, and the detection limit of the traditional colloidal gold immunochromatographic assay is higher than that of methods such as ELISA and the like.
In the colloidal gold immunochromatography assay, proteins are immobilized on a nitrocellulose membrane (NC membrane) as a capture reagent for a sample to be detected. Since the detection result completely depends on the good adsorption effect of the capture reagent on the membrane, the uniform and good adsorption of the protein on the membrane is very important for the detection result of the colloidal gold. If the amount of protein bound to the NC membrane is insufficient or the binding force of protein is not strong enough, a considerable problem occurs, and it is very obvious on the detection line of the detection result. If the amount of protein bound to the membrane is too low, the color development of the detection line is weak and the detection sensitivity is reduced in the result. If the protein is not firmly adsorbed to the NC membrane, the protein diffuses before adsorbing to the NC membrane, so that the detection line is wide, the color development is weak, the detection line is bright and clear, and the detection result is difficult to explain. Under extreme conditions, if the physical adsorption of the protein to the NC membrane is too weak, the protein assay and surfactant solution flowing through may wash the immobilized protein off the NC membrane, thereby revealing a wider or not clear detection line at all, making it difficult to interpret the detection results.
Disclosure of Invention
The invention provides a gastric function and gastric cancer occurrence risk detection device and a preparation method thereof, and aims to solve the problems of insufficient NC membrane protein adsorption amount and weak binding force in the prior art. The five-in-one combined detection device for human gastrin 17, pepsinogen I, pepsinogen II, helicobacter pylori urease IgG and helicobacter pylori urease IgM, which is prepared by the invention, can realize sensitive, specific and rapid detection of markers for gastric function assessment and gastric cancer risk assessment, improve reasonable comprehensive judgment on gastric function and gastric cancer risk, and can rapidly and accurately carry out early warning and disease condition risk judgment.
The technical scheme adopted by the invention is as follows: the sample pad 1, the immune colloidal gold glass fiber membrane 2, the nitrocellulose membrane 3 and the absorption pad 4 are respectively stuck on the plastic plate 5, two ends of the nitrocellulose membrane 3 are respectively lapped with the absorption pad 4 and the immune colloidal gold glass fiber membrane 2, and the other end of the immune colloidal gold glass fiber membrane 2 is lapped with the sample pad 1; a first detection line T1, a second detection line T2, a third detection line T3, a fourth detection line T4, a fifth detection line T5, a quality control line C1 and a quality control line C2 are arranged on the nitrocellulose membrane 3; the solid phase on the first detection line T1 is provided with a high-specificity gastrin 17 antibody; the solid phase on the second detection line T2 is provided with a high-specificity pepsinogen I antibody; a solid phase on the third detection line T3 is provided with a high-specificity pepsinogen II antibody; a high-specificity anti-human IgG antibody is arranged on a solid phase on the fourth detection line T4; a high-specificity anti-human IgM antibody is arranged on a solid phase on the fifth detection line T5; the detection lines T1, T2, T3 and the quality control line C1 can be longitudinally arranged on the same nitrocellulose membrane 3, meanwhile, T4, T5 and the quality control line C2 are longitudinally arranged on the other nitrocellulose membrane 3, and the two nitrocellulose membranes 3 are arranged in parallel to form a combined detection device; or the detection lines T1, T2, T3, T4 and T5 can be arranged on five nitrocellulose membranes 3 in parallel, three quality control lines C1 are arranged and correspond to the detection lines T1, T2 and T3, and two quality control lines C2 are arranged and correspond to the detection lines T4 and T5, so that a combined detection device is formed together; goat anti-mouse IgG polyclonal antibody is sprayed on the quality control line C1; and an anti-helicobacter pylori urease polyclonal antibody is sprayed on the quality control line C2.
A method for preparing a device for detecting the occurrence risk of gastric function and gastric cancer comprises the following steps:
(a) Preparing colloidal gold by a trisodium citrate reduction method;
(b) Adopting the colloidal gold prepared in the step (a) to mark a gastrin 17 antibody, a pepsinogen I antibody, a pepsinogen II antibody and a helicobacter pylori urease antigen to obtain immune colloidal gold;
(c) Diluting the immune colloidal gold obtained in the step (b) by adopting a gold spraying buffer solution to obtain an immune colloidal gold solution, and spraying the immune colloidal gold solution on a glass fiber pad to prepare an immune colloidal gold glass fiber membrane;
(d) Pretreating the nitrocellulose membrane by using a polyethylene glycol glycerol treatment solution, spraying a gastrin 17 antibody, a pepsinogen I antibody, a pepsinogen II antibody, an anti-human IgG and an anti-human IgM which are combined with zinc sulfide nano particles modified by oleic acid as detection lines, spraying a goat anti-mouse IgG antibody as a quality control line C1 and a urease polyclonal antibody for resisting helicobacter pylori as a quality control line C2 to prepare an immune nitrocellulose membrane;
(e) And (3) sequentially sticking the pretreated sample pad, the immune colloidal gold glass fiber membrane prepared in the step (c), the immune nitrocellulose membrane prepared in the step (d) and absorbent paper on a rubber plate, cutting to obtain a detection reagent strip, and finally filling the detection reagent strip into a plastic shell.
