Dry-type detection and analysis assembly for pancreatic amylase
Through the use of two-layer coating process and environmentally friendly yarn mesh, the problems of complex and high production of existing pancreatic amylase dry analysis components have been solved, and process simplification, cost reduction and detection accuracy have been achieved, suitable for clinical diagnosis .
Patent Information
- Application Number
- CN202411811515.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-10
- Publication Date
- 2025-02-18
AI Technical Summary
The existing pancreatic amylase dry analytical components have complex production processes, high costs, and are used with organic solvents that are harmful to the human body and the environment, making it difficult to achieve the goal of simplifying and reducing costs.
Using two-layer coating process, the first reagent layer uses a modified 30% binder and a modified gelatin to connect the active material, the second reagent layer contains D4 hydrogel, and the diffusion layer uses an environmentally friendly pore ratio of 200 or 300. Gauze mesh.
It has achieved simplification of the process, reduced production costs, preservation at room temperature, improved penetration effect and color uniformity, ensuring high detection accuracy and wide measurement range, and is suitable for clinical diagnosis.
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Figure CN119470307A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of in vitro detection, and in particular to a pancreatic amylase dry-type detection and analysis component. Background Art
[0002] Pancreatic amylase (P-AMY) mainly acts on the a-1,4 glycosidic bonds of polysaccharide molecules to decompose polysaccharides. The activity of pancreatic amylase is often used as an indicator for disease diagnosis. The activity of pancreatic amylase increases in certain chronic diseases such as chronic pancreatitis, mumps, and salivary gland suppuration; and in various liver diseases such as hepatitis, cirrhosis, liver abscess, and cholecystitis, the activity of pancreatic amylase decreases. Since the activity of pancreatic amylase increases significantly during the acute attack of pancreatitis, the pancreatic amylase in urine will also increase 12-24 hours after the onset of the disease. Therefore, blood pancreatic amylase determination is mainly used to diagnose acute pancreatitis.
[0003] At present, the commonly used dry analysis components for detecting pancreatic amylase are to coat the shielding layer, diffusion layer, reagent layer, color development layer, etc. on a transparent support in sequence according to different functions, and detect the concentration of the object to be tested by measuring the change of reflected light. This type of multilayer film dry analysis component has accurate test results and is easy to operate, but its production process is complicated and the production cost is high, because: its production process requires multilayer coating, shielding light source and dispersing samples to make the sample diffuse evenly; the prepared analysis component test area needs to be large to meet the requirements of subsequent testing by reflectance photometry (Kubelka-Munk theory), avoiding the problem of weak light receiving signal and insufficient sensitivity caused by small test area; the prepared components need to be stored at low temperature, because the storage conditions of the raw material fish gelatin used in the production process are 2-8 ° C, and the gel effect of fish gelatin is poor at room temperature. In addition, the organic solvents used in the production process (such as using organic solvents to prepare diffusion layers, etc.) have high requirements for materials and equipment, and there is a risk of endangering the health of operators and polluting the environment. Therefore, under the premise of ensuring the accuracy of the test results, a dry analysis component of pancreatic amylase with a simpler preparation process and lower cost is needed. Summary of the invention
[0004] In order to solve the above problems, the present invention provides a pancreatic amylase dry detection and analysis component.
[0005] The present invention adopts the following technical solutions:
[0006] A pancreatic amylase dry detection and analysis component comprises, from top to bottom, an upper support layer, a diffusion layer, a first reagent layer, a second reagent layer, a support and a lower support layer, wherein the upper support layer and the lower support layer are connected at both ends by snaps, a sample loading hole is provided in the middle of the upper support layer, and a test hole is provided in the middle of the lower support layer; the reagents contained in the diffusion layer include a buffer solution, an anti-salivary amylase monoclonal antibody, bovine serum albumin, and a non-ionic polymer compound; the reagents contained in the first reagent layer include a buffer solution, a modified 30% binder, modified gelatin, white gelatin, bovine serum albumin, PNPG7, a surfactant, a soluble cellulose ether, an activator, a defoaming agent and an active material; and the reagents contained in the second reagent layer include a D4 hydrogel with a concentration of 0.3 g / L.
[0007] Furthermore, the diffusion layer is made of a mesh with an opening rate of 200 or 300;
[0008] The first reagent layer and the second reagent layer are transparent plastic substrates or frosted plastic substrates with a light transmittance of more than 80% under a wavelength of 300-800nm, including any one of polyethylene terephthalate, polycarbonate, polypropylene, polyethylene, and polycarbonate sheets, and the thickness thereof is 30-200μm;
[0009] The support body is made of PET material with a thickness of 100-200 um, including any one of polyethylene terephthalate, polybutylene terephthalate, and polyvinyl chloride.
