CN111398268A - Peroxidase detection test paper and preparation method and application thereof - Google Patents
Peroxidase detection test paper and preparation method and application thereof Download PDFInfo
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- CN111398268A CN111398268A CN202010331659.0A CN202010331659A CN111398268A CN 111398268 A CN111398268 A CN 111398268A CN 202010331659 A CN202010331659 A CN 202010331659A CN 111398268 A CN111398268 A CN 111398268A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/10—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using catalysis
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Abstract
The invention provides a peroxidase detection test paper, a preparation method and an application thereof, relating to the technical field of in vitro immunoassay. The basic reagent comprises a buffer system, a dye, a metal ion chelating agent and an optional surfactant, and the components are matched for use, so that the basic reagent has a good buffer function on the basis of providing a basic color for the detection test paper, and meanwhile, the false result caused by metal ions can be avoided, and the detection accuracy is improved; the core reagent comprises tetramethylbenzidine, peroxide, an optional surfactant and an optional catalyst, the peroxidase can decompose the peroxide to release oxygen atoms, and the oxygen atoms oxidize the tetramethylbenzidine indicator to enable the indicator to generate color change, so that the detection of the peroxidase is realized.
Description
Technical Field
The invention relates to the technical field of in vitro immunodetection, in particular to a peroxidase detection test paper and a preparation method and application thereof.
Background
Pleural effusion is a common clinical disease, and distinguishing whether pleural effusion is effusion or effusion greatly helps to diagnose the cause of disease and select a treatment scheme.
The exudate is inflammatory effusion, and can be caused by infectious (tuberculous, purulent pleurisy) or non-infectious (tumor, connective tissue disease) diseases. The effusion is non-inflammatory effusion and is mostly caused by systemic diseases, such as hydrostatic pressure rise in capillary vessels during heart failure and colloid osmotic pressure drop of hypoproteinemia during nephropathy and malnutrition to cause fluid accumulation in the thoracic cavity. Peroxidase generated by inflammatory cells in the effusion can be used as a key factor for distinguishing, and whether bubbles appear or not can be observed clinically through adding 30% hydrogen peroxide at present to be used as a mark for detecting the peroxidase, so that whether the pleural effusion is the effusion or the effusion is the effusion. However, the method has low sensitivity, non-visual result and large influence of subjective factors of observers.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
It is a first object of the present invention to provide a test strip for detecting peroxidase, which alleviates at least one of the technical problems of the prior art.
The second object of the present invention is to provide a method for preparing the above-mentioned test paper for detecting peroxidase.
The third purpose of the invention is to provide the application of the test paper for detecting peroxidase.
The invention provides a test paper for detecting peroxidase, which comprises test paper and a peroxidase detection reagent fixed on the test paper, wherein the peroxidase detection reagent comprises a basic reagent and a core reagent;
the basic agent comprises a buffer system, a dye, a surfactant and a metal ion chelating agent;
the core reagent comprises tetramethylbenzidine, peroxide, surfactant and optionally a catalyst.
Further, the solvent of the base reagent includes water;
preferably, in the basic agent, every 1000ml of water contains 50-100g of buffer system, 0.7-1.5g of dye, 20-50g of surfactant and 2-4g of metal ion chelating agent;
preferably, in the base reagent, 64g of the buffer system, 1.0g of the dye, 3g of the metal ion chelating agent and 24g of the surfactant are contained per 1000ml of water.
Further, the buffer system comprises a tris buffer, a phosphate buffer or a citric acid buffer, and the pH of the buffer system is preferably 7-10;
preferably, the dye comprises one or more of hydrazine yellow, methyl orange or sunset yellow;
preferably, the surfactant comprises a non-ionic surfactant, preferably comprising one or more of polyvinylpyrrolidone, polyethylene glycol, polyoxyethylene lauryl ether or polyvinyl alcohol;
preferably, the metal ion chelating agent comprises an organometallic ion chelating agent, preferably comprising one or more of ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid dipotassium salt or diethylenetriaminepentaacetic acid.
Further, the solvent of the core reagent comprises an organic solvent;
preferably, in the core reagent, every 1000ml of organic solvent contains 2-4g of tetramethyl benzidine, 3.5-15g of peroxide, 20-80g of surfactant and 0.6-3g of catalyst;
preferably, in the core reagent, 3g of tetramethylbenzidine, 10g of peroxide, 72g of surfactant and 1.5g of catalyst are contained per 1000ml of organic solvent.
Further, the peroxide comprises cumene hydroperoxide and/or a derivative of cumene hydroperoxide;
preferably, the surfactant comprises one or more of polyvinylpyrrolidone, polyethylene glycol, polyoxyethylene lauryl ether or polyvinyl alcohol;
preferably, the catalyst comprises quinoline and/or a derivative of quinoline;
preferably, the organic solvent comprises a lower alcohol solvent or a lower alcohol derivative solvent, preferably comprising methanol, ethanol, a methanol derivative or an ethanol derivative.
Further, the test paper comprises filter paper, preferably qualitative filter paper, quantitative filter paper or chromatography filter paper.
Furthermore, the test paper for detecting peroxidase also comprises a substrate, and the test paper is fixed on the surface of the substrate.
The invention also provides a preparation method of the peroxidase detection test paper, wherein a basic reagent and a core reagent are sequentially fixed on the test paper to obtain the peroxidase detection test paper.
Further, soaking the test paper in a basic reagent, drying for the first time, soaking in the core reagent, and drying for the second time to obtain the detection test paper of the peroxidase;
preferably, the primary drying satisfies at least one of the following conditions:
the temperature is 70-100 ℃, and preferably 80 ℃; the time is 30-60min, preferably 50 min;
preferably, the secondary drying satisfies at least one of the following conditions:
the temperature is 40-70 ℃, and the preferred temperature is 55 ℃; the time is 20-40min, preferably 30 min.
In addition, the invention also provides application of the detection test paper of the peroxidase in identifying pleural effusion and pleural effusion.
The invention provides the detection test paper of peroxidase with the basic reagent and the core reagent fixed according to different functions and different solubilities of the basic reagent and the core reagent. The basic reagent comprises a buffer system, a dye, a metal ion chelating agent and an optional surfactant, and the components are matched for use, so that the basic reagent has a good buffer function on the basis of providing a basic color for the detection test paper, and meanwhile, the false result caused by metal ions can be avoided, and the detection accuracy is improved; the core reagent comprises tetramethylbenzidine, peroxide, an optional surfactant and an optional catalyst, the peroxidase can decompose the peroxide to release oxygen atoms, and the oxygen atoms oxidize the tetramethylbenzidine indicator to enable the indicator to generate color change, so that the detection of the peroxidase is realized.
The invention has the following advantages by matching the basic reagent and the core reagent:
1. the sensitivity is high. The method is characterized in that a tetramethylbenzidine method is used for detecting peroxidase, the peroxidase can decompose peroxide to release oxygen atoms, the oxygen atoms oxidize a tetramethylbenzidine indicator to enable the indicator to have color change, and trace oxygen atoms can oxidize the tetramethylbenzidine to have color change, so that a bubble reaction is converted into a color reaction by using a dry chemical method, and the reaction sensitivity can be greatly improved.
2. And the detection result is visual. When detecting a relatively turbid sample, such as a pleural fluid or ascites sample, the bubble reaction detection result is not easy to observe, and an erroneous detection result is easily obtained. The detection result of the detection test paper provided by the invention is color change, and the reaction carrier is the test paper substrate, so that the detection result is simple, convenient and visual and is easier to observe.
3. The result can be judged by a combined machine, and the influence of subjective factors of an observer is reduced. The detection result of the detection test paper provided by the invention can be accurately analyzed by a machine through the change of the absorbance value, so that the accuracy of the detection result is further improved, and the influence caused by subjective factors of observers is reduced.
In conclusion, the peroxidase detection test paper provided by the invention has obvious advantages and is expected to be popularized in relevant in vitro diagnosis projects.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to one aspect of the invention, a peroxidase detection test paper is provided, which comprises a test paper and a peroxidase detection reagent fixed on the test paper, wherein the peroxidase detection reagent comprises a basic reagent and a core reagent;
the base reagent comprises a buffer system, a dye, a metal ion chelating agent and an optional surfactant;
the core reagent comprises tetramethylbenzidine, peroxide, an optional surfactant, and an optional catalyst.
The invention provides the detection test paper of peroxidase with the basic reagent and the core reagent fixed according to different functions and different solubilities of the basic reagent and the core reagent.
The base reagent comprises a buffer system, a dye, a metal ion chelating agent and an optional surfactant:
the buffer system has the function of providing a buffer environment for the reaction, and preferably comprises a tris buffer, a phosphate buffer or a citric acid buffer, and the pH is preferably 7-10; the dye has the function of providing basic color, preferably, the dye comprises one or more of hydrazine yellow, methyl orange or sunset yellow, and as blue and yellow are complementary colors, the final positive result is blue, and the best observation effect can be achieved under the contrast of the basic color yellow; the metal ion chelating agent is used for chelating metal ions in a sample to solve false results caused by the metal ions, and preferably, the metal ion chelating agent comprises one or more of disodium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate or diethylenetriaminepentaacetic acid. The components are matched for use, so that the basic reagent has a good buffering function on the basis of providing basic color for the detection test paper, and meanwhile, the false result caused by metal ions can be avoided, and the detection accuracy is improved.
The core reagent comprises tetramethyl benzidine, peroxide, optional surfactant and optional catalyst: the peroxide acts as an oxygen donor for the reaction, and the peroxidase decomposes the peroxide to release an oxygen atom, and preferably, the peroxide comprises cumene hydroperoxide and/or a derivative of cumene hydroperoxide; the tetramethylbenzidine is used as an indicator of reaction, and the oxygen atom oxidizes the tetramethylbenzidine indicator to cause the indicator to have color change, thereby realizing the detection of peroxidase.
The invention has the following advantages by matching the basic reagent and the core reagent:
1. the sensitivity is high. The method is characterized in that a tetramethylbenzidine method is used for detecting peroxidase, the peroxidase can decompose peroxide to release oxygen atoms, the oxygen atoms oxidize a tetramethylbenzidine indicator to enable the indicator to have color change, and trace oxygen atoms can oxidize the tetramethylbenzidine to have color change, so that a bubble reaction is converted into a color reaction by using a dry chemical method, and the reaction sensitivity can be greatly improved.
2. And the detection result is visual. When detecting a relatively turbid sample, such as a pleural fluid or ascites sample, the bubble reaction detection result is not easy to observe, and an erroneous detection result is easily obtained. The detection result of the detection test paper provided by the invention is color change, and the reaction carrier is the test paper substrate, so that the detection result is simple, convenient and visual and is easier to observe.
3. The result can be judged by a combined machine, and the influence of subjective factors of an observer is reduced. The detection result of the detection test paper provided by the invention can be accurately analyzed by a machine through the change of the absorbance value, so that the accuracy of the detection result is further improved, and the influence caused by subjective factors of observers is reduced.
In conclusion, the peroxidase detection test paper provided by the invention has obvious advantages and is expected to be popularized in relevant in vitro diagnosis projects.
In the present invention, "one or more" means that one of them may be selected and added as a component, or two, three, or more of them may be selected and added as a component. Taking the metal ion chelating agent as an example, the metal ion chelating agent in the present invention may be ethylenediamine tetraacetic acid disodium salt alone, ethylenediamine tetraacetic acid dipotassium salt alone, diethylenetriamine pentaacetic acid alone, ethylenediamine tetraacetic acid disodium salt and ethylenediamine tetraacetic acid dipotassium salt, ethylenediamine tetraacetic acid disodium salt and diethylenetriamine pentaacetic acid, ethylenediamine tetraacetic acid dipotassium salt and diethylenetriamine pentaacetic acid, or a combination of ethylenediamine tetraacetic acid disodium salt, ethylenediamine tetraacetic acid dipotassium salt and diethylenetriamine pentaacetic acid.
The basic reagent in the invention can contain a surfactant or not, preferably contains a surfactant, and when the surfactant is contained, the components in the basic reagent can be blended, so that the components can be uniformly distributed on the test paper. Preferably, the surfactant comprises one or more of polyvinylpyrrolidone, polyethylene glycol, polyoxyethylene lauryl ether or polyvinyl alcohol.
The core reagent in the present invention may or may not contain a surfactant and/or a catalyst, for example, may contain a surfactant but not a catalyst, contains a catalyst but not a surfactant, contains no surfactant and no catalyst, or contains both a surfactant and a catalyst, preferably contains both a surfactant and a catalyst, and when the surfactant is contained, can adjust each component in the core reagent so that each component can be uniformly distributed on the test paper, preferably, the surfactant includes a nonionic surfactant, preferably, one or more of polyvinylpyrrolidone, polyethylene glycol, polyoxyethylene lauryl ether or polyvinyl alcohol; when a catalyst is included, it is capable of catalyzing the reaction, resulting in a shorter reaction time and improved sensitivity, and preferably, the catalyst comprises quinoline and/or a derivative of quinoline.
In the present invention, the test paper preferably includes filter paper, and more preferably includes qualitative filter paper, quantitative filter paper or chromatographic filter paper.
In some preferred embodiments, the components of the base reagent according to the present invention are all soluble in water, and the solvent of the base reagent comprises water.
Preferably, in the base reagent, every 1000ml of water contains 50-100g of buffer system, such as but not limited to 50g, 60g, 70g, 80g, 90g or 100 g; contains 0.7-1.5g of dye, such as but not limited to 0.7g, 0.8g, 1g, 1.2g or 1.5 g; 2-4g of metal ion chelating agent, such as but not limited to 2g, 3g or 4 g; contains 20-50g of optional surfactant, such as but not limited to 20g, 30g, 40g or 50 g.
In order to further improve the detection sensitivity of the peroxidase detection test paper provided by the invention, it is preferable that the basic reagent contains 64g of a buffer system, 1.0g of a dye, 3g of a metal ion chelating agent and optionally 24g of a surfactant per 1000ml of water.
In some preferred embodiments, each component of the core reagent according to the present invention has a high solubility in an organic solvent, and the solvent of the core reagent comprises an organic solvent, preferably the organic solvent comprises a low carbon alcohol solvent, preferably methanol, ethanol, a methanol derivative or an ethanol derivative.
Preferably, in the core reagent, 2-4g of tetramethylbenzidine is contained per 1000ml of organic solvent, and may be, for example, but not limited to, 2g, 3g or 4 g; contains 3.5-15g of peroxide, such as but not limited to 3.5g, 4g, 5g, 8g, 10g, 12g or 5 g; contains 20-80g of optional surfactant, such as but not limited to 20g, 30g, 40g, 50g, 60g, 70g or 80 g; the optional catalyst is present in an amount of 0.6 to 3g, and may be, for example, but not limited to, 0.6g, 1g, 1.5g, 2g, 2.5g or 3 g.
In order to further improve the detection sensitivity of the peroxidase detection test paper provided by the invention, preferably, the core reagent contains tetramethylbenzidine Xg, peroxide Xg, an optional surfactant Xg and an optional catalyst Xg per 1000ml of organic solvent.
In some preferred embodiments, the peroxidase test strip further comprