CN109580927B - Liver function test card and preparation method thereof - Google Patents
Liver function test card and preparation method thereof Download PDFInfo
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- CN109580927B CN109580927B CN201910008631.0A CN201910008631A CN109580927B CN 109580927 B CN109580927 B CN 109580927B CN 201910008631 A CN201910008631 A CN 201910008631A CN 109580927 B CN109580927 B CN 109580927B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
Abstract
The invention discloses a liver function test card and a preparation method thereof, wherein the test card comprises the following components: the kit comprises an upper shell, a lower shell, a sampling hole arranged on the upper shell, and a glutamic-pyruvic transaminase detection unit, a total bilirubin detection unit and a glutamic-oxalacetic transaminase detection unit which are arranged between the upper shell and the lower shell; the glutamic-pyruvic transaminase detection unit, the total bilirubin detection unit and the glutamic-oxalacetic transaminase detection unit comprise: the detection device comprises a bottom plate arranged at the upper end of a lower shell, a lower shell detection hole arranged on the lower shell, a bottom plate detection hole arranged on the bottom plate, a diffusion membrane arranged at the lower end of an upper shell and connected to the bottom plate, a whole blood separation layer arranged under the diffusion membrane, and a reaction layer arranged between the whole blood separation layer and the bottom plate detection hole; the test card is matched with a dry biochemical analyzer for use, is simple to operate, can quickly and simultaneously detect the contents of three items of glutamic-pyruvic transaminase, total bilirubin and glutamic-oxaloacetic transaminase without professionals, and has good matching of the detection result and the hospital detection result.
Description
Technical Field
The invention relates to the field of detection of dry chemistry, in particular to a liver function test card and a preparation method thereof.
Background
Viral hepatitis is widely existed all over the world and seriously threatens human health, and is particularly important for detecting liver functions, and liver function detection mainly comprises four indexes: transaminase, bilirubin, albumin and liver fiber are basically examined by major biochemistry in hospitals at present, the operation is complex, the charge is expensive, and the operation is required by professionals.
Liver function examination, items which are examined by adopting a dry chemical method on the market at present are only transaminase, such as Aikan biological ALT and AST test strips, the corresponding patent number is 201010542684.X, ALT/GPT test strips of Nanjing big trees, the corresponding patent number is CN 206146942U and the like, although several families in China have related product information, such as patent CN 102419366A, the product which is really sold on the market only has Aikan biological and only has alanine transaminase ALT test strips, only one item can be detected, and the liver function can not be well reflected; there are also related patents abroad such as US4591553, US4897347, US5462858, US65657381B1, US570545, etc., but there is no actual product on the market, and there are also only products of both ALT and AST items. The detection of liver function in blood is mainly a kit. The kit is mainly used together with a large-scale biochemical analyzer, has higher detection cost, needs professional technicians to operate, has poor mobility and is difficult to popularize. Therefore, the liver function index is the trend of future development by adopting a dry chemical method, the dry chemical test card has the characteristics of simple and convenient operation, easy use and low cost in the test process, is convenient to carry, bears the function of a laboratory, does not need traditional hospital laboratory equipment, has no liquid pollution treatment requirement, can be directly burnt, reduces the biological pollution risk and the treatment cost, is very suitable for being used in primary hospitals and blood stations, and can serve patients in all directions without being limited by time and place. Meanwhile, the method is operated in time, so that experimental results can be obtained more quickly, the occupation of resources is reduced, the medical cost of the society is reduced, and the expenditure of patients who do not need further treatment is reduced.
The market needs a test card that can test multiple items at one time, suitable for each pharmacy, clinic, research institute, community hospital, pet hospital, inspection center or other government screening, and the present invention addresses such a problem.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a test card for liver functions and a preparation method thereof, the test card is matched with a dry biochemical analyzer for use, the operation is simple, the contents of three items of glutamic-pyruvic transaminase, total bilirubin and glutamic-oxalacetic transaminase in whole blood, serum/plasma and other body fluids of a human body or an animal can be rapidly and simultaneously detected without professionals, and the matching of the detection result and the hospital detection result is good.
In order to achieve the above object, the present invention adopts the following technical solutions:
a liver function test card comprising: the kit comprises an upper shell, a lower shell, a sampling hole arranged on the upper shell, and a glutamic-pyruvic transaminase detection unit, a total bilirubin detection unit and a glutamic-oxalacetic transaminase detection unit which are arranged between the upper shell and the lower shell; the glutamic-pyruvic transaminase detection unit, the total bilirubin detection unit and the glutamic-oxalacetic transaminase detection unit comprise: set up in the bottom plate of the upper end of inferior valve, set up the inferior valve inspection hole on the inferior valve, set up the bottom plate inspection hole on the bottom plate, set up in the epitheca lower extreme and connect in the diffusion barrier of bottom plate, set up in whole blood separating layer under the diffusion barrier, set up in reaction layer between whole blood separating layer and the bottom plate inspection hole.
In the liver function test card, the glutamic-pyruvic transaminase reaction solution comprises: 10-40KU pyruvate oxidase, 1-20KU peroxidase, 1-20KU ascorbic acid, 0.1-2g L-alanine, 1-4g alpha-ketoglutaric acid, 0.1-3g thiamine pyrophosphate, 0.1-2g flavin adenine dinucleotide, 0.5-4g film forming agent, 2-20g stabilizer, 0.1-5g surfactant, 0.1-10g color developing agent, and 0.1-1M buffer solution.