[0010] Furthermore, in the diffusion layer, the buffer solution includes any one or more of phosphate buffer, Hepes buffer (4-hydroxyethylpiperazineethanesulfonic acid buffer), and MES buffer (2-(N-morpholino)ethanesulfonic acid buffer), with a concentration of 10-20 mmol / L and a pH of 5-9; the anti-salivary amylase monoclonal antibody is added in an amount of 1-200 mg per unit square meter or 5-100 mg per unit square meter; the bovine serum albumin is added in an amount of 1-15 g per unit square meter or 1-5 g per unit square meter; the non-ionic polymer chemical includes any one or two of PVPK90, PVPK30, and PVPK15, and is added in an amount of 1-10 g per unit square meter or 1-7 g per unit square meter.
[0011] Further, the modified 30% binder is added to the first reagent layer in an amount of 1-50 g per unit square meter or 1-40 g per unit square meter;
[0012] The preparation method of the modified 30% binder is as follows:
[0013] S1. Weigh acrylamide and N-vinyl pyrrolidone in a mass ratio of 8:2 and dissolve them in 100 g of ultrapure water. After dissolution, weigh 2% of N,N'-methylenebisacrylamide and 1% of N,N,N',N'-tetramethylethylenediamine and stir them evenly in the solution.
[0014] S2, transferring the solution obtained in step S1 to a polymerization reactor, introducing nitrogen for 10 min, raising the temperature to 40° C. after the nitrogen is introduced, and maintaining the temperature for 30 min under the protection of nitrogen;
[0015] S3. Add 0.4% potassium persulfate and sodium bisulfite, continue to react at a constant temperature for 10 minutes, then react for 4 hours under sealed conditions, take out, and test the viscosity with a viscometer. The binder that passes the test is the desired binder.
[0016] Furthermore, in the first reagent layer, the buffer includes any one or more of phosphate buffer, Hepes buffer, and MES buffer, and its pH is 5-9; the white glue is added to the first reagent layer in an amount of 0.05-30g per unit square meter or 0.1-15g per unit square meter; the PNPG7 is added to the first reagent layer in an amount of 0.05-5g per unit square meter or 0.1-2.5g per unit square meter; the bovine serum albumin is added to the first reagent layer in an amount of 0.05-10g per unit square meter or 0.1-7g per unit square meter; the activator uses Nacl or Mgcl, and is added to the first reagent layer in an amount of 0.01-1g per unit square meter or 0.05-1g per unit square meter.
[0017] Further, the surfactant is added to the first reagent layer in an amount of 0.05-5 g per unit square meter or in an amount of 0.07-2 g per unit square meter;
[0018] The surfactant is a zwitterionic surfactant, a cationic surfactant or an anionic surfactant; the zwitterionic surfactant includes any one of Tween 20, Tween 80, Triton X-405 and Triton X-100; the cationic surfactant includes any one of hexadecyltrimethylammonium bromide and dodecyltrimethylammonium bromide; the anionic surfactant includes any one or more of sodium dodecylbenzene sulfonate and sodium dodecyl sulfate.
[0019] Furthermore, the soluble cellulose ether is added to the first reagent layer in an amount of 1-30 g per unit square meter or 1-20 g per unit square meter; the soluble cellulose ether includes any one of methyl cellulose, carboxymethyl cellulose, ethyl cellulose, benzyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, cyanoethyl cellulose, benzyl cyanoethyl cellulose, carboxymethyl hydroxyethyl cellulose and phenyl cellulose.
[0020] Furthermore, the defoamer is added to the first reagent layer in an amount of 0.1-5 ml per unit square meter or 0.5-2 ml per unit square meter; the defoamer includes any one or more of silicone defoamers, polyether defoamers, alcohol defoamers, and composite defoamers.
[0021] Furthermore, the active material is added to the first reagent layer in an amount of 1-50 g per unit square meter or 10-40 g per unit square meter; the active material includes any one or more of diatomaceous earth, volcanic ash, silica gel, fly ash, microcrystalline cellulose, bentonite, titanium dioxide, burnt clay, and cellulose powder.
