CN111197071A - Test card for detecting glutamic-pyruvic transaminase by photochemical method and preparation method thereof - Google Patents
Test card for detecting glutamic-pyruvic transaminase by photochemical method and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a test card for detecting glutamic-pyruvic transaminase by a photochemical method and a preparation method thereof, wherein the test card comprises an upper test card shell, a lower test card shell, a main test hole, a comparison test hole, a sample adding hole, a diffusion membrane, a first blood filtering membrane, two second blood filtering membranes, a first reaction membrane, a second reaction membrane and a single-sided adhesive, wherein the first reaction liquid comprises L-alanine, pyruvate oxidase, α -ketoglutaric acid, peroxidase, thiamine pyrophosphate, magnesium chloride, 4-aminoantipyrine, a color developing agent, a film forming agent, a stabilizing agent and a buffer solution, and the second reaction liquid comprises pyruvate oxidase, peroxidase, thiamine pyrophosphate, magnesium chloride, 4-aminoantipyrine, a color developing agent, a film forming agent, a stabilizing agent and a buffer solution.
Description
Technical Field
The invention relates to the field of detection, in particular to a test card for detecting glutamic-pyruvic transaminase by a photochemical method and a preparation method thereof.
Background
Glutamate pyruvate transaminase (ALT) is mainly present in various cells, most preferably hepatocytes, and has an intracellular transaminase content of about 100 times that of blood. In normal conditions, the activity of the enzyme in the serum can be significantly increased by only a small amount of release into the blood. ALT is released into blood in large quantity in acute stage of various viral hepatitis and when toxic liver cells in medicine are destroyed, so that it is an important index for diagnosing viral hepatitis and toxic hepatitis. As long as 1% of hepatocytes are necrotic, the enzyme activity in blood is increased by 1-fold, and transaminases (especially ALT) are therefore sensitive markers of acute hepatocyte damage.
The detection of glutamic-pyruvic transaminase is mainly carried out by a wet chemical method, a kit is matched with a large biochemical analyzer for detection, the time is long, the operation is complex, and the detection is carried out by professional persons. Causing some inconvenience to the user or patient. The dry detection mainly comprises a test strip or a test card, and a user only needs to drop a small amount of blood on the test strip or the test card to be used together with a matched instrument, so that a result is obtained within a few minutes. However, several companies have related products in the market, and the test result is not uniform due to the fact that the interference of endogenous pyruvic acid in a sample cannot be removed, so that the market cannot be widely used, the market needs a test card which can avoid the interference of endogenous pyruvic acid and accurately and rapidly quantitatively detect the content of glutamic-pyruvic transaminase in body fluid, and the problem is solved by the invention.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a test card for detecting glutamic-pyruvic transaminase by a photochemical method and a preparation method thereof, which can avoid endogenous pyruvic acid interference, and can be matched with a dry biochemical analyzer for use to quickly and quantitatively detect the content of the glutamic-pyruvic transaminase in human or animal body fluid; the detection result is well matched with the hospital detection result, the use is simple, and the operation of professional persons is not needed.
In order to achieve the above object, the present invention adopts the following technical solutions:
a test card for detecting glutamic-pyruvic transaminase by a photochemical method comprises the following components: the device comprises a test card upper shell, a test card lower shell, a main test hole and a contrast test hole which are arranged on the test card lower shell, a sample adding hole which is arranged on the test card upper shell, a diffusion membrane which is arranged below the test card upper shell, a first blood filtering membrane which is arranged below the diffusion membrane, two second blood filtering membranes which are arranged below the first blood filtering membrane and respectively correspond to the main test hole and the contrast test hole, a first reaction membrane and a second reaction membrane which are arranged below the two second blood filtering membranes, and a single-sided adhesive which is arranged below the first reaction membrane and the second reaction membrane;
the first reaction liquid on the first reaction film has the formula comprising L-alanine 50-500mM, pyruvate oxidase 50-200KU/L, α -oxoglutarate 5-100mM, peroxidase 50-200KU/L, thiamine pyrophosphate 0.1-1mM, magnesium chloride 0.1-5g/L, 4-aminoantipyrine 1-50mM, developer 1-50mM, film forming agent 0.1-10g/L, stabilizer 1-50g/L, and buffer solution 0.1-2M;
the formula of the second reaction liquid on the second reaction film comprises: 50-200KU/L pyruvate oxidase, 50-200KU peroxidase, 0.1-1mM thiamine pyrophosphate, 0.1-5g/L magnesium chloride, 1-50mM 4-aminoantipyrine, 1-50mM color developing agent, 0.1-10g/L film forming agent, 1-50g/L stabilizer and 0.1-2M buffer solution.