In the above test card for liver function, the total bilirubin reaction solution includes: 1-50g of alkaline sodium tartrate, 0.1-5g of surfactant and 0.1-1M buffer solution.
In the liver function test card, the glutamic oxalacetic transaminase reaction solution includes: 5-50KU pyruvate oxidase, 5-40KU oxaloacetate decarboxylase, 1-50KU peroxidase, 5-40KU ascorbic acid, 0.1-5g L-sodium aspartate, 0.1-5g alpha-ketoglutarate, 0.1-5g thiamine pyrophosphate, 0.5-4g film forming agent, 2-20g stabilizer, 0.1-5g surfactant, 0.1-10g color developing agent and 0.1-1M buffer solution.
The above-mentioned test card for liver function, the glutamic-pyruvic transaminase detecting unit, the total bilirubin detecting unit, the glutamic-oxalacetic transaminase detecting unit further include: and the auxiliary layer is arranged between the whole blood separation layer and the reaction layer.
In the test card for liver function, the total bilirubin auxiliary liquid includes: 1-20g of caffeine, 1-20g of sodium benzoate, 1-10g of sodium nitrite, 1-10g of sulfanilic acid, 1-10g of sodium azide, 0.1-5gOP-10 g of sodium acetate and 2-40g of sodium acetate are added into 1L of pure water.
In the liver function test card, the glutamic-pyruvic transaminase auxiliary liquid and the glutamic-oxalacetic transaminase auxiliary liquid include: 5-50g of polyvinylpyrrolidone, 1-20g of magnesium chloride and 0.1-5gOP-10 were added to 1L of 0.1-1MpH phosphoric acid buffer solution with a value of 7.20.
In the liver function test card, the whole blood separation layer is a porous cellulose membrane.
A method for preparing a test card for liver function comprises the following steps:
preparing a glutamic-pyruvic transaminase reaction liquid, namely 10-40KU pyruvate oxidase, 1-20KU peroxidase, 1-20KU ascorbic acid, 0.1-2g L-alanine, 1-4g alpha-ketoglutaric acid, 0.1-3g thiamine pyrophosphate, 0.1-2g flavin adenine dinucleotide, 0.5-4g film forming agent, 2-20g stabilizer, 0.1-5g surfactant, 0.1-10g developer and 0.1-1M buffer solution, stirring for one hour for later use;
preparing total bilirubin reaction liquid, namely 1-50g of alkaline sodium tartrate, 0.1-5g of surfactant and 0.1-1M buffer solution, and stirring for one hour for later use;
preparing a glutamic-oxaloacetic transaminase reaction solution, wherein the glutamic-oxaloacetic transaminase reaction solution comprises the following components: 5-50KU pyruvate oxidase, 5-40KU oxaloacetate decarboxylase, 1-50KU peroxidase, 5-40KU ascorbic acid, 0.1-5g L-sodium aspartate, 0.1-5g alpha-ketoglutarate, 0.1-5g thiamine pyrophosphate, 0.5-4g film forming agent, 2-20g stabilizer, 0.1-5g surfactant, 0.1-10g color developing agent and 0.1-1M buffer solution are stirred for one hour for standby application;
preparing total bilirubin auxiliary liquid: 1-20g of caffeine, 1-20g of sodium benzoate, 1-10g of sodium nitrite, 1-10g of sulfanilic acid, 1-10g of sodium azide and 0.1-5gOP-10 g of sodium acetate, and 2-40g of sodium acetate are added into 1L of pure water and stirred for two hours for later use;
preparing glutamic-pyruvic transaminase auxiliary liquid and glutamic-oxalacetic transaminase auxiliary liquid: adding 30g of polyvinylpyrrolidone, 10g of magnesium chloride and 0.5-0.5 gOP-10 into 1L of 0.1MpH phosphoric acid buffer solution with the value of 7.20, and stirring for two hours for later use;
preparing a reaction layer: soaking the glutamic-pyruvic transaminase reaction layer in glutamic-pyruvic transaminase reaction solution for 1-10min, taking out, and drying at 25-50 deg.C for 5-50 min; soaking the total bilirubin reaction layer in the total bilirubin reaction liquid for 1-10min, taking out, and drying at 25-50 deg.C for 5-50 min; soaking the reaction layer in the reaction solution for 1-10min, taking out, and drying at 25-50 deg.C for 5-50 min;
manufacturing an auxiliary layer: soaking the auxiliary layer in auxiliary liquid for 1-10min, taking out, and drying at 25-50 deg.C for 5-50 min; soaking glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase auxiliary layers in glutamic-pyruvic transaminase auxiliary solution and glutamic-oxalacetic transaminase auxiliary solution for 1-10min, respectively, taking out, and drying at 25-50 deg.C for 5-50 min;
respectively stacking the bottom plate, the reaction layer, the auxiliary layer, the whole blood separation membrane and the diffusion membrane in the plastic lower shell, and tightly covering the plastic upper shell to obtain a finished test card.
The invention has the advantages that:
the test card is matched with a dry biochemical analyzer for use, the operation is simple, and no professional is needed;
the contents of three items of glutamic-pyruvic transaminase, total bilirubin and glutamic-oxalacetic transaminase in whole blood, serum/plasma and other body fluids of a human body or an animal can be rapidly and simultaneously detected;
the detection result has good matching with the hospital test result.