[0022] Furthermore, the preparation method of the modified gelatin is as follows:
[0023] A1. Mix 10 g of 3,4-dihydroxybenzoic acid and 50 ml of ethyl acetate solvent and stir until completely dissolved, then add 20 g of acetyl chloride for reflux reaction, the reflux temperature is controlled at 60° C., the reflux time is 3 h, after the reflux reaction is complete, distill and separate, the distillation temperature is controlled at 70° C., the ethyl acetate solvent is separated, the product obtained after separation is modifier 1, and the modifier 1 is placed in a 50° C. drying oven for drying;
[0024] A2, add trichloropropane to modifier 1 and stir and disperse, then add dimethyl sulfoxide and reflux, the reflux time is 3h, after the reaction is complete, distill and separate at 75°C to separate the solvent dimethyl sulfoxide, the product obtained after separation is modifier 2, place modifier 2 in a drying oven at 50°C to dry, grind the dried modifier 2 into powder for standby use;
[0025] A3. Take 10g of gelatin and place it in 100ml of distilled water. Heat and dissolve it in a water bath for 30min. After it is completely dissolved, adjust the pH of the solution to 8.5 with 1M NaOH. At the same time, evenly add the powdered modifier 2 while stirring until the solution is completely mixed. The final solution obtained is the modified gelatin solution.
[0026] A4. After the modified gelatin solution is precipitated and washed with 10M sulfuric acid, it is melted with distilled water. After melting, the pH is adjusted to 7.0 with 1M NaOH. After freeze-drying, the desired modified gelatin is obtained.
[0027] After adopting the above technical solution, the present invention has the following advantages compared with the background technology:
[0028] 1. The present invention optimizes and innovates the formula, coating process and reagent cost. The first reagent layer is combined with modified 30% binder and modified gelatin, and connected with active materials to prepare a reagent layer with fast diffusion, penetration and uniform color development. The pancreatic amylase dry detection and analysis component of the present invention only needs to be coated with two layers (the first reagent layer and the second reagent layer), which greatly improves the problem of multi-layer coating in the prior art and improves the penetration effect, realizes the purpose of room temperature storage, simplifies the number of coatings, simplifies the process, and reduces the production cost. The test results are highly accurate and have a wide measurement range, which meets the needs of instant diagnosis and on-site diagnosis and can be applied to clinical value;
[0029] 2. In addition, the present invention uses a gauze mesh as a diffusion layer, which is environmentally friendly and safe, and can achieve rapid penetration and accurate detection of samples to be analyzed containing pancreatic amylase, thereby solving the problems of organic reagent pollution in the diffusion layer and harm to the human body and the environment in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 It is a structural schematic diagram of the components of the present invention;
[0031] Figure 2 is a linear correlation diagram of the component test results of Example 2 and the biochemical analyzer test results;
[0032] Figure 3 is a linear correlation diagram of the component test results of Example 3 and the biochemical analyzer test results;
[0033] Figure 4 It is a linear correlation diagram of the component test results of Example 4 and the biochemical analyzer test results.
[0034] Description of reference numerals:
[0035] 1. Upper support layer; 2. Diffusion layer; 3. First reagent layer; 4. Second reagent layer; 5. Support; 6. Lower support layer; 7. First card position; 8. Second card position; a. Sample loading hole; b. Test hole. DETAILED DESCRIPTION
[0036] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0037] Example 1
[0038] like Figure 1 As shown, a pancreatic amylase dry detection and analysis component, the component includes an upper support layer 1, a diffusion layer 2, a first reagent layer 3, a second reagent layer 4, a support body 5 and a lower support layer 6 from top to bottom, the upper support layer 1 and the lower support layer 6 are respectively connected at both ends by a first clamping position 7 and a second clamping position 8, which play the role of fixing and protecting the diffusion layer 2, the first reagent layer 3, the second reagent layer 4, and the support body 5; the middle position of the upper support layer 1 is provided with a sample addition hole a, and the middle position of the lower support layer 6 is provided with a test hole b; the support body 5 plays a supporting role, the diffusion layer 2 contains Buffer solution, anti-salivary amylase monoclonal antibody (Hbb antibody), bovine serum albumin, non-ionic polymer compound; the reagents contained in the first reagent layer 3 include buffer, modified 30% binder (i.e. poly (acrylamide-N-vinyl pyrrolidone)), modified gelatin, white gelatin, bovine serum albumin, PNPG7 (4,6-ethylene-p-nitrobenzene-Α-D-maltoheptaglycoside), surfactant, soluble cellulose ether, activator, defoamer and active material; the reagents contained in the second reagent layer 4 include D4 hydrogel with a concentration of 0.3g / L.