The aforementioned test card for detecting alanine aminotransferase by a photochemical method includes: TOOS, DAOS, MAOS, TODB.
The aforementioned test card for detecting glutamic-pyruvic transaminase by photochemical method comprises: hydroxymethyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, sodium alginate, and cyclamine.
The test card for detecting alanine aminotransferase by a photochemical method and the preparation method thereof comprise the following stabilizers: polyethylene glycol 6000, polyvinylpyrrolidone, trehalose, sucrose and mannitol.
The aforementioned test card for detecting alanine aminotransferase by photochemical method, wherein the buffer solution comprises: phosphate buffer, TRIS buffer, MES buffer.
In the aforementioned test card for detecting alanine aminotransferase by a photochemical method, the first reaction film and the second reaction film include: polysulfone membranes, nylon membranes.
In the aforementioned test card for detecting alanine aminotransferase by a photochemical method, the diffusion membrane includes: hydrophilic polyester gauze, hydrophobic polyester gauze and nylon gauze.
The formula of the first reaction solution comprises 50mM of L-alanine, 100KU/L of pyruvate oxidase, 50mM of α -oxoglutarate, 100KU/L of peroxidase, 0.2mM of thiamine pyrophosphate, 0.2g/L of magnesium chloride, 10mM of 4-aminoantipyrine, 10mM of MAOS, 0.1-10g/L of sodium alginate, 1-50g/L of trehalose, and 0.05M of phosphate buffer solution with the pH of 6.
In the aforementioned test card for detecting alanine aminotransferase by a photochemical method, the formula of the second reaction solution includes: pyruvate oxidase 100KU/L, peroxidase 100KU/L, thiamine pyrophosphate 0.2mM, magnesium chloride 0.2g/L, 4-aminoantipyrine 10mM, MAOS10mM, sodium alginate 0.1-10g/L, trehalose 1-50g/L, and 0.05M phosphate buffer solution with pH of 6.
A preparation method of a test card for detecting glutamic-pyruvic transaminase by a photochemical method comprises the following steps:
the method comprises the following steps: the first reaction solution was prepared according to the following formulation,
the formula comprises 50-500mM of L-alanine, 50-200KU/L of pyruvate oxidase, 5-100mM of α -ketoglutaric acid, 50-200KU/L of peroxidase, 0.1-1mM of thiamine pyrophosphate, 0.1-5g/L of magnesium chloride, 1-50mM of 4-aminoantipyrine, 1-50mM of color developing agent, 0.1-10g/L of film forming agent, 1-50g/L of stabilizing agent and 0.1-2M of buffer solution, and the buffer solution is stirred for standby after one hour;
step two: the second reaction solution was prepared according to the following formulation,
the formula comprises the following components: 50-200KU/L pyruvate oxidase, 50-200KU peroxidase, 0.1-1mM thiamine pyrophosphate, 0.1-5g/L magnesium chloride, 1-50mM 4-aminoantipyrine, 1-50mM color developing agent, 0.1-10g/L film forming agent, 1-50g/L stabilizer and 0.1-2M buffer solution;
step three: the first reaction film is prepared by the following steps,
soaking the reaction membrane in the first reaction solution for 1-10min, taking the reaction membrane, and drying at 25-50 deg.C for 10-60 min;
step four: the second reaction film is prepared by the following steps,
soaking the reaction membrane in the second reaction solution for 1-10min, taking the reaction membrane, and drying at 25-50 ℃ for 10-60min for later use;
step five: and (3) sequentially sticking a first reaction film and a second reaction film on the single-sided adhesive, then sequentially sticking a second blood filtering film, a first blood filtering film and a diffusion film, loading the test card into a lower shell of the test card after sticking, and then loading the test card onto an upper shell cover to obtain the finished test card.
The invention has the advantages that:
according to the invention, the test card is improved in formula and structure, so that the interference of endogenous pyruvic acid can be avoided, and the matching of the detection result and the hospital test result is good;
the test card provided by the invention can be used with an analyzer in a matched manner, can be used for rapidly and quantitatively detecting the content of the glutamic-pyruvic transaminase in the body fluid of a human or animal, is simple to use, does not need to be operated by professional persons, and is suitable for general investigation of various drugstores, clinics, research institutes, community hospitals, pet hospitals, inspection centers or other governments.
Drawings
FIG. 1 is an exploded schematic view of one embodiment of the present invention;
FIG. 2 is a schematic cross-sectional view of one embodiment of the present invention;
FIG. 3 is a graph showing the test results of example 1 of the present invention;
FIG. 4 is a graph showing the test results of example 2 of the present invention;
FIG. 5 is a graph showing the test results of example 3 of the present invention;
FIG. 6 is a graph showing the test results of example 4 of the present invention;
FIG. 7 is a graph showing the test results of example 5 of the present invention.
The meaning of the reference symbols in the figures:
100 test card lower shell, 101 main test hole, 102 contrast test hole, 200 single-sided glue, 301 first reaction membrane, 302 second reaction membrane, 401, 402 second blood filtering membrane, 501 first blood filtering membrane, 601 diffusion membrane, 700 test card upper shell, 701 sample adding hole.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
As shown in fig. 1 and 2, a test card for detecting glutamic-pyruvic transaminase by a photochemical method comprises: the test card comprises a test card upper shell 700, a test card lower shell 100, a main test hole 101 and a contrast test hole 102 which are arranged on the test card lower shell 100, a sample adding hole 701 which is arranged on the test card upper shell 700, a diffusion membrane 601 which is arranged below the test card upper shell 700, a first blood filtering membrane 501 which is arranged below the diffusion membrane 601, two second blood filtering membranes 401 and 402 which are arranged below the first blood filtering membrane 501 and respectively correspond to the main test hole 101 and the contrast test hole 102, a first reaction membrane 301 and a second reaction membrane 302 which are arranged below the two second blood filtering membranes 401 and 402, and a single-sided adhesive 200 which is arranged below the first reaction membrane 301 and the second reaction membrane 302; as a preference, the single-sided adhesive 200 includes: polyvinyl chloride, polycarbonate, polyesteramide, polyester; the first and second reaction films 301 and 302 include: polysulfone membranes, nylon membranes; the first and second blood filtration membranes 501, 401, 402 comprise: modified polysulfone membrane, nylon membrane, polypropylene filter membrane, filter paper and glass fiber filter membrane; the diffusion film 601 includes: hydrophilic polyester gauze, hydrophobic polyester gauze and nylon gauze.
The method comprises the steps of adding a sample into a sample adding hole 701, uniformly distributing the sample on a diffusion membrane 601 through a flow guide groove on a test card upper shell 700, uniformly infiltrating down to a first blood filtering membrane 501, filtering out more than 80% of red blood cells, wherein about 20% of the red blood cells penetrate through the first blood filtering membrane 501 and respectively enter second blood filtering membranes 401 and 402, uniformly infiltrating down to a reaction membrane, detecting the concentration of glutamic-pyruvic transaminase and the concentration of endogenous pyruvic acid in the sample through the enzyme and chemical substances of the first reaction membrane 301, detecting the concentration of endogenous pyruvic acid in the sample through the enzyme and chemical substances of the second reaction membrane 302, and directly calculating the concentration of the glutamic-pyruvic transaminase in the sample through a matched analyzer.