Drawings
FIG. 1 is an exploded view of one embodiment of the test card of the present invention;
FIG. 2 is a schematic cross-sectional view of one embodiment of a test card of the present invention;
FIG. 3 is a linear relationship of glutamic-pyruvic transaminase value to hospital value for finished product 2 of the invention;
FIG. 4 is a linear relationship of total bilirubin values and hospital values for finished product 2 of the present invention;
FIG. 5 is a linear relationship between the glutamic-oxaloacetic transaminase value of the product 2 of the present invention and the hospital value.
The meaning of the reference symbols in the figures:
100 inferior shells, 101 glutamic-pyruvic transaminase detection holes, 102 total bilirubin detection holes, 103 glutamic-oxalacetic transaminase detection holes, 200 bottom plates, 201 glutamic-pyruvic transaminase bottom plate detection holes, 202 total bilirubin bottom plate detection holes, 203 glutamic-oxalacetic transaminase bottom plate detection holes, 300 reaction layers, 301 glutamic-oxalacetic transaminase reaction layers, 302 total bilirubin reaction layers, 303 glutamic-oxalacetic transaminase reaction layers, 400 auxiliary layers, 401 glutamic-pyruvic transaminase auxiliary layers, 402 total bilirubin auxiliary layers, 403 glutamic-oxalacetic transaminase auxiliary layers, 500 whole blood separation layers, 501 glutamic-pyruvic transaminase whole blood separation layers, 502 glutamic-pyruvic transaminase whole blood separation layers, 503 glutamic-oxalacetic transaminase whole blood separation layers, 600 diffusion membranes, 700 superior shells and 701 sample adding holes.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
A liver function test card comprising: an upper shell 700, a lower shell 100, a sample adding hole 701 arranged on the upper shell 700, a glutamic-pyruvic transaminase detecting unit, a total bilirubin detecting unit and a glutamic-oxalacetic transaminase detecting unit arranged between the upper shell 700 and the lower shell 100; the glutamic-pyruvic transaminase detection unit, the total bilirubin detection unit and the glutamic-oxalacetic transaminase detection unit comprise: the bottom plate 200 that sets up in the upper end of inferior valve 100, the inferior valve inspection hole that sets up on inferior valve 100, the bottom plate inspection hole that sets up on bottom plate 200, set up in the diffusion barrier 600 that goes up the 700 lower extreme of epitheca and be connected to bottom plate 200, set up the whole blood separation layer under the diffusion barrier 600, set up the reaction layer 300 between whole blood separation layer and bottom plate inspection hole, set up in auxiliary layer between whole blood separation layer and the reaction layer 300. Inferior valve inspection socket includes: glutamic-pyruvic transaminase detection hole 101, total bilirubin detection hole 102 and glutamic-oxalacetic transaminase detection hole 103. The bottom plate inspection hole includes: a glutamic-pyruvic transaminase bottom plate detection hole 201, a total bilirubin bottom plate detection hole 202 and a glutamic-oxalacetic transaminase bottom plate detection hole 203. The auxiliary layer 400 includes: glutamic-pyruvic transaminase auxiliary layer 401, total bilirubin auxiliary layer 402 and glutamic-oxaloacetic transaminase auxiliary layer 403. The whole blood separation layer 500 includes: a glutamic-pyruvic transaminase whole blood separation layer 501, a glutamic-pyruvic transaminase whole blood separation layer 502 and a glutamic-oxaloacetic transaminase whole blood separation layer 503. As an example, the bottom plate 200 is made of single-sided adhesive made of PET or PVC; the reaction layer 300 adopts a cellulose film or a nylon film; the auxiliary layer adopts a glass fiber film or filter paper or a nylon film; the whole blood separation layer adopts a cellulose membrane with a plurality of pore diameters; the diffusion film 600 is a nylon film.
The glutamic-pyruvic transaminase reaction solution comprises: 10-40KU pyruvate oxidase, 1-20KU peroxidase, 1-20KU ascorbic acid, 0.1-2g L-alanine, 1-4g alpha-ketoglutaric acid, 0.1-3g thiamine pyrophosphate, 0.1-2g flavin adenine dinucleotide, 0.5-4g film forming agent, 2-20g stabilizer, 0.1-5g surfactant, 0.1-10g color developing agent, and 0.1-1M buffer solution.
The total bilirubin reaction solution comprises: 1-50g of alkaline sodium tartrate, 0.1-5g of surfactant and 0.1-1M buffer solution.
The glutamic-oxaloacetic transaminase reaction liquid comprises: 10-40KU pyruvate oxidase, 1-20KU peroxidase, 1-20KU ascorbic acid, 0.1-2g L-alanine, 1-4g alpha-ketoglutaric acid, 0.1-3g thiamine pyrophosphate, 0.1-2g flavin adenine dinucleotide, 0.5-4g film forming agent, 2-20g stabilizer, 0.1-5g surfactant, 0.1-10g color developing agent, and 0.1-1M buffer solution.
As an example, the film-forming agent comprises: one or more of hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, chitosan, cyclodextrin and glucose sulfate.
The stabilizer comprises: proteins: bovine serum albumin, casein, and the like; polysaccharide substance: sucrose, trehalose, mannitol, and the like; polyethylene glycol stabilizer: polyethylene glycol 10000, polyethylene glycol 6000, polyethylene glycol 1000, and the like; one or more of these.
The surfactant includes: one or more of tween-20, tween-80, triton X-100, triton X-405, Emulgen B66, OP-10, etc.
The color developing agent comprises: 3,3',5,5' -tetramethylbenzidine, N, N-bis (4-sulfobutyl) -3-methylaniline disodium salt, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-bismethylaniline sodium salt, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3' 5-dimethoxyaniline sodium salt, benzoquinone,: one or more of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt, methyl red, bromothymol blue and methyl blue.
The buffer solution comprises: one or more of phosphate buffer, TRIS buffer and MES buffer.
A method for preparing a test card for liver function comprises the following steps:
preparing a glutamic-pyruvic transaminase reaction liquid, namely 10-40KU pyruvate oxidase, 1-20KU peroxidase, 1-20KU ascorbic acid, 0.1-2g L-alanine, 1-4g alpha-ketoglutaric acid, 0.1-3g thiamine pyrophosphate, 0.1-2g flavin adenine dinucleotide, 0.5-4g film forming agent, 2-20g stabilizer, 0.1-5g surfactant, 0.1-10g developer and 0.1-1M buffer solution, stirring for one hour for later use;
preparing total bilirubin reaction liquid, namely stirring 1-50g of alkaline sodium tartrate, 0.1-5g of surfactant and 0.1-1M buffer solution for one hour for later use;
preparing a glutamic-oxaloacetic transaminase reaction solution, wherein the glutamic-oxaloacetic transaminase reaction solution comprises the following components: 5-50KU pyruvate oxidase, 5-40KU oxaloacetate decarboxylase, 1-50KU peroxidase, 5-40KU ascorbic acid, 0.1-5g L-sodium aspartate, 0.1-5g alpha-ketoglutarate, 0.1-5g thiamine pyrophosphate, 0.5-4g film forming agent, 2-20g stabilizer, 0.1-5g surfactant, 0.1-10g color developing agent and 0.1-1M buffer solution are stirred for one hour for standby application;
preparing total bilirubin auxiliary liquid: adding 1-20g of caffeine, 1-20g of sodium benzoate, 1-10g of sodium nitrite, 1-10g of sulfanilic acid, 1-10g of sodium azide and 0.1-5gOP-10 g of sodium acetate into 1L of pure water, and stirring for two hours for later use;
preparing glutamic-pyruvic transaminase auxiliary liquid and glutamic-oxalacetic transaminase auxiliary liquid: adding 30g of polyvinylpyrrolidone, 10g of magnesium chloride and 0.5gOP-10 into 1L of 0.1MpH phosphoric acid buffer solution with the value of 7.20, and stirring for two hours for later use;
preparation of the reaction layer 300: soaking the glutamic-pyruvic transaminase reaction layer 300 in glutamic-pyruvic transaminase reaction solution for 1-10min, taking out, and drying at 25-50 deg.C for 5-50 min; soaking the total bilirubin reaction layer 300 in the total bilirubin reaction liquid for 1-10min, taking the total bilirubin reaction liquid, and drying at 25-50 ℃ for 5-50min for later use; soaking the glutamic-oxaloacetic transaminase reaction layer 300 in the glutamic-oxaloacetic transaminase reaction solution for 1-10min, taking out, and drying at 25-50 deg.C for 5-50 min;
manufacturing an auxiliary layer: soaking the auxiliary layer in auxiliary liquid for 1-10min, taking out, and drying at 25-50 deg.C for 5-50 min; soaking glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase auxiliary layers in glutamic-pyruvic transaminase auxiliary solution and glutamic-oxalacetic transaminase auxiliary solution for 1-10min, respectively, taking out, and drying at 25-50 deg.C for 5-50 min;
the base plate 200, the reaction layer 300, the auxiliary layer, the whole blood separation membrane and the diffusion membrane 600 are respectively stacked in the plastic lower case 100, and then the plastic upper case 700 is tightly covered to manufacture a finished product of the test card.
And (3) a verification experiment part:
finished products 1, 2, 3, 4 were made according to the following example.