[0039] Specifically, the diffusion layer 2 uses a gauze with an opening rate of 300; in the diffusion layer 2, the buffer solution is a phosphate buffer solution with a concentration of 15 mmol / L and a pH of 7; the anti-salivary amylase monoclonal antibody is added in an amount of 80 mg per unit square meter; the bovine serum albumin is added in an amount of 4 g per unit square meter; the non-ionic polymer chemical uses PVPK90 and is added in an amount of 6 g per unit square meter.
[0040] The first reagent layer 3 and the second reagent layer 4 are made of a transparent plastic substrate polyethylene terephthalate with a light transmittance of more than 80% under a wavelength of 500 nm, and a thickness of 100 μm;
[0041] Among them, in the first reagent layer 3, the buffer is a phosphate buffer with a pH of 7; the modified 30% binder is added in an amount of 30 g per unit square meter; the white glue is added in an amount of 13 g per unit square meter to the first reagent layer; the PNPG7 (4,6-ethylene-p-nitrobenzene-Α-D-maltoheptaglycoside) is added in an amount of 2 g per unit square meter to the first reagent layer; the bovine serum albumin is added in an amount of 6 g per unit square meter to the first reagent layer; The activator is NaCl and is added to the first reagent layer at a rate of 0.8 g per square meter or per square meter; the surfactant is a zwitterionic surfactant Tween 20 and is added at a rate of 0.1 g per square meter; the soluble cellulose ether methyl cellulose is added at a rate of 15 g per square meter; the defoaming agent polyether defoaming agent is added at a rate of 0.7 ml per square meter; the active material diatomaceous earth is added at a rate of 30 g per square meter.
[0042] The support 5 is made of PET material with a thickness of 100 um.
[0043] The preparation method of the modified 30% binder is as follows:
[0044] The preparation method of the modified 30% binder is as follows:
[0045] S1. Weigh acrylamide and N-vinyl pyrrolidone in a mass ratio of 8:2 and dissolve them in 100 g of ultrapure water. After dissolution, weigh 2% of N,N'-methylenebisacrylamide and 1% of N,N,N',N'-tetramethylethylenediamine and stir them evenly in the solution.
[0046] S2, transferring the solution obtained in step S1 to a polymerization reactor, introducing nitrogen for 10 min, raising the temperature to 40° C. after the nitrogen is introduced, and maintaining the temperature for 30 min under the protection of nitrogen;
[0047] S3. Add 0.4% potassium persulfate and sodium bisulfite, continue to react at a constant temperature for 10 minutes, then react for 4 hours under sealed conditions, take out, and test the viscosity with a viscometer. The binder that passes the test is the desired binder.
[0048] The preparation method of the above modified gelatin is as follows:
[0049] A1. Mix 10 g of 3,4-dihydroxybenzoic acid and 50 ml of ethyl acetate solvent and stir until completely dissolved, then add 20 g of acetyl chloride for reflux reaction, the reflux temperature is controlled at 60° C., the reflux time is 3 h, after the reflux reaction is complete, distill and separate, the distillation temperature is controlled at 70° C., the ethyl acetate solvent is separated, the product obtained after separation is modifier 1, and the modifier 1 is placed in a 50° C. drying oven for drying;
[0050] A2, add trichloropropane to modifier 1 and stir and disperse, then add dimethyl sulfoxide and reflux, the reflux time is 3h, after the reaction is complete, distill and separate at 75°C to separate the solvent dimethyl sulfoxide, the product obtained after separation is modifier 2, place modifier 2 in a drying oven at 50°C to dry, grind the dried modifier 2 into powder for standby use;
[0051] A3. Take 10g of gelatin and place it in 100ml of distilled water. Heat and dissolve it in a water bath for 30min. After it is completely dissolved, adjust the pH of the solution to 8.5 with 1M NaOH. At the same time, evenly add the powdered modifier 2 while stirring until the solution is completely mixed. The final solution obtained is the modified gelatin solution.
[0052] A4. After the modified gelatin solution is precipitated and washed with 10M sulfuric acid, it is melted with distilled water. After melting, the pH is adjusted to 7.0 with 1M NaOH. After freeze-drying, the desired modified gelatin is obtained.
[0053] The detection method of the pancreatic amylase dry detection and analysis component of the present invention is as follows:
[0054] 10ul of the serum sample to be tested is vertically dripped into the sample well a, and the sample diffuses to the diffusion layer 2, the first reagent layer 3 and the second reagent layer 4 in turn, and incubated at about 37°C for 200s. During this process, PNPG7 is hydrolyzed by a-glycosidase to produce PNP and glucose. This substance PNP has a maximum absorption peak at a wavelength of 405nm, and the activity of pancreatic amylase is calculated by the change in the reflectivity of the absorption peak. The equation for the corresponding substance reaction of the pancreatic amylase dry analysis reagent is as follows. The corresponding substance reaction of the pancreatic amylase dry analysis reagent is as follows:
[0055] (1) PNPG7+H2O generates PNPG3 and PNPG2 under P-AMY conditions;
[0056] (2) PNPG3+PNPG2+H2O generates PNP and Glucose under a-glucosidase conditions.
[0057] The dry analytical reagent for detecting pancreatic amylase of the present invention has good stability, is easy to store and preserve, has high detection accuracy and a wide linear range.
[0058] Example 2
[0059] This example specifically introduces the preparation of a pancreatic amylase dry detection and analysis component and the test results are as follows:
[0060] (1) Solution preparation of the second reagent layer: Weigh 30 g of D4 hydrogel into 100 ml of mixed alcohol, where the ratio of alcohol to water is 9:1, and wait until it is completely dissolved to obtain the solution of the second reagent layer;
[0061] (2) Solution preparation of the first reagent layer: Weigh Hepes (4-hydroxyethylpiperazineethanesulfonic acid-Hepes), BSA (bovine serum albumin), PNPG7, Nacl, Mgcl, and white glue in accordance with the amount and add them to the aqueous solution in sequence. After they are completely dissolved, add active materials, modified gelatin, modified 30% binder, surfactant, and soluble cellulose ether to the solution in sequence. Stir with a spiral stirrer at a speed of 500 r / min for 1 h. When the time is up, continue to add defoaming agent to the slurry. At this time, adjust the rotation speed from 500 r / min to 400 r / min. Stir for 1 h before coating.
[0062] (3) Preparation of diffusion layer solution: Weigh a certain mass of buffer solution, add BSA, non-ionic polymer compound PVPK30, and anti-salivary amylase monoclonal antibody in sequence, stir evenly, and use scraping technology to soak, scrape and dry the gauze with an opening rate of 200 or 300. The drying temperature is controlled at 60°C and the drying time is controlled at 10-15min;
[0063] (4) Coating: Place the transparent 125um thick PET film support 4 on the coating machine, press both ends, return the micrometer to zero, set the coating machine wet film thickness to 80um, transfer the D4 hydrogel of the second reagent layer to the bottom of the scraper, press the coating button, and the scraper starts coating. The coating speed is controlled at 50cm / min. Place the coated second reagent layer in a 50℃ oven for constant temperature drying, and the drying time is 5min. After drying, place it back on the coating machine, set the coating machine wet film thickness to 360um, pour the slurry of the first reagent layer to the bottom of the scraper, and press the coating button to start coating. Place the coated reagent layer 2 in a 50℃ oven for drying, and the drying time is 20min. The reagent layer after drying is cut with a long strip cutter with a specification of 294*9.5mm. The gauze is then cut into the same size and then cut into small pieces of 9.5*9.5mm2. Finally, a black PET card sleeve is used as the upper support layer, lower support layer and middle support body to fix and assemble into a dry detection and analysis component.
[0064] Table 1 Formula of the first reagent layer in Example 2
[0065] Reagents <![CDATA[Coating amount / m 2 > Hepes buffer PH7.0 10% white glue 10ml Hexadecyltrimethylammonium bromide 200mg Modified Gelatin 12g PNPG7 1.5g Nacl 250mg Mgcl 150mg Modified 30% Binder 25g Alcohol defoamer 1g Methylcellulose 5g Cellulose powder 30g Volcanic ash 5g Twain 80 0.5g
[0066] Table 2 Diffusion layer formulation in Example 2
[0067]
[0068]
[0069] 25 serum samples of different gradient concentrations were prepared in proportion by Randox quality control, and the correlation of multiple test samples was analyzed by the method of this embodiment and the biochemical analyzer test method. The results are as follows: Figure 2 As shown, the test results of the method of this embodiment and the test results of the biochemical analyzer method show a good linear correlation.