The particle size of the colloidal gold particles prepared by the trisodium citrate reduction method in the step (a) is 20-60 nm.
The metal spraying buffer solution in the step (c) of the invention consists of Tris-HCl solution, sucrose, trehalose and bovine serum albumin BSA, and has a pH value of 8.5, wherein the concentration of Tris-HCl is 0.02mol/L, the concentration of sucrose is 5-20%, the concentration of trehalose is 1-5%, and the concentration of bovine serum albumin BSA is 0.5-1%.
The pretreatment of the nitrocellulose membrane with the polyethylene glycol glycerol treatment solution in the step (d) of the invention is as follows: soaking the nitrocellulose membrane for 1h by using polyethylene glycol glycerol treatment liquid, oscillating and shaking at a low speed, taking out, washing for 3 times by using distilled water, and finally drying in a vacuum drying oven;
the zinc sulfide nanoparticles in the step (d) are combined with gastrin 17, pepsinogen I, pepsinogen II, anti-human IgG and anti-human IgM respectively, and the combination ratio is as follows: taking ZnS modified by oleic acid as a carrier, taking 1mL of gastrin 17, pepsinogen I, pepsinogen II, anti-human IgG and anti-human IgM solutions, stirring for 1 hour, centrifuging for 10 minutes at 12000rpm,8500rpm and 7000rpm respectively, collecting, and washing for 2 times by deionized water respectively.
The polyethylene glycol glycerol treatment solution in the step (d) is formed by diluting polyethylene glycol glycerol to the concentration of 0.5%, and is filtered by a filter membrane of 0.22 mu m for later use.
The polyethylene glycol glycerol treatment liquid in the step (d) is formed by mixing polyethylene glycol glycerol and polylysine (SIGMA, 150 KD-300 KD), wherein the concentration of the polyethylene glycol glycerol is 0.5 percent, the concentration of the polylysine is 0.5 percent, and the polyethylene glycol glycerol treatment liquid is filtered by a filter membrane of 0.22 mu m for standby.
The polyethylene glycol glycerol treatment liquid in the step (d) is prepared by mixing polyethylene glycol glycerol, polylysine (SIGMA, 150 KD-300 KD) and PEG20000, wherein the concentration of the polyethylene glycol glycerol is 0.5%, the concentration of the polylysine is 0.5%, the concentration of the PEG20000 is 0.1%, and the mixture is filtered by a 0.22 mu m filter membrane for later use.
The preparation method of the oleic acid modified zinc sulfide nano-particles comprises the following steps: adding 15ml of oleic acid absolute ethyl alcohol solution into 15ml of zinc acetate aqueous solution with the concentration of 0.3mol/L, stirring in a water bath at 40 ℃, adjusting the pH value by using ammonia water, adding 15ml of sodium sulfide aqueous solution with the concentration of 0.3mol/L, reacting for 5min, adding 5ml of SDS aqueous solution, and pouring the reaction solution into a 90ml hydrothermal kettle after uniformly mixing. The hydrothermal kettle is sealed and then placed into a constant temperature drying box for constant temperature reaction for a certain time at a certain temperature. And (4) after the reaction is finished, cooling to 50 ℃, and taking out a product. Washing with acetone, deionized water and ethanol, centrifuging, vacuum drying at 50 deg.C for 2 hr to obtain ZnS powder, and storing.
The sample pad treatment liquid adopted by the pretreated sample pad in the step (e) consists of Tris-HCL liquid, bovine serum albumin BSA, casein and surfactant alkylphenol ethoxylates, wherein the concentration of the Tris-HCL liquid is 0.1mol/L, the concentration of the bovine serum albumin BSA is 0.5-1%, the concentration of the casein is 0.1-0.2%, and the concentration of the surfactant is 0.5-1%.
The reaction of glycol and epichlorohydrin is catalyzed by alkali, the product is neutralized by dilute hydrochloric acid, extracted by carbon tetrachloride and distilled under reduced pressure, thus obtaining polyethylene glycol glycerol (PEGG) which is light yellow sticky matter. PEGG can be mixed with water in any proportion, can also be dissolved in common organic solvents such as ethanol, acetone, tetrahydrofuran and chloroform, and has certain surface activity. The polyethylene glycol glycerol has a structure containing a plurality of hydroxyl groups for coupling, the activation process is simple, and the protein can be conveniently fixed on the surface of the NC membrane. Under the conventional conditions, the number of the combined antibodies on the NC membrane per unit area is limited, and after the treatment by adopting the polyethylene glycol glycerol, the number of the combined antibodies on the NC membrane per unit area can be increased, so that higher detection sensitivity can be realized.