Example 1
The manufacturing method of the finished product 1 comprises the following steps:
the method comprises the following steps: preparing glutamic-pyruvic transaminase reaction liquid, namely adding 10KU pyruvate oxidase, 7KU peroxidase, 5KU ascorbic acid 0.3g of L-alanine, 1g of alpha-ketoglutaric acid, 0.5g of thiamine pyrophosphate, 0.5g of flavin adenine dinucleotide, 20ml of 1.5% polyvinyl alcohol, 1g of bovine serum albumin, 1g of trehalose, 0.1gtriton X-100,0.2g of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3' 5-dimethoxyaniline sodium salt and 1g of 4-aminoantipyrine into 1L of TRIS buffer solution with the value of 6.8 and 0.2MpH, and stirring for one hour for later use;
step two: preparing total bilirubin reaction liquid, namely adding 5g of alkaline sodium tartrate and 0.1g of Emulgen B66 into 1L of 0.2MpH phosphoric acid buffer solution with the value of 7.2, and stirring for one hour for later use;
step three: preparing glutamic oxaloacetic transaminase reaction liquid, namely adding 10KU pyruvate oxidase, 5KU oxaloacetate decarboxylase, 5KU peroxidase, 5KU ascorbic acid 0.2g of sodium aspartate, 0.5g of alpha-ketoglutarate, 0.2g of thiamine pyrophosphate, 50ml of 1.5% polyvinyl alcohol, 1g of bovine serum albumin, 1g of trehalose, 0.5gtriton X-100,1g of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3' 5-dimethoxyaniline sodium salt and 1.5g of 4-aminoantipyrine into 1L of TRIS buffer solution with the value of 6.8 and 0.2MpH, and stirring for one hour for later use;
step four: preparing total bilirubin auxiliary liquid: adding 5g of caffeine, 5g of sodium benzoate, 2g of sodium nitrite, 2g of sulfanilic acid, 2g of sodium azide and 5g of sodium acetate into 1L of 0.2gOP-10 pure water, and stirring for two hours for later use;
step five: preparing glutamic-pyruvic transaminase auxiliary liquid and glutamic-oxalacetic transaminase auxiliary liquid: adding 10g of polyvinylpyrrolidone, 1g of magnesium chloride and 0.5gOP-10 into 1L of 0.5MpH phosphoric acid buffer solution with the value of 7.20, and stirring for two hours for later use;
step six: the glutamic-pyruvic transaminase reaction layer 300 is prepared by: soaking the glutamic-pyruvic transaminase reaction layer 300 in glutamic-pyruvic transaminase reaction solution for 2min, taking the glutamic-pyruvic transaminase reaction solution, and drying at 50 deg.C for 8 min; soaking the total bilirubin reaction layer 300 in the total bilirubin reaction liquid for 2min, taking the total bilirubin reaction liquid, and drying the total bilirubin reaction layer at 50 ℃ for 8min for later use; soaking the glutamic-oxaloacetic transaminase reaction layer 300 in the glutamic-oxaloacetic transaminase reaction solution for 2min, taking the solution, and drying at 50 ℃ for 8min for later use;
step seven: manufacturing an auxiliary layer: soaking the auxiliary layer in auxiliary liquid for 2min, taking out, and drying at 50 deg.C for 8 min; soaking the auxiliary layers in glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase auxiliary solution for 2min, taking out, and drying at 50 deg.C for 8 min;
step eight: in the manner shown in fig. 2, the base plate 200, the treated reaction layer 300, the treated auxiliary layer, the whole blood separation membrane and the diffusion membrane 600 are stacked in the plastic lower case 100, and the plastic upper case 700 is tightly covered to obtain the test card product 1.
Example 2
The manufacturing method of the finished product 2 comprises the following steps:
the method comprises the following steps: preparing glutamic-pyruvic transaminase reaction liquid, namely adding 25KU pyruvate oxidase, 10KU peroxidase, 10KU ascorbic acid 1 gL-alanine, 2g alpha-ketoglutaric acid, 1g thiamine pyrophosphate, 1g flavin adenine dinucleotide, 100ml 1.5% polyvinyl alcohol, 10g bovine serum albumin, 2g trehalose, 1gtriton X-100,2g N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3', 5-dimethoxyaniline sodium salt and 3g 4-aminoantipyrine into 1L 0.1MpH TRIS buffer solution with the value of 6.8, and stirring for one hour for later use;
step two: preparing total bilirubin reaction liquid, namely adding 20g of alkaline sodium tartrate and 0.5g of Emulgen B66 into 1L of 0.1MpH phosphoric acid buffer solution with the value of 7.2, and stirring for one hour for later use;
step three: preparing glutamic oxaloacetic transaminase reaction liquid, namely adding 25KU pyruvate oxidase, 15KU oxaloacetate decarboxylase, 20KU peroxidase, 15KU ascorbic acid 1g L-sodium aspartate, 2g alpha-ketoglutarate, 1g thiamine pyrophosphate, 100ml of 1.5% polyvinyl alcohol, 10g bovine serum albumin, 2g trehalose, 1gtriton X-100,2g N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3' 5-dimethoxyaniline sodium salt and 3g 4-aminoantipyrine into 1L of 0.1MpH TRIS buffer solution with the value of 6.8, and stirring for one hour for later use;
step four: preparing total bilirubin auxiliary liquid: adding 10g of caffeine, 10g of sodium benzoate, 5g of sodium nitrite, 5g of sulfanilic acid, 5g of sodium azide and 20g of sodium acetate into 1L of 0.5gOP-10 g of pure water, and stirring for two hours for later use;
step five: preparing glutamic-pyruvic transaminase auxiliary liquid and glutamic-oxalacetic transaminase auxiliary liquid: adding 30g of polyvinylpyrrolidone, 10g of magnesium chloride and 0.5-0.5 gOP-10 into 1L of 0.1MpH phosphoric acid buffer solution with the value of 7.20, and stirring for two hours for later use;
step six: the glutamic-pyruvic transaminase reaction layer 300 is prepared by: soaking the glutamic-pyruvic transaminase reaction layer 300 in glutamic-pyruvic transaminase reaction solution for 5min, taking the glutamic-pyruvic transaminase reaction solution, and drying at 35 deg.C for 30 min; soaking the total bilirubin reaction layer 300 in the total bilirubin reaction liquid for 5min, taking the total bilirubin reaction liquid, and drying the total bilirubin reaction layer for 30min at the temperature of 35 ℃ for later use; soaking the glutamic-oxaloacetic transaminase reaction layer 300 in the glutamic-oxaloacetic transaminase reaction liquid for 5min, taking the glutamic-oxaloacetic transaminase reaction liquid, and drying the glutamic-oxaloacetic transaminase reaction liquid for 30min at 35 ℃ for later use;
step seven: manufacturing an auxiliary layer: soaking the auxiliary layer in auxiliary liquid for 5min, taking out, and drying at 35 deg.C for 30 min; soaking the auxiliary layers in glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase auxiliary solution for 5min, taking out, and drying at 35 deg.C for 30 min;
step eight: in the manner shown in fig. 2, the base plate 200, the treated reaction layer 300, the treated auxiliary layer, the whole blood separation membrane and the diffusion membrane 600 are stacked in the plastic lower case 100, and the plastic upper case 700 is tightly covered to obtain the test card product 2.
Example 3:
the manufacturing method of the finished product 3 comprises the following steps:
the method comprises the following steps: preparing glutamic-pyruvic transaminase reaction liquid, namely adding 40KU pyruvate oxidase, 18KU peroxidase, 18KU ascorbic acid 1.5g of L-alanine, 4g of alpha-ketoglutaric acid, 2.5g of thiamine pyrophosphate, 1.8g of flavin adenine dinucleotide, 250ml of 1.5% polyvinyl alcohol, 10g of bovine serum albumin, 5g of trehalose, 3gtriton X-100,2g of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3' 5-dimethoxyaniline sodium salt and 3g of 4-aminoantipyrine into 1L of 0.5 TRIS buffer solution with the value of 0.5MpH of 6.8, and stirring for one hour for later use;
step two: preparing total bilirubin reaction liquid, namely adding 10g of alkaline sodium tartrate and 0.8g of Emulgen B66 into 1L of phosphate buffer solution with the value of 1MpH of 7.2, and stirring for one hour for later use;
step three: preparing glutamic oxaloacetic transaminase reaction liquid, namely adding 30KU pyruvate oxidase, 20KU oxaloacetate decarboxylase, 20KU peroxidase, 20KU ascorbic acid, 1.5g of sodium L-aspartate, 3g of alpha-ketoglutarate, 1g of thiamine pyrophosphate, 250ml of 1.5% polyvinyl alcohol, 10g of bovine serum albumin, 5g of trehalose, 2gtriton X-100,3g of N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3' 5-dimethoxyaniline sodium salt and 5g of 4-aminoantipyrine into 1L of 0.5-0.5 MpH TRIS buffer solution with the value of 6.8, and stirring for one hour for later use;
step four: preparing total bilirubin auxiliary liquid: adding 10g of caffeine, 10g of sodium benzoate, 6g of sodium nitrite, 4g of sulfanilic acid, 4g of sodium azide and 10g of sodium acetate into 1L of 0.5gOP-10 g of pure water, and stirring for two hours for later use;
step five: preparing glutamic-pyruvic transaminase auxiliary liquid and glutamic-oxalacetic transaminase auxiliary liquid: adding 20g of polyvinylpyrrolidone, 1.5g of magnesium chloride and 1gOP-10 into 1L of phosphate buffer solution with the value of 1MpH being 7.20, and stirring for two hours for later use;
step six: the glutamic-pyruvic transaminase reaction layer 300 is prepared by: soaking the glutamic-pyruvic transaminase reaction layer 300 in glutamic-pyruvic transaminase reaction solution for 10min, taking the glutamic-pyruvic transaminase reaction solution, and drying at 25 deg.C for 50 min; soaking the total bilirubin reaction layer 300 in the total bilirubin reaction liquid for 10min, taking the total bilirubin reaction liquid, and drying the total bilirubin reaction layer for 50min at 25 ℃ for later use; soaking the glutamic-oxaloacetic transaminase reaction layer 300 in the glutamic-oxaloacetic transaminase reaction solution for 10min, taking the solution out, and drying the solution for 50min at 25 ℃ for later use;
step seven: manufacturing an auxiliary layer: soaking the auxiliary layer in the auxiliary liquid for 10min, taking out, and drying at 25 deg.C for 50 min; soaking the auxiliary layers in glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase auxiliary solution for 10min, taking out, and drying at 25 deg.C for 50 min;
step eight: in the manner shown in fig. 2, the base plate 200, the treated reaction layer 300, the treated auxiliary layer, the whole blood separation membrane and the diffusion membrane 600 are stacked in the plastic lower case 100, and the plastic upper case 700 is tightly covered to obtain the finished test card 4.
Example 4:
the manufacturing method of the comparison finished product comprises the following steps:
the method comprises the following steps: preparing glutamic-pyruvic transaminase reaction liquid, namely adding 25KU pyruvate oxidase, 10KU peroxidase, 10KU ascorbic acid 1 gL-alanine, 2g alpha-ketoglutaric acid, 1g thiamine pyrophosphate, 1g flavin adenine dinucleotide, 100ml 1.5% polyvinyl alcohol, 10g bovine serum albumin, 2g trehalose, 1gtriton X-100,2g N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3', 5-dimethoxyaniline sodium salt and 3g 4-aminoantipyrine into 1L 0.1MpH TRIS buffer solution with the value of 6.8, and stirring for one hour for later use;
step two: preparing total bilirubin reaction liquid, namely adding 20g of alkaline sodium tartrate and 0.5g of Emulgen B66 into 1L of 0.1MpH phosphoric acid buffer solution with the value of 7.2, and stirring for one hour for later use;
step three: preparing glutamic oxaloacetic transaminase reaction liquid, namely adding 25KU pyruvate oxidase, 15KU oxaloacetate decarboxylase, 20KU peroxidase, 15KU ascorbic acid 1g L-sodium aspartate, 2g alpha-ketoglutarate, 1g thiamine pyrophosphate, 100ml of 1.5% polyvinyl alcohol, 10g bovine serum albumin, 2g trehalose, 1gtriton X-100,2g N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3' 5-dimethoxyaniline sodium salt and 3g 4-aminoantipyrine into 1L of 0.1MpH TRIS buffer solution with the value of 6.8, and stirring for one hour for later use;
step four: the glutamic-pyruvic transaminase reaction layer 300 is prepared by: soaking the glutamic-pyruvic transaminase reaction layer 300 in glutamic-pyruvic transaminase reaction solution for 5min, taking the glutamic-pyruvic transaminase reaction solution, and drying at 35 deg.C for 30 min; soaking the total bilirubin reaction layer 300 in the total bilirubin reaction liquid for 5min, taking the total bilirubin reaction liquid, and drying the total bilirubin reaction layer for 30min at the temperature of 35 ℃ for later use; soaking the glutamic-oxaloacetic transaminase reaction layer 300 in the glutamic-oxaloacetic transaminase reaction liquid for 5min, taking the glutamic-oxaloacetic transaminase reaction liquid, and drying the glutamic-oxaloacetic transaminase reaction liquid for 30min at 35 ℃ for later use;
step five: the base plate 200, the treated reaction layer 300, the whole blood separation membrane and the diffusion membrane 600 are respectively stacked in the plastic lower case 100, and the plastic upper case 700 is tightly covered to obtain the test card product 4.
Example 4 has no auxiliary layer compared to the first three examples.
And evaluating the test cards of the finished products 1, 2, 3 and 4 by comparing with the hospital results.
The test card is tested with a self-made dry biochemical analyzer DBM-101, 20 venous blood samples with gradient are extracted at any time for testing, and then the venous blood samples are compared with hospital values for evaluation.
The results of the data for finished product 1 are shown in the table below,
and (4) analyzing results: the R square values are all between 0.90 and 0.93, and the detection result has better matching with the hospital inspection result. The data results for finished product 2 are shown in the following table, fig. 3, fig. 4, fig. 5;
and (4) analyzing results: the R square values are all above 0.96, and the matching of the detection result and the hospital test result is good.
Data results for finished product 3 are shown in the following table:
and (4) analyzing results: the R square values are all about 0.85, and the detection result and the hospital inspection result are linear.
Comparing the results of the finished products 1, 2 and 3, it is understood that example 2 is the most preferable example.
The results of comparing the data of the finished product are shown in the following table;
and (4) analyzing results: the comparative product had no auxiliary layer added, the R-square values were all relatively poor, the total bilirubin program was essentially non-linear, and glutamate pyruvate transaminase (ALT) and glutamate oxaloacetate transaminase (AST) were somewhat linear.
The invention provides a test card for liver function and a preparation method thereof, the test card is matched with a dry biochemical analyzer for use, the operation is simple, the contents of three items of glutamic-pyruvic transaminase, total bilirubin and glutamic-oxalacetic transaminase in whole blood, serum/plasma and other body fluids of a human body or an animal can be rapidly and simultaneously detected without professional persons, and the matching of the detection result and the hospital detection result is good.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Claims (3)
1. A liver function test card, comprising: the kit comprises an upper shell, a lower shell, a sampling hole arranged on the upper shell, and a glutamic-pyruvic transaminase detection unit, a total bilirubin detection unit and a glutamic-oxalacetic transaminase detection unit which are arranged between the upper shell and the lower shell; the glutamic-pyruvic transaminase detection unit, the total bilirubin detection unit and the glutamic-oxalacetic transaminase detection unit comprise: the detection device comprises a bottom plate arranged at the upper end of a lower shell, a lower shell detection hole arranged on the lower shell, a bottom plate detection hole arranged on the bottom plate, a diffusion membrane arranged at the lower end of an upper shell and connected to the bottom plate, a whole blood separation layer arranged under the diffusion membrane, and a reaction layer arranged between the whole blood separation layer and the bottom plate detection hole; the glutamic-pyruvic transaminase reaction solution comprises: 10-40KU pyruvate oxidase, 1-20KU peroxidase, 1-20KU ascorbic acid, 0.1-2g L-alanine, 1-4g alpha-ketoglutaric acid, 0.1-3g thiamine pyrophosphate, 0.1-2g flavin adenine dinucleotide, 0.5-4g film forming agent, 2-20g stabilizer, 0.1-5g surfactant, 0.1-10g color developing agent, 0.1-1M buffer solution; the glutamic-oxaloacetic transaminase reaction liquid comprises: 5-50KU pyruvate oxidase, 5-40KU oxaloacetate decarboxylase, 1-50KU peroxidase, 5-40KU ascorbic acid, 0.1-5g L-sodium aspartate, 0.1-5g alpha-ketoglutarate, 0.1-5g thiamine pyrophosphate, 0.5-4g film former, 2-20g stabilizer, 0.1-5g surfactant, 0.1-10g color developer and 0.1-1M buffer solution; the glutamic-pyruvic transaminase detection unit, the total bilirubin detection unit and the glutamic-oxalacetic transaminase detection unit further comprise: the auxiliary layer is arranged between the whole blood separation layer and the reaction layer; the total bilirubin adjuvant liquid comprises: 1-20g of caffeine, 1-20g of sodium benzoate, 1-10g of sodium nitrite, 1-10g of sulfanilic acid, 1-10g of sodium azide, 0.1-5gOP-10 g of sodium acetate and 2-40g of sodium acetate are added into 1L of pure water; the glutamic-pyruvic transaminase auxiliary liquid and the glutamic-oxalacetic transaminase auxiliary liquid comprise: 5-50g of polyvinylpyrrolidone, 1-20g of magnesium chloride and 0.1-5gOP-10 were added to 1L of 0.1-1MpH phosphoric acid buffer solution with a value of 7.20.
2. The liver function test card of claim 1, wherein the whole blood separation layer is a porous cellulose membrane.
3. A preparation method of a liver function test card is characterized by comprising the following steps:
preparing a glutamic-pyruvic transaminase reaction solution, namely 10-40KU pyruvate oxidase, 1-20KU peroxidase, 1-20KU ascorbic acid, 0.1-2g L-alanine, 1-4g alpha-ketoglutaric acid, 0.1-3g thiamine pyrophosphate, 0.1-2g flavin adenine dinucleotide, 0.5-4g film forming agent, 2-20g stabilizer, 0.1-5g surfactant, 0.1-10g developer and 0.1-1M buffer solution, stirring for one hour for later use;
preparing total bilirubin reaction liquid, namely 1-50g of alkaline sodium tartrate, 0.1-5g of surfactant and 0.1-1M buffer solution, and stirring for one hour for later use;
preparing a glutamic-oxaloacetic transaminase reaction solution, wherein the glutamic-oxaloacetic transaminase reaction solution comprises the following components: 5-50KU pyruvate oxidase, 5-40KU oxaloacetate decarboxylase, 1-50KU peroxidase, 5-40KU ascorbic acid, 0.1-5g L-sodium aspartate, 0.1-5g alpha-ketoglutarate, 0.1-5g thiamine pyrophosphate, 0.5-4g film forming agent, 2-20g stabilizer, 0.1-5g surfactant, 0.1-10g color developing agent and 0.1-1M buffer solution are stirred for one hour for standby application;
preparing total bilirubin auxiliary liquid: 1-20g of caffeine, 1-20g of sodium benzoate, 1-10g of sodium nitrite, 1-10g of sulfanilic acid, 1-10g of sodium azide and 0.1-5gOP-10 g of sodium acetate, and 2-40g of sodium acetate are added into 1L of pure water and stirred for two hours for later use;
preparing glutamic-pyruvic transaminase auxiliary liquid and glutamic-oxalacetic transaminase auxiliary liquid: adding 30g of polyvinylpyrrolidone, 10g of magnesium chloride and 0.5-0.5 gOP-10 into 1L of 0.1MpH phosphoric acid buffer solution with the value of 7.20, and stirring for two hours for later use;
preparing a reaction layer: soaking the glutamic-pyruvic transaminase reaction layer in glutamic-pyruvic transaminase reaction solution for 1-10min, taking out, and drying at 25-50 deg.C for 5-50 min; soaking the total bilirubin reaction layer in the total bilirubin reaction liquid for 1-10min, taking out, and drying at 25-50 deg.C for 5-50 min; soaking the reaction layer in the reaction solution for 1-10min, taking out, and drying at 25-50 deg.C for 5-50 min;
manufacturing an auxiliary layer: soaking the auxiliary layer in auxiliary liquid for 1-10min, taking out, and drying at 25-50 deg.C for 5-50 min; soaking glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase auxiliary layers in glutamic-pyruvic transaminase auxiliary solution and glutamic-oxalacetic transaminase auxiliary solution for 1-10min, respectively, taking out, and drying at 25-50 deg.C for 5-50 min;
respectively stacking the bottom plate, the reaction layer, the auxiliary layer, the whole blood separation membrane and the diffusion membrane in the plastic lower shell, and tightly covering the plastic upper shell to obtain a finished test card.
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| CN111197071A (en) * | 2020-01-10 | 2020-05-26 | 杭州联晟生物科技有限公司 | Test card for detecting glutamic-pyruvic transaminase by photochemical method and preparation method thereof |
| CN113588747B (en) * | 2021-07-12 | 2024-01-19 | 成都云芯医联科技有限公司 | Electrochemical test card for simultaneously measuring glutamic pyruvic transaminase and glutamic oxaloacetic transaminase |
| CN113670910A (en) * | 2021-08-24 | 2021-11-19 | 四川成电医联科技咨询有限公司 | Total bilirubin test card and manufacturing method thereof |
| CN116121336B (en) * | 2023-04-17 | 2023-06-27 | 复星诊断科技(长沙)有限公司 | Blood fat three-item test strip and preparation method thereof |
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