[0070] Example 3
[0071] This example specifically introduces the preparation of a pancreatic amylase dry detection and analysis component and the test results are as follows:
[0072] (1) Solution preparation of the second reagent layer: Weigh 30 g of D4 hydrogel into 100 ml of mixed alcohol (where the ratio of alcohol to water is 9:1) and wait for dissolution;
[0073] (2) Solution preparation for the first reagent layer: Weigh the buffer solution according to the amount, add BSA, PNPG7, Nacl, Mgcl, and white glue in the reagent group to the solution, and add active materials, modified gelatin, modified 30% binder, surfactant, and soluble cellulose ether in sequence after the solution is completely dissolved. Stir with a spiral stirrer at a speed of 400 r / min for 2 h. When the time is up, continue to add defoaming agent to the slurry. At this time, the speed is adjusted from 400 r / min to 300 r / min. Stir for 1 h before coating;
[0074] (3) Preparation of diffusion layer solution: Weigh a certain mass of buffer solution, add BSA, non-ionic polymer compound PVPK90, and hbb antibody in turn, stir evenly, and use scraping technology to soak, scrape and dry the gauze with an opening rate of 200 or 300. The drying temperature is controlled at 60°C and the drying time is controlled at 10-15 minutes;
[0075] (4) Coating: Place the transparent PET film support 4 on the coating machine, press the two ends, return the micrometer to zero, set the wet film thickness of the coating machine to 100um, transfer the D4 hydrogel of the reagent layer 1 to the bottom of the scraper, press the coating button, and the scraper starts coating. The coating speed is controlled at 80cm / min. Place the coated reagent layer 1 in a 60℃ oven for constant temperature drying. The drying time is 5min. After drying, place it back on the coating machine, set the wet film thickness of the coating machine to 340um, pour the slurry of the reagent layer 2 to the bottom of the scraper, and press the coating button to start coating. Put the coated reagent layer 2 into a 60℃ oven for drying, and the drying time is 15min. The reagent layer to be dried is cut with a long strip cutter, and the cutting specification is 294*9.5mm. The gauze was then cut into the same size, and then cut into small pieces of 9.5*9.5 mm2. Finally, a black PET card sleeve was used as the upper support layer, the lower support layer and the middle support body to fix and assemble them into a dry detection and analysis component, and then used for measurement.
[0076] Table 3 Formula of the first reagent layer in Example 3
[0077] Reagents <![CDATA[Coating amount / m 2 > Phosphate buffered saline PH7.2 10% white glue 10ml Hexadecyltrimethylammonium bromide 250mg Modified Gelatin 10g Nacl 300mg Mgcl 100mg PNPG7 1.5g Modified 30% Binder 22g Alcohol defoamer 1g Methylcellulose 10g Silicic acid gel 30g Volcanic ash 5g Twain 20 1g
[0078] Table 4 Diffusion layer formulation in Example 3
[0079]
[0080]
[0081] 25 serum samples of different gradient concentrations were prepared in proportion by Randox quality control, and the correlation of multiple test samples was analyzed by the method of this embodiment and the biochemical analyzer test method. The results are as follows: Figure 3 As shown, the test results of the method of this embodiment and the test results of the biochemical analyzer method show a good linear correlation.
[0082] Example 4
[0083] This example specifically introduces the preparation of a pancreatic amylase dry detection and analysis component and the test results are as follows:
[0084] (1) Solution preparation of the second reagent layer: Weigh 30 g of D4 hydrogel into 100 ml of mixed alcohol (where the ratio of alcohol to water is 9:1) and wait for dissolution;
[0085] (2) Solution preparation for the first reagent layer: Buffer solution. Add BSA, PNPG7, Nacl, Mgcl, and white glue reagent in the reagent group to the solution in sequence. After the solution is completely dissolved, add active materials, modified gelatin, modified 30% binder, surfactant, and soluble cellulose ether in sequence. Stir with a spiral stirrer at a speed of 550 r / min for 2 h. When the time is up, continue to add defoaming agent to the slurry. At this time, adjust the speed from 550 r / min to 400 r / min. Stir for 1 h before coating.
[0086] (3) Preparation of diffusion layer solution: Weigh a certain mass of buffer solution, add BSA, non-ionic polymer compound PVPK30 and hbb antibody in turn, stir evenly, and use scraping technology to soak, scrape and dry the gauze with an opening rate of 200 or 300. The drying temperature is controlled at 60°C and the drying time is controlled at 10-15min;
[0087] (4) Coating: Place the transparent 125um thick PET film support 4 on the coating machine, press the two ends, return the micrometer to zero, set the coating machine wet film thickness to 90um, transfer the D4 hydrogel of the reagent layer 1 to the bottom of the scraper, press the coating button, and the scraper starts coating. The coating speed is controlled at 70cm / min. Place the coated reagent layer 1 in a 70℃ oven for constant temperature drying. The drying time is 5min. After drying, place it back on the coating machine, set the coating machine wet film thickness to 350um, pour the slurry of the reagent layer 2 to the bottom of the scraper, and press the coating button to start coating. Place the coated reagent layer 2 in a 70℃ oven for drying, and the drying time is 10min. The reagent layer to be dried is cut with a long strip cutter, and the cutting specification is 294*9.5mm. The gauze was then cut into the same size, and then cut into small pieces of 9.5*9.5 mm2. Finally, a black PET card sleeve was used as the upper support layer, the lower support layer and the middle support body to fix and assemble them into a dry detection and analysis component, and then used for measurement.
[0088] Table 5: Formula of the first reagent layer in Example 4
[0089] Reagents <![CDATA[Coating amount / m 2 > Phosphate buffered saline PH6.8 10% white glue 10ml Dodecyltrimethylammonium bromide 250mg Modified Gelatin 8g PNPG7 1.5g Nacl 450mg Mgcl 75mg Modified 30% Binder 22g Alcohol defoamer 1g Ethyl cellulose 12g Microcrystalline Cellulose 30g Bentonite 5g Twain 20 2g
[0090] Table 6 Diffusion layer formulation in Example 4
[0091] Reagents Coating amount / m2 Phosphate buffered saline PH6.8 BSA 3g PVPk30 1.5g Hbb antibody 50mg
[0092] 25 serum samples of different gradient concentrations were prepared in proportion by Randox quality control, and the correlation of multiple test samples was analyzed by the method of this embodiment and the biochemical analyzer test method. The results are as follows: Figure 4As shown, the test results of the method of this embodiment and the test results of the biochemical analyzer method show a good linear correlation.
[0093] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.
Claims
1. A pancreatic amylase dry type detection and analysis component, characterized in that: The component comprises an upper support layer, a diffusion layer, a first reagent layer, a second reagent layer, a support and a lower support layer in order from top to bottom; the two ends of the upper support layer and the lower support layer are respectively connected by snaps; a sample loading hole is arranged in the middle of the upper support layer, and a test hole is arranged in the middle of the lower support layer; the reagents contained in the diffusion layer include a buffer solution, an anti-salivary amylase monoclonal antibody, bovine serum albumin, and a non-ionic polymer compound; the reagents contained in the first reagent layer include a buffer solution, a modified 30% binder, modified gelatin, white gelatin, bovine serum albumin, PNPG7, a surfactant, a soluble cellulose ether, an activator, a defoaming agent and an active material; the reagents contained in the second reagent layer include a D4 hydrogel with a concentration of 0.3 g / L.
2. A pancreatic amylase dry type detection and analysis component as claimed in claim 1, characterized in that: The diffusion layer is made of a mesh with an opening rate of 200 or 300; The first reagent layer and the second reagent layer are transparent plastic substrates or frosted plastic substrates with a light transmittance of more than 80% under a wavelength of 300-800nm, including any one of polyethylene terephthalate, polycarbonate, polypropylene, polyethylene, and polycarbonate sheets, and the thickness thereof is 30-200μm; The support body is made of PET material with a thickness of 100-200 um, including any one of polyethylene terephthalate, polybutylene terephthalate, and polyvinyl chloride.
3. A pancreatic amylase dry type detection and analysis component as claimed in claim 2, characterized in that: In the diffusion layer, the buffer solution includes any one or more of phosphate buffer, Hepes buffer, and MES buffer, with a concentration of 10-20 mmol / L and a pH of 5-9; the anti-salivary amylase monoclonal antibody is added in an amount of 1-200 mg per unit square meter or 5-100 mg per unit square meter; the bovine serum albumin is added in an amount of 1-15 g per unit square meter or 1-5 g per unit square meter; the non-ionic polymer chemical includes any one or two of PVPK90, PVPK30, and PVPK15, and is added in an amount of 1-10 g per unit square meter or 1-7 g per unit square meter.
4. A pancreatic amylase dry type detection and analysis component as claimed in claim 3, characterized in that: The modified 30% binder is added to the first reagent layer in an amount of 1-50 g per unit square meter or 1-40 g per unit square meter; The preparation method of the modified 30% binder is as follows: S1. Weigh acrylamide and N-vinyl pyrrolidone in a mass ratio of 8:2 and dissolve them in 100 g of ultrapure water. After dissolution, weigh 2% of N,N'-methylenebisacrylamide and 1% of N,N,N',N'-tetramethylethylenediamine and stir them evenly in the solution. S2, transferring the solution obtained in step S1 to a polymerization reactor, introducing nitrogen for 10 min, raising the temperature to 40° C. after the nitrogen is introduced, and maintaining the temperature for 30 min under the protection of nitrogen; S3. Add 0.4% potassium persulfate and sodium bisulfite, continue to react at a constant temperature for 10 minutes, then react for 4 hours under sealed conditions, take out, and test the viscosity with a viscometer. The binder that passes the test is the desired binder.
5. A pancreatic amylase dry type detection and analysis component as claimed in claim 4, characterized in that: In the first reagent layer, the buffer includes any one or more of phosphate buffer, Hepes buffer, and MES buffer, and its pH is 5-9; the white glue is added to the first reagent layer in an amount of 0.05-30g per unit square meter or 0.1-15g per unit square meter; the PNPG7 is added to the first reagent layer in an amount of 0.05-5g per unit square meter or 0.1-2.5g per unit square meter; the bovine serum albumin is added to the first reagent layer in an amount of 0.05-10g per unit square meter or 0.1-7g per unit square meter; the activator uses Nacl or Mgcl, and is added to the first reagent layer in an amount of 0.01-1g per unit square meter or 0.05-1g per unit square meter.
6. A pancreatic amylase dry type detection and analysis component as claimed in claim 5, characterized in that: The surfactant is added to the first reagent layer in an amount of 0.05-5 g per unit square meter or 0.07-2 g per unit square meter; The surfactant is a zwitterionic surfactant, a cationic surfactant or an anionic surfactant; the zwitterionic surfactant includes any one of Tween 20, Tween 80, Triton X-405 and Triton X-100; the cationic surfactant includes any one of hexadecyltrimethylammonium bromide and dodecyltrimethylammonium bromide; the anionic surfactant includes any one or more of sodium dodecylbenzene sulfonate and sodium dodecyl sulfate.
7. A pancreatic amylase dry type detection and analysis component as claimed in claim 6, characterized in that: The soluble cellulose ether is added to the first reagent layer in an amount of 1-30 g per unit square meter or 1-20 g per unit square meter; the soluble cellulose ether includes any one of methyl cellulose, carboxymethyl cellulose, ethyl cellulose, benzyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, cyanoethyl cellulose, benzyl cyanoethyl cellulose, carboxymethyl hydroxyethyl cellulose and phenyl cellulose.
8. A pancreatic amylase dry type detection and analysis component as claimed in claim 7, characterized in that: The defoamer is added to the first reagent layer in an amount of 0.1-5 ml per unit square meter or 0.5-2 ml per unit square meter; the defoamer includes any one or more of silicone defoamers, polyether defoamers, alcohol defoamers, and composite defoamers.
9. A pancreatic amylase dry type detection and analysis component as claimed in claim 8, characterized in that: The active material is added to the first reagent layer in an amount of 1-50 g per unit square meter or 10-40 g per unit square meter; the active material includes any one or more of diatomaceous earth, volcanic ash, silica gel, fly ash, microcrystalline cellulose, bentonite, titanium dioxide, burnt clay, and cellulose powder.
10. A pancreatic amylase dry type detection and analysis component according to any one of claims 1 to 9, characterized in that: The preparation method of the modified gelatin is as follows: A1. Mix 10 g of 3,4-dihydroxybenzoic acid and 50 ml of ethyl acetate solvent and stir until completely dissolved, then add 20 g of acetyl chloride for reflux reaction, the reflux temperature is controlled at 60° C., the reflux time is 3 h, after the reflux reaction is complete, distill and separate, the distillation temperature is controlled at 70° C., the ethyl acetate solvent is separated, the product obtained after separation is modifier 1, and the modifier 1 is placed in a 50° C. drying oven for drying; A2, add trichloropropane to modifier 1 and stir and disperse, then add dimethyl sulfoxide and reflux, the reflux time is 3h, after the reaction is complete, distill and separate at 75°C to separate the solvent dimethyl sulfoxide, the product obtained after separation is modifier 2, place modifier 2 in a drying oven at 50°C to dry, grind the dried modifier 2 into powder for standby use; A3. Take 10g of gelatin and place it in 100ml of distilled water. Heat and dissolve it in a water bath for 30min. After it is completely dissolved, adjust the pH of the solution to 8.5 with 1M NaOH. At the same time, evenly add the powdered modifier 2 while stirring until the solution is completely mixed. The final solution obtained is the modified gelatin solution. A4. After the modified gelatin solution is precipitated and washed with 10M sulfuric acid, it is melted with distilled water. After melting, the pH is adjusted to 7.0 with 1M NaOH. After freeze-drying, the desired modified gelatin is obtained.