The first reaction liquid on the first reaction film 301 comprises 50-500mM of L-alanine, 50-200KU/L of pyruvate oxidase, 5-100mM of α -oxoglutarate, 50-200KU/L of peroxidase, 0.1-1mM of thiamine pyrophosphate, 0.1-5g/L of magnesium chloride, 1-50mM of 4-aminoantipyrine, 1-50mM of color developing agent, 0.1-10g/L of film forming agent, 1-50g/L of stabilizing agent and 0.1-2M of buffer solution;
the formulation of the second reaction solution on the second reaction film 302 includes: 50-200KU/L pyruvate oxidase, 50-200KU peroxidase, 0.1-1mM thiamine pyrophosphate, 0.1-5g/L magnesium chloride, 1-50mM 4-aminoantipyrine, 1-50mM color developing agent, 0.1-10g/L film forming agent, 1-50g/L stabilizer and 0.1-2M buffer solution.
As an example, the developing agent includes: TOOS, DAOS, MAOS, TODB; the film forming agent comprises: hydroxymethyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, sodium alginate, cyclamine; the stabilizer comprises: polyethylene glycol 6000, polyvinylpyrrolidone, trehalose, sucrose and mannitol; the buffer solution comprises: phosphate buffer, TRIS buffer, MES buffer.
A preparation method of a test card for detecting glutamic-pyruvic transaminase by a photochemical method comprises the following steps:
the method comprises the following steps: the first reaction solution was prepared according to the following formulation,
the formula comprises 50-500mM of L-alanine, 50-200KU/L of pyruvate oxidase, 5-100mM of α -ketoglutaric acid, 50-200KU/L of peroxidase, 0.1-1mM of thiamine pyrophosphate, 0.1-5g/L of magnesium chloride, 1-50mM of 4-aminoantipyrine, 1-50mM of color developing agent, 0.1-10g/L of film forming agent, 1-50g/L of stabilizing agent and 0.1-2M of buffer solution, and the buffer solution is stirred for standby after one hour;
step two: the second reaction solution was prepared according to the following formulation,
the formula comprises the following components: 50-200KU/L pyruvate oxidase, 50-200KU peroxidase, 0.1-1mM thiamine pyrophosphate, 0.1-5g/L magnesium chloride, 1-50mM 4-aminoantipyrine, 1-50mM color developing agent, 0.1-10g/L film forming agent, 1-50g/L stabilizer and 0.1-2M buffer solution;
step three: a first reaction film 301 is prepared and,
soaking the reaction membrane in the first reaction solution for 1-10min, taking the reaction membrane, and drying at 25-50 deg.C for 10-60 min;
step four: a second reactive film 302 is prepared and,
soaking the reaction membrane in the second reaction solution for 1-10min, taking the reaction membrane, and drying at 25-50 ℃ for 10-60min for later use;
step five: and sequentially sticking a first reaction film 301 and a second reaction film 302 on the single-sided adhesive 200, sequentially sticking second blood filtering films 401 and 402, a first blood filtering film 501 and a diffusion film 601, putting the films into the lower test card shell 100 after sticking, and covering the upper test card shell 700 to obtain the finished test card.
Experimental verification part:
finished products 1, 2, 3, 4 and 5 were obtained according to the following method
Example 1
Preparing a first reaction solution, namely stirring 50mM of L-alanine, 100KU/L of pyruvate oxidase, 50mM of α -ketoglutaric acid, 100KU/L of peroxidase, 0.2mM of thiamine pyrophosphate, 0.2g/L of magnesium chloride, 10mM of 4-aminoantipyrine, 10mM of MAOS10mM, 0.1-10g/L of sodium alginate, 1-50g/L of seaweed and 0.05M of phosphate buffer solution with the pH value of 6 for standby after one hour;
step two: production of the first reaction film 301: soaking the reaction membrane in the first reaction solution for 5min, taking the reaction membrane out, and drying the reaction membrane for 20min at the temperature of 45 ℃ for later use;
step three: taking polycarbonate single-sided adhesive 200 with the thickness of 0.1mm, attaching a first reaction film 301 to a mounting hole of the single-sided adhesive 200, pressing tightly, attaching an MF1 polypropylene filter membrane 501 to a second blood filter membrane 401, attaching a hydrophilic polyester gauze 601 right above the MF1 polypropylene filter membrane 501, cutting a single strip, then placing the strip into a lower test card shell 100, covering the upper test card shell, and pressing tightly to obtain a finished product 1.
Example 2
Preparing a first reaction solution, namely stirring 50mM of L-alanine, 100KU/L of pyruvate oxidase, 50mM of α -ketoglutaric acid, 100KU/L of peroxidase, 0.2mM of thiamine pyrophosphate, 0.2g/L of magnesium chloride, 10mM of 4-aminoantipyrine, 10mM of MAOS (maleic anhydride) 10mM, 0.1-10g/L of sodium alginate, 1-50g/L of trehalose and 0.05M of phosphate buffer solution with the pH value of 6 for one hour for later use;
step two: production of the first reaction film 301: soaking the reaction membrane in the first reaction solution for 5min, taking the reaction membrane out, and drying the reaction membrane for 20min at the temperature of 45 ℃ for later use;
step three: taking a polycarbonate single-sided adhesive 200 with the thickness of 0.1mm, attaching a first reaction film 301 to a mounting hole of the single-sided adhesive 200, attaching a cut second blood filtering film 401 (a modified polysulfone film PES 0.65) above the first reaction film 301, pressing the edge tightly, attaching a first blood filtering film (MF1 polypropylene filter film) 501 above the second blood filtering film 401, attaching a hydrophilic polyester gauze 601 right above the first blood filtering film (MF1 polypropylene filter film) 501, cutting a single strip, then loading into a lower test card shell 100, covering the upper test card shell, and pressing to obtain a finished product 2.
The manufacturing method of the finished product 3 comprises the following steps:
preparing a first reaction solution, namely stirring 50mM of L-alanine, 100KU/L of pyruvate oxidase, 50mM of α -ketoglutaric acid, 100KU/L of peroxidase, 0.2mM of thiamine pyrophosphate, 0.2g/L of magnesium chloride, 10mM of 4-aminoantipyrine, 10mM of MAOS (maleic anhydride) 10mM, 0.1-10g/L of sodium alginate, 1-50g/L of trehalose and 0.05M of phosphate buffer solution with the pH value of 6 for one hour for later use;
step two: preparing a second reaction solution: phosphate buffer solution with pyruvate oxidase 100KU/L, peroxidase 100KU/L, thiamine pyrophosphate 0.2mM, magnesium chloride 0.2g/L, 4-aminoantipyrine 10mM, MAOS10mM, sodium alginate 0.1-10g/L, trehalose 1-50g/L, and pH 6 of 0.05M; stirring for one hour for later use;
step three: production of the first reaction film 301: soaking the reaction membrane in the first reaction solution for 5min, taking the reaction membrane out, and drying the reaction membrane for 20min at the temperature of 45 ℃ for later use;
step four: second reaction film 302 fabrication: soaking the reaction membrane in the second reaction solution for 5min, taking the reaction membrane out, and drying the reaction membrane for 20min at the temperature of 45 ℃ for later use;
step five: taking a polycarbonate single-sided adhesive 200 with the thickness of 0.1mm, attaching a first reaction film 301 to a mounting hole of the single-sided adhesive 200, attaching a second reaction film 302 to the mounting hole of the single-sided adhesive 200, pressing, respectively attaching a cut blood filtration film modified polysulfone film PES 0.65 right above the first reaction film 301 and the second reaction film 302, and pressing the edge; then, the first blood filtering membrane 501(MF1 polypropylene filter membrane) is attached to the second blood filtering membranes 401 and 402, the diffusion membrane 601 (hydrophilic polyester gauze) is attached to the position right above the first blood filtering membrane 501, and then the test card lower shell 100 is filled with the cut single strips, covered by the test card upper shell and compressed to obtain the finished product 3.
In the case of the example 4, the following examples are given,
preparing a first reaction solution, namely stirring 200mM of L-alanine, 50KU/L of pyruvate oxidase, 10mM of α -ketoglutarate, 50KU/L of peroxidase, 0.1mM of thiamine pyrophosphate, 0.1g/L of magnesium chloride, 2mM of 4-aminoantipyrine, MAOS1mM, 0.1/L of sodium alginate, 2g/L of trehalose and 0.05M of phosphate buffer solution with the pH value of 6 for standby after one hour;
step two, preparing a second reaction solution, namely stirring a phosphate buffer solution of 50KU/L pyruvate oxidase, 10mM α -oxoglutarate, 50KU/L peroxidase, 0.1mM thiamine pyrophosphate, 0.1g/L magnesium chloride, 2mM 4-aminoantipyrine, 1mM MAOS, 0.1/L sodium alginate, 2g/L trehalose and 0.05M pH 6 for later use after one hour;
step three: production of the first reaction film 301: soaking the reaction membrane in the first reaction solution for 2min, taking the reaction membrane out, and drying at 50 ℃ for 10min for later use;
step four: second reaction film 302 fabrication: soaking the reaction membrane in the second reaction solution for 2min, taking the reaction membrane out, and drying at 50 ℃ for 10min for later use;
step five: taking a polycarbonate single-sided adhesive 200 with the thickness of 0.1mm, attaching a first reaction film 301 to a mounting hole of the single-sided adhesive 200, attaching a second reaction film 302 to the mounting hole of the single-sided adhesive 200, compressing, then respectively attaching cut second blood filtration films 401 and 402 (modified polysulfone films PES 0.65) right above the first reaction film 301 and the second reaction film 302, and compressing the edges; then, the first blood filtering membrane 501(MF1 polypropylene filter membrane) is attached to the second blood filtering membranes 401 and 402, then the hydrophilic polyester gauze (the diffusion membrane 601) is attached to the position right above the first blood filtering membrane 501, then the test card lower shell 100 is filled with the single strips, the test card upper shell is covered, and the finished product 4 is obtained by pressing.
In the case of the example 5, the following examples were conducted,
preparing a first reaction solution, namely stirring 500mM of L-alanine, 200KU/L of pyruvate oxidase, 100mM of α -ketoglutaric acid, 200KU/L of peroxidase, 1mM of thiamine pyrophosphate, 5g/L of magnesium chloride, 50mM of 4-aminoantipyrine, 50mM of MAOS50mM, 10g/L of sodium alginate, 50g/L of trehalose and 0.05M of phosphate buffer solution with the pH of 6 for standby after one hour;
preparing a second reaction solution, namely stirring a phosphate buffer solution of which the pH value is 6 of pyruvate oxidase 200KU/L, α -ketoglutaric acid 100mM, peroxidase 200KU/L, thiamine pyrophosphate 1mM, magnesium chloride 5g/L, 4-aminoantipyrine 50mM, MAOS50mM, sodium alginate 10g/L, trehalose 50g/L and 0.05M for standby after one hour;
step three: production of the first reaction film 301: soaking the reaction membrane in the first reaction solution for 10min, taking the reaction membrane out, and drying the reaction membrane for 60min at 25 ℃ for later use;
step four: second reaction film 302 fabrication: soaking the reaction membrane in the second reaction solution for 10min, taking the reaction membrane out, and drying the reaction membrane for 60min at 25 ℃ for later use;
step five: taking a polycarbonate single-sided adhesive 200 with the thickness of 0.1mm, attaching a first reaction film 301 to a mounting hole of the single-sided adhesive 200, attaching a second reaction film 302 to a mounting hole of the other single-sided adhesive 200, compressing, respectively attaching cut second blood filtration films 401 and 402 (modified polysulfone films PES 0.65) right above the first reaction film 301 and the second reaction film 302, and compressing the edges; then, the first blood filtering membrane 501(MF1 polypropylene filter membrane) is attached to the second blood filtering membranes 401 and 402, the diffusion membrane 601 (hydrophilic polyester gauze) is attached to the position right above the first blood filtering membrane 501, and then the test card lower shell 100 is filled with the cut single strips, covered by the test card upper shell and compressed to obtain the finished product 5.
The test card was tested with a home-made analyzer developed by this company, and 10 venous blood samples having a gradient were randomly sampled for testing and then compared with hospital values for evaluation.
The experimental results are as follows:
the results of the data for finished product 1 are shown in table 1 below and fig. 3;
TABLE 1
And (4) analyzing results: the finished product 1 is used for testing with an analyzer manufactured by the company, 10 venous blood samples with gradient are randomly drawn for testing, and then compared with a hospital value, the R square value is 0.807, which indicates that the matching of the detection result and the hospital inspection result is poor.
The results of the data for finished product 2 are shown in table 2 below and fig. 4;
and (4) analyzing results: the finished product 2 is used for testing with an analyzer manufactured by the company, 10 venous blood samples with gradient are randomly drawn for testing, and then compared with a hospital value, the R square value is 0.908, which indicates that the detection result is generally matched with the hospital test result.
The results for the data for finished product 3 are shown in table 3 below and fig. 5;
and (4) analyzing results: the finished product 3 is used for testing with an analyzer manufactured by the company, 10 venous blood samples with gradient are randomly drawn for testing, and then compared with a hospital value, the R square value is 0.997, which indicates that the matching of the detection result and the hospital test result is better.
The results for data for finished product 4 are shown in table 4 below and fig. 6;
TABLE 4
And (4) analyzing results: the finished product 4 is used for testing with an analyzer manufactured by the company, 10 venous blood samples with gradient are randomly drawn for testing, and then compared with hospital values, the R square value is 0.965, which shows that the matching of the detection result and the hospital test result is poorer than that of the finished product 3.
The results for the data for finished product 5 are shown in table 5 below and fig. 7;
and (4) analyzing results: the finished product 5 is used for testing with an analyzer manufactured by the company, 10 venous blood samples with gradient are randomly drawn for testing, and then compared with a hospital value, the R square value is 0.918, which shows that the matching of the detection result and the hospital test result is poorer than that of the finished product 3.
And (3) comprehensive analysis: from the results of comparison between examples 3-5 and examples 1-2, it can be seen that: the detection result of the test card adopting the structure and the formula of the invention has better matching property with the hospital inspection result; the formulation of example 3 is optimal.
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 (10)
1. A test card for detecting glutamic-pyruvic transaminase by a photochemical method is characterized by comprising: the device comprises a test card upper shell, a test card lower shell, a main test hole and a contrast test hole which are arranged on the test card lower shell, a sample adding hole which is arranged on the test card upper shell, a diffusion membrane which is arranged under the test card upper shell, a first blood filtering membrane which is arranged under the diffusion membrane, two second blood filtering membranes which are arranged under the first blood filtering membrane and respectively correspond to the main test hole and the contrast test hole, a first reaction membrane and a second reaction membrane which are arranged under the two second blood filtering membranes, and a single-sided adhesive which is arranged under the first reaction membrane and the second reaction membrane;
the formula of the first reaction liquid on the first reaction membrane comprises 50-500mM of L-alanine, 50-200KU/L of pyruvate oxidase, 5-100mM of α -oxoglutarate, 50-200KU/L of peroxidase, 0.1-1mM of thiamine pyrophosphate, 0.1-5g/L of magnesium chloride, 1-50mM of 4-aminoantipyrine, 1-50mM of color developing agent, 0.1-10g/L of film forming agent, 1-50g/L of stabilizing agent and 0.1-2M of buffer solution;
the formula of the second reaction liquid on the second reaction film comprises: 50-200KU/L pyruvate oxidase, 50-200KU peroxidase, 0.1-1mM thiamine pyrophosphate, 0.1-5g/L magnesium chloride, 1-50mM 4-aminoantipyrine, 1-50mM color developing agent, 0.1-10g/L film forming agent, 1-50g/L stabilizer and 0.1-2M buffer solution.
2. The test card for photochemically detecting glutamic-pyruvic transaminase according to claim 1, wherein the indicator comprises: TOOS, DAOS, MAOS, TODB.
3. The test card for detecting glutamic-pyruvic transaminase by a photochemical method according to claim 1, wherein the film-forming agent comprises: hydroxymethyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, sodium alginate, and cyclamine.
4. The test card for photochemically detecting glutamic-pyruvic transaminase and the preparation method thereof as claimed in claim 1, wherein the stabilizer comprises: polyethylene glycol 6000, polyvinylpyrrolidone, trehalose, sucrose and mannitol.
5. The test card for photochemically detecting glutamic-pyruvic transaminase of claim 1, wherein the buffer solution comprises: phosphate buffer, TRIS buffer, MES buffer.
6. The test card for photochemically detecting glutamic-pyruvic transaminase according to claim 1, wherein the first and second reaction films comprise: polysulfone membranes, nylon membranes.
7. The test card for photochemically detecting glutamic-pyruvic transaminase of claim 1, wherein the diffusion membrane comprises: hydrophilic polyester gauze, hydrophobic polyester gauze and nylon gauze.
8. The test card for detecting glutamate pyruvate transaminase by a photochemical method according to claim 1, wherein the formulation of the first reaction solution comprises 50mM of L-alanine, 100KU/L of pyruvate oxidase, 50mM of α -oxoglutarate, 100KU/L of peroxidase, 0.2mM of thiamine pyrophosphate, 0.2g/L of magnesium chloride, 10mM of 4-aminoantipyrine, 10mM of MAOS, 0.1-10g/L of sodium alginate, 1-50g/L of trehalose, and 0.05M of phosphate buffer solution with pH of 6.
9. The test card for detecting glutamic-pyruvic transaminase by a photochemical method according to claim 1, wherein the second reaction solution comprises: pyruvate oxidase 100KU/L, peroxidase 100KU/L, thiamine pyrophosphate 0.2mM, magnesium chloride 0.2g/L, 4-aminoantipyrine 10mM, MAOS10mM, sodium alginate 0.1-10g/L, trehalose 1-50g/L, and 0.05M phosphate buffer solution with pH of 6.
10. A preparation method of a test card for detecting glutamic-pyruvic transaminase by a photochemical method is characterized by comprising the following steps:
the method comprises the following steps: the first reaction solution was prepared according to the following formulation,
the formula comprises 50-500mM of L-alanine, 50-200KU/L of pyruvate oxidase, 5-100mM of α -ketoglutaric acid, 50-200KU/L of peroxidase, 0.1-1mM of thiamine pyrophosphate, 0.1-5g/L of magnesium chloride, 1-50mM of 4-aminoantipyrine, 1-50mM of color developing agent, 0.1-10g/L of film forming agent, 1-50g/L of stabilizing agent and 0.1-2M of buffer solution, and the buffer solution is stirred for standby after one hour;
step two: the second reaction solution was prepared according to the following formulation,
the formula comprises the following components: 50-200KU/L pyruvate oxidase, 50-200KU peroxidase, 0.1-1mM thiamine pyrophosphate, 0.1-5g/L magnesium chloride, 1-50mM 4-aminoantipyrine, 1-50mM color developing agent, 0.1-10g/L film forming agent, 1-50g/L stabilizer and 0.1-2M buffer solution;
step three: the first reaction film is prepared by the following steps,
soaking the reaction membrane in the first reaction solution for 1-10min, taking the reaction membrane, and drying at 25-50 deg.C for 10-60 min;
step four: the second reaction film is prepared by the following steps,
soaking the reaction membrane in the second reaction solution for 1-10min, taking the reaction membrane, and drying at 25-50 ℃ for 10-60min for later use;
step five: and (3) sequentially sticking a first reaction film and a second reaction film on the single-sided adhesive, then sequentially sticking a second blood filtering film, a first blood filtering film and a diffusion film, loading the test card into a lower shell of the test card after sticking, and then loading the test card onto an upper shell cover to obtain the finished test card.
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