On the basis of improving the protein adsorption of the NC membrane, the study on the protein adsorption effect of the NC membrane is another way to improve the sensitivity of the colloidal gold. The ZnS modified by the oleic acid/sodium dodecyl sulfate not only has a nano-scale particle size, but also has good water solubility and biocompatibility, and can be uniformly dispersed in an aqueous medium by utilizing functional groups on the outer surface of the ZnS, so that the ZnS modified by the oleic acid/sodium dodecyl sulfate can be combined with biological macromolecules. The zinc sulfide nano-particles have the advantages of good stability, easy preparation, good biocompatibility, low immunogenicity and the like, and are widely researched in the field of biomedicine. However, the application of the method in the colloidal gold immunochromatography technology has not been reported. The research discusses the influence of zinc sulfide nanoparticles on NC membrane coated antibodies, firstly, the antibodies for membrane scribing are combined with the zinc sulfide nanoparticles, sealed, centrifugally purified, unbound antibodies are removed, then, the antibodies are redissolved to a certain proportion, and then, the membrane scribing is carried out, so that one zinc sulfide particle can be combined with a plurality of antibodies, the efficiency of coating the antibodies is increased, and the sensitivity is greatly improved.
In order to improve the sensitivity of the colloidal gold immunochromatography technology, the nitrocellulose membrane is pretreated by polyethylene glycol glycerol treatment fluid, and an antibody coated with NC is combined with zinc sulfide nanoparticles, so that the aim of improving the sensitivity of the test paper is fulfilled.
The invention has the beneficial effects that:
1. the detection device provided by the invention has a simple structure and a novel concept, and the gastrin 17, the pepsinogen I, the pepsinogen II, the antihuman IgG and the antihuman IgM are coated on the nitrocellulose membrane, so that the specificity is strong, the gastrin 17, the pepsinogen I, the pepsinogen II, the helicobacter pylori urease IgG and the helicobacter pylori urease IgM in the sample can be detected simultaneously, and the complexity of production operation is not increased.
2. In the immune colloidal gold preparation step, the immune colloidal gold can be completely released by matching with a proper gold spraying buffer solution and a sample pad treatment solution, the reaction sensitivity is effectively improved, the using amount of the immune colloidal gold can be reduced under the same threshold value, and the cost is saved.
3. The invention pretreats the nitrocellulose membrane, modifies the antibody coated with the nitrocellulose membrane, and improves the sensitivity and specificity of the test paper.
4. The detection device does not need any special instrument and equipment, and has low detection cost.
5. The detection device is simple and convenient to operate, and does not need to be operated by professional staff. The practicability is strong.
Drawings
FIG. 1 is a schematic structural diagram of the present invention, in which detection lines T1, T2, T3 and C1 are longitudinally arranged on the same nitrocellulose membrane 3, while T4, T5 and C2 are longitudinally arranged on another nitrocellulose membrane 3, and the two nitrocellulose membranes 3 are arranged side by side to form a joint detection device;
FIG. 2 isbase:Sub>A cross-sectional view A-A of FIG. 1;
FIG. 3 is a sectional view taken along line B-B of FIG. 1;
FIG. 4 is another schematic structural diagram of the present invention, in which detection lines T1, T2, T3, T4, and T5 can be arranged in parallel on five nitrocellulose membranes 3, three quality control lines C1 are provided and correspond to T1, T2, and T3, and two quality control lines C2 are provided and correspond to T4 and T5, which together form a joint detection device;
FIG. 5 is a cross-sectional view C-C of FIG. 4;
FIG. 6 is a cross-sectional view D-D of FIG. 4;
FIG. 7 is a cross-sectional view E-E of FIG. 4;
FIG. 8 is a cross-sectional view F-F of FIG. 4;
fig. 9 is a sectional view taken along line G-G of fig. 4.
Detailed Description
Example 1:
the device comprises a sample pad 1, an immune colloidal gold glass fiber membrane 2, an immune nitrocellulose membrane 3, an absorption pad 4 and a plastic plate 5, wherein the sample pad 1, the immune colloidal gold glass fiber membrane 2, the immune nitrocellulose membrane 3 and the absorption pad 4 are respectively stuck on the plastic plate 5, two ends of the nitrocellulose membrane 3 are respectively lapped with the absorption pad 4 and the immune colloidal gold glass fiber membrane 2, and the other end of the immune colloidal gold glass fiber membrane 2 is lapped with the sample pad 1; a first detection line (T1), a second detection line (T2), a third detection line (T3), a third detection line (T4), a third detection line (T5), a quality control line (C1) and a quality control line (C2) are arranged on the nitrocellulose membrane (3); the high specificity gastrin 17 antibody is arranged on the solid phase of the first detection line (T1); the solid phase on the second detection line (T2) is provided with a pepsinogen I antibody with high specificity; the solid phase on the third detection line (T3) is provided with a high-specificity pepsinogen II antibody; a high-specificity anti-human IgG antibody is arranged on a solid phase on the fourth detection line (T4); a high-specificity anti-human IgM antibody is arranged on a solid phase on the fifth detection line (T5); the detection lines T1, T2 and T3 and the quality control line C1 are arranged on the nitrocellulose membrane (3) longitudinally and arranged on the same nitrocellulose membrane (3), meanwhile, the detection lines T4 and T5 and the quality control line C2 are arranged on the other nitrocellulose membrane (3) longitudinally and arranged on the nitrocellulose membrane (3), and the two nitrocellulose membranes (3) are arranged in parallel to form a combined detection device; or the detection lines T1, T2, T3, T4 and T5 arranged on the nitrocellulose membrane (3) can be arranged on five nitrocellulose membranes (3) in parallel, three quality control lines (C1) are arranged and correspond to the detection lines T1, T2 and T3, and two quality control lines (C2) are arranged and correspond to the detection lines T4 and T5, so that a combined detection device is formed; goat anti-mouse IgG polyclonal antibody is spotted on the quality control line (C1); and an anti-helicobacter pylori urease polyclonal antibody is sprayed on the quality control line (C2).
The preparation method comprises the following steps:
(a) Preparation of colloidal gold by trisodium citrate reduction method
Gold chloride was added rapidly to heated 100ml of purified water, and after the solution boiled again, trisodium citrate, gold chloride: trisodium citrate 1:0.5, continuously boiling, observing that the color of the solution changes from yellow to black and then to purple, and finally changing to stable wine red, and continuously heating for 10 minutes at regular time, wherein the particle size of the colloidal gold is 20nm;
(b) Immune colloidal gold preparation
1) Respectively taking 100ml of 20nm colloidal gold solution, adding 140 mu l of pH regulator, and uniformly mixing; standing for 5min;
2) Adding gastrin 17, pepsinogen I and pepsinogen II in a proportion of 14 mu g per ml of colloidal gold solution into 20nm colloidal gold solution, wherein the total amount is 1.4mg, and uniformly mixing; standing for 5min;
3) Respectively adding 0.4 ml of colloidal gold stabilizer according to the proportion of 0.4%, uniformly mixing, and standing for 5 minutes;
4) Centrifuging at 10000, 12000 and 14000rpm for 10min, respectively collecting precipitates, and combining the three collected precipitates;
(c) Diluting immune colloidal gold by adopting an optimized gold spraying buffer solution to obtain an immune colloidal gold solution, and spraying the immune colloidal gold solution on a glass fiber pad to prepare an immune colloidal glass fiber membrane; the gold spraying buffer solution comprises: tris-HCl solution with concentration of 20mM, sucrose concentration of 5%, trehalose concentration of 1%, BSA concentration of 0.51%, pH of 8.5;
(d) Solid phase nitrocellulose membrane
1) Cellulose nitrate membrane pretreated by polyethylene glycol glycerol treatment liquid
Preparing a polyethylene glycol glycerol treatment fluid: filtering with 0.22 μm filter membrane to obtain polyethylene glycol glycerol with concentration of 0.5%;
pretreating a nitrocellulose membrane by using polyethylene glycol glycerol treatment liquid: soaking the nitrocellulose membrane in a polyethylene glycol glycerol treatment solution for 1h, shaking at a low speed, taking out, washing with distilled water for 3 times, and finally drying in a vacuum drying oven;
2) Zinc sulfide nanoparticles modified gastrin 17, pepsinogen I and pepsinogen II antibodies, anti-human IgG and anti-human IgM
Preparing oleic acid modified zinc sulfide nanoparticles: adding 15ml of oleic acid absolute ethyl alcohol solution into 15ml of zinc acetate aqueous solution with the concentration of 0.3mol/L, stirring in a water bath at 40 ℃, adjusting the pH value by using ammonia water, adding 15ml of sodium sulfide aqueous solution with the concentration of 0.3mol/L, reacting for 5min, adding 5ml of SDS aqueous solution, and pouring the reaction solution into a 90ml hydrothermal kettle after mixing uniformly. And (3) sealing the hydrothermal kettle, putting the sealed hydrothermal kettle into a constant-temperature drying box, and reacting at a constant temperature for a certain time. And (4) cooling to 50 ℃ after the reaction is finished, and taking out the product. Washing with acetone, deionized water and ethanol, centrifuging, vacuum drying at 50 deg.C for 2 hr to obtain ZnS powder, and storing.
Zinc sulfide nanoparticles modify gastrin 17, pepsinogen I and pepsinogen II antibodies, anti-human IgG and anti-human IgM: taking ZnS modified by oleic acid as a carrier, taking 1mL of gastrin 17, pepsinogen I, pepsinogen II antibody, anti-human IgG and anti-human IgM solutions, stirring for 1 hour, centrifuging for 10 minutes at 12000rpm,8500rpm and 7000rpm respectively, and collecting, and washing 2 times by deionized water respectively.
3) Coating of nitrocellulose membrane detection line and quality control line antibody
When the film spraying amount is 1.4 mu l/cm, diluting a zinc sulfide nanoparticle-gastrin 17 antibody, a zinc sulfide nanoparticle-pepsinogen I antibody, a zinc sulfide nanoparticle-pepsinogen II antibody, a zinc sulfide nanoparticle-antihuman IgG antibody and a zinc sulfide nanoparticle-antihuman IgM antibody to 1.5mg/ml, diluting a quality control line 1 goat anti-mouse IgG antibody and a quality control line 2 anti-helicobacter pylori urease polyclonal antibody to 1mg/ml, respectively coating a detection line and a quality control line of a nitrocellulose membrane, drying at room temperature overnight, and storing for later use;
(e) Sample pad pretreatment
Soaking glass fiber in a sample pad treatment solution for 10min, wherein the sample pad treatment solution comprises: the concentration of Tris-HCL solution is 0.1M, the concentration of bovine serum albumin BSA is 0.5%, the concentration of casein is 0.1%, the concentration of surfactant is 0.5%, the drying is carried out for standby at 37 ℃, and the reaction sensitivity can be improved by the sample pad after the treatment;
and (3) sequentially sticking the pretreated sample pad, the immune colloidal gold glass fiber membrane, the immune nitrocellulose membrane and the absorbent paper on a rubber plate, cutting to obtain a detection reagent strip, and finally filling the detection reagent strip into a plastic shell.
And (3) quantitative detection: through the detection of a colloidal gold detector, the combined detection device detects that the minimum detection concentration of human gastrin 17 is 0.5pg/ml, the minimum detection concentration of pepsinogen I is 5ng/ml, the minimum detection concentration of pepsinogen II is 5ng/ml, the minimum detection concentration of helicobacter pylori urease IgG antibody is 1ng/ml, and the minimum detection concentration of helicobacter pylori urease IgM antibody is 1ng/ml.
Example 2:
the gold spraying buffer solution comprises: tris-HCl solution with concentration of 20mM, sucrose concentration of 12%, trehalose concentration of 3%, BSA concentration of 0.7%, pH of 8.5;
preparing a polyethylene glycol glycerol treatment solution: is prepared by mixing polyethylene glycol glycerol with 0.5% concentration and polylysine (SIGMA, 150 KD) with 0.5% concentration, and filtering with 0.22 μm filter membrane;
the concentration of Tris-HCL solution is 0.1mol/L, the concentration of bovine serum albumin BSA is 0.7%, the concentration of casein is 0.15%, and the concentration of surfactant is 0.7%;
the rest is the same as example 1.
Example 3:
the gold spraying buffer solution comprises: tris-HCl solution with concentration of 20mM, sucrose concentration of 20%, trehalose concentration of 5%, BSA concentration of 1%, pH of 8.5;
preparing a polyethylene glycol glycerol treatment fluid: is prepared by mixing polyethylene glycol glycerol, polylysine (SIGMA, 150 KD) and PEG2000, wherein the concentration of polyethylene glycol glycerol is 0.5%, the concentration of polylysine is 0.5%, the concentration of PEG20000 is 0.1%, and filtering with 0.22 μm filter membrane;
the concentration of Tris-HCL solution is 0.1mol/L, the concentration of bovine serum albumin BSA is 1%, the concentration of casein is 0.2%, and the concentration of surfactant is 1%;
the rest is the same as example 1.
The following experiment further illustrates the effects of the present invention.
Experimental example 1
1. Comparison of adsorption capacity of polyethylene glycol glycerol treatment solution on nitrocellulose membrane protein
1.1 materials and methods
1.1 materials: nitrocellulose membrane, pore size 4.5um, available from general electric company of USA
1.2 nitrocellulose Membrane treatment
1.2.1 preparing polyethylene glycol glycerol treating fluid
Preparing three polyethylene glycol glycerol treatment liquids: polyethylene glycol glycerol group, the concentration of polyethylene glycol glycerol is 0.5%; the polyethylene glycol glycerol treatment fluid polylysine group comprises polyethylene glycol glycerol with the concentration of 0.5 percent and polylysine with the concentration of 0.5 percent; polyethylene glycol glycerol, polylysine and PEG20000, wherein the concentration of polyethylene glycol glycerol is 0.5%, the concentration of polylysine is 0.5%, the concentration of PEG20000 is 0.1%, and the three groups of treatment solutions are filtered with 0.22 μm filter membrane for use.
1.2.2 nitrocellulose Membrane treatment
And (3) putting the nitrocellulose membrane into the polyethylene glycol glycerol treatment solution, soaking for 1h, shaking at a low speed, taking out, washing for 3 times by using distilled water, and finally drying in a vacuum drying oven.
1.3 Experimental methods
The combined test paper for detecting gastrin 17, pepsinogen I, pepsinogen II, helicobacter pylori urease IgG and helicobacter pylori urease IgM is prepared by respectively using the untreated and treated nitrocellulose membranes according to the process flows of the above embodiments, the test flows refer to the specification of the test paper, and the differences of the adsorption force and the stability index of the untreated and treated nitrocellulose membranes are compared.
1.4 results
1.4.1 comparison of protein adsorption Capacity
And (3) taking the test paper of the treatment group and the test paper of the untreated group, respectively adding the test paper into a sample to be detected, and judging the protein adsorption capacity of the treated membrane by observing the color development condition, wherein the results are shown in a table 1. The result shows that the treated nitrocellulose membrane is obviously better than the untreated membrane in the aspect of solution wettability, the color of the positive strip of the treated membrane is slightly dark, and particularly when the concentration is lower, the reaction sensitivity is improved, which shows that the protein adsorption capacity is obviously enhanced, and the reaction sensitivity is improved. The adsorption effect of the polyethylene glycol glycerol-polylysine-PEG 20000 treatment group is obviously better than that of the polyethylene glycol glycerol group and the polyethylene glycol glycerol-polylysine group.
TABLE 1 comparison of the adsorption Capacity of nitrocellulose membranes
1.4.2 comparison of stability of nitrocellulose membranes
The stability of the adsorbed protein on the nitrocellulose membrane after treatment was judged by taking 3 groups of test paper of the treatment group and the untreated group and observing the color development through an accelerated experiment at 37 ℃, and the results are shown in table 2. The results in Table 2 and the results in Table 1 show that the color change of the nitrocellulose membrane after the treatment is basically consistent with that before 10 days, and the stability is good.
TABLE 2 accelerated stability comparison of nitrocellulose membranes (10 days at 37 ℃ C.)
Experimental example 2:
2. zinc sulfide nanoparticles modified with oleic acid for modification of gastrin 17, pepsinogen I and pepsinogen II antibodies, anti-human IgG and anti-human IgM
2.1 materials and methods
2.1.1 materials: nitrocellulose membrane, pore size 4.5um, available from general electric company of USA
2.1.2 preparation of oleic acid-modified Zinc sulfide nanoparticles
Adding 15ml of oleic acid absolute ethyl alcohol solution into 15ml of zinc acetate aqueous solution with the concentration of 0.3mol/L, stirring in a water bath at 40 ℃, adjusting the pH value by using ammonia water, adding 15ml of sodium sulfide aqueous solution with the concentration of 0.3mol/L, reacting for 5min, adding 5ml of SDS aqueous solution, and pouring the reaction solution into a 90ml hydrothermal kettle after uniformly mixing. And (3) sealing the hydrothermal kettle, putting the sealed hydrothermal kettle into a constant-temperature drying box, and reacting at a constant temperature for a certain time. And (4) cooling to 50 ℃ after the reaction is finished, and taking out the product. Washing with acetone, deionized water and ethanol, centrifuging, vacuum drying at 50 deg.C for 2 hr to obtain ZnS powder, and storing.
2.1.3 Zinc sulfide nanoparticles modification of Gastrin 17, progastrin I and Progastrin II antibodies, anti-human IgG and anti-human IgM
Taking ZnS modified by oleic acid as a carrier, taking 1mL of gastrin 17, pepsinogen I and pepsinogen II antibodies, anti-human IgG and anti-human IgM solutions, stirring for 1 hour, centrifuging for 10 minutes at 12000rpm,8500rpm and 7000rpm respectively, collecting, and washing for 2 times by deionized water respectively.
2.2 Experimental methods
Respectively mixing the zinc sulfide nanoparticles modified gastrin 17, pepsinogen I and pepsinogen II antibodies, anti-human IgG and anti-human IgM with the unmodified gastrin 17, pepsinogen I and pepsinogen II antibodies, anti-human IgG and anti-human IgM
The combined test paper is prepared according to the process flow of the embodiment, and the difference of protein adsorption force and stability indexes of zinc sulfide nanoparticle treatment and non-treatment is compared according to the test paper specification in the test flow.
2.3 results
2.3.1 comparison of protein adsorption Capacity
The test paper of the zinc sulfide nanoparticle treatment group and the test paper of the untreated group are respectively added into a sample to be detected, and the protein adsorption capacity of the treated membrane is judged by observing the color development condition, and the result is shown in a table 3. The result shows that the positive strip of the zinc sulfide nanoparticle modified membrane is slightly dark in color, and particularly when the concentration is low, the reaction sensitivity is improved, which shows that the protein adsorption capacity is obviously enhanced, and the reaction sensitivity is improved.
TABLE 3 comparison of protein adsorption Capacity for Zinc sulfide nanoparticle modification
2.3.2 stability comparison
Test paper of a zinc sulfide nanoparticle treatment group and test paper of an untreated group are taken, the stability of the protein adsorbed on the nitrocellulose membrane after zinc sulfide modification is judged by observing the color development condition through an accelerated experiment at 37 ℃, and the result is shown in table 4. The results in Table 4 and the results in Table 3 show that the color change of the nitrocellulose membrane after the treatment is basically consistent with that before 10 days, and the stability is good.
TABLE 2 accelerated stability comparison (10 days at 37 ℃ C.)
The above description is only a preferred example of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like of the present invention shall be included in the protection scope of the present invention.
Claims (10)
1. A preparation method of a device for detecting the occurrence risk of gastric function and gastric cancer is characterized by comprising the following steps: the method comprises the following steps:
(a) Preparing colloidal gold by a trisodium citrate reduction method;
(b) Adopting the colloidal gold prepared in the step (a) to mark a gastrin 17 antibody, a pepsinogen I antibody, a pepsinogen II antibody and a helicobacter pylori urease antigen to obtain immune colloidal gold;
(c) Diluting the immune colloidal gold obtained in the step (b) by adopting a gold spraying buffer solution to obtain an immune colloidal gold solution, and spraying the immune colloidal gold solution on a glass fiber pad to prepare an immune colloidal gold glass fiber membrane;
(d) Pretreating a nitrocellulose membrane with a polyethylene glycol glycerol treatment solution, spraying a gastrin 17 antibody, a pepsinogen I antibody, a pepsinogen II antibody, an anti-human IgG and an anti-human IgM which are combined with zinc sulfide nano particles modified by oleic acid as detection lines, and spraying a goat anti-mouse IgG antibody and an anti-helicobacter pylori urease polyclonal antibody as quality control lines to prepare an immune nitrocellulose membrane;
(e) And (3) sequentially sticking the pretreated sample pad, the immune colloidal gold glass fiber membrane prepared in the step (c), the immune nitrocellulose membrane prepared in the step (d) and the absorption pad on a plastic plate, cutting to obtain a detection reagent strip, and finally filling the detection reagent strip into a plastic shell.
2. The method for manufacturing a device for detecting the occurrence risk of gastric function and gastric cancer according to claim 1, wherein: the particle size of the colloidal gold particles prepared by the trisodium citrate reduction method in the step (a) is 20-60nm.
3. The method for manufacturing a device for detecting the occurrence risk of gastric function and gastric cancer according to claim 1, wherein: the gold spraying buffer solution in the step (c) consists of Tris-HCl solution, sucrose, trehalose and bovine serum albumin BSA, and has a pH value of 8.5, wherein the concentration of Tris-HCl is 0.02mol/L, the concentration of sucrose is 5 to 20%, the concentration of trehalose is 1 to 5%, and the concentration of bovine serum albumin BSA is 0.5 to 1%.
4. The method for manufacturing a device for detecting the occurrence risk of gastric function and gastric cancer according to claim 1, wherein: the step (d) of pretreating the nitrocellulose membrane with a polyethylene glycol glycerol treatment solution comprises the following steps: soaking the nitrocellulose membrane for 1h by using polyethylene glycol glycerol treatment liquid, oscillating and shaking at a low speed, taking out, washing for 3 times by using distilled water, and finally drying in a vacuum drying oven;
the oleic acid-modified zinc sulfide nanoparticles obtained in the step (d) are respectively combined with a gastrin 17 antibody, a pepsinogen I antibody, a pepsinogen II antibody, an anti-human IgG antibody and an anti-human IgM antibody, and the combination ratio is as follows: taking 1mL of gastrin 17 antibody, pepsinogen I antibody, pepsinogen II antibody, anti-human IgG and anti-human IgM antibody solutions by taking ZnS modified by oleic acid as a carrier, stirring for 1 hour, centrifuging for 10 minutes at 12000rpm,8500rpm and 7000rpm respectively, collecting, and washing 2 times by using deionized water.
5. The method for manufacturing a device for detecting gastric function and risk of developing gastric cancer according to claim 1 or 4, wherein: the polyethylene glycol glycerol treatment solution in the step (d) is formed by diluting polyethylene glycol glycerol to the concentration of 0.5%, and is filtered by a filter membrane of 0.22 mu m for standby.
6. The method for manufacturing a device for detecting gastric function and risk of developing gastric cancer according to claim 1 or 4, wherein: the polyethylene glycol glycerol treatment liquid in the step (d) is prepared by mixing polyethylene glycol glycerol with the concentration of 0.5 percent and polylysine with the concentration of 0.5 percent, and the polyethylene glycol glycerol treatment liquid is prepared by mixing the polyethylene glycol glycerol with the polylysine SIGMA with the concentration of 150 KD-300 KD and filtering the mixture through a filter membrane with the diameter of 0.22 mu m for later use.
7. The method for manufacturing a device for detecting gastric function and risk of developing gastric cancer according to claim 1 or 4, wherein: the polyethylene glycol glycerol treatment liquid in the step (d) is prepared by mixing polyethylene glycol glycerol, polylysine SIGMA,150 KD-300 KD and PEG20000, wherein the concentration of the polyethylene glycol glycerol is 0.5%, the concentration of the polylysine is 0.5%, the concentration of the PEG20000 is 0.1%, and the mixture is filtered by a filter membrane of 0.22 mu m for standby.
8. The method for manufacturing a device for detecting gastric function and risk of developing gastric cancer according to claim 1 or 4, wherein: the preparation method of the oleic acid modified zinc sulfide nano-particles comprises the following steps: adding 15ml of oleic acid absolute ethyl alcohol solution into 15ml of zinc acetate aqueous solution with the concentration of 0.3mol/L, stirring in a water bath at 40 ℃, adjusting the pH value by using ammonia water, adding 15ml of sodium sulfide aqueous solution with the concentration of 0.3mol/L, reacting for 5min, adding 5ml of SDS aqueous solution, mixing uniformly, pouring the reaction solution into a 90ml hydrothermal kettle, sealing the hydrothermal kettle, placing the hydrothermal kettle into a constant-temperature drying box for constant-temperature reaction, cooling to 50 ℃ after the reaction is finished, taking out a product, washing with acetone, deionized water, ethanol, performing centrifugal separation, performing vacuum drying at 50 ℃ for 2 hours to obtain powder ZnS, and storing for later use.
9. The method for manufacturing a device for detecting the occurrence risk of gastric function and gastric cancer according to claim 1, wherein: the sample pad treatment liquid adopted by the pretreated sample pad in the step (e) consists of Tris-HCl liquid, bovine serum albumin BSA, casein and surfactant alkylphenol ethoxylates, wherein the concentration of the Tris-HCl liquid is 0.1mol/L, the concentration of the bovine serum albumin BSA is 0.5-1%, the concentration of the casein is 0.1-0.2%, and the concentration of the surfactant is 0.5-1%.
10. The device for detecting the gastric function and the occurrence risk of gastric cancer, which is prepared by the method according to any one of claims 1 to 9, is characterized in that: the sample pad (1), the immune colloidal gold glass fiber membrane (2), the nitrocellulose membrane (3) and the absorption pad (4) are respectively stuck on the plastic plate (5), two ends of the nitrocellulose membrane (3) are respectively lapped with the absorption pad (4) and the immune colloidal gold glass fiber membrane (2), and the other end of the immune colloidal gold glass fiber membrane (2) is lapped with the sample pad (1); the nitrocellulose membrane (3) is provided with a first detection line T1, a second detection line T2, a third detection line T3, a fourth detection line T4, a fifth detection line T5, a quality control line C1 and a quality control line C2; the solid phase on the first detection line T1 is provided with a high-specificity gastrin 17 antibody; a high-specificity pepsinogen I antibody is arranged on a solid phase on the second detection line T2; the solid phase on the third detection line T3 is provided with a high-specificity pepsinogen II antibody; a high-specificity anti-human IgG antibody is arranged on a solid phase on the fourth detection line T4; a high-specificity anti-human IgM antibody is arranged on a solid phase on the fifth detection line T5; the first detection line T1, the second detection line T2, the third detection line T3 and the quality control line C1 are longitudinally arranged on the same nitrocellulose membrane (3), meanwhile, the fourth detection line T4, the fifth detection line T5 and the quality control line C2 are longitudinally arranged on the other nitrocellulose membrane (3), and the two nitrocellulose membranes (3) are arranged in parallel to form a combined detection device; or the first detection line T1, the second detection line T2, the third detection line T3, the fourth detection line T4 and the fifth detection line T5 are arranged on five nitrocellulose membranes (3) in parallel, three quality control lines C1 are arranged and correspond to the first detection line T1, the second detection line T2 and the third detection line T3, and two quality control lines C2 are arranged and correspond to the fourth detection line T4 and the fifth detection line T5, so that a combined detection device is formed together; goat anti-mouse IgG polyclonal antibody is spotted on the quality control line C1; and an anti-helicobacter pylori urease polyclonal antibody is sprayed on the quality control line C2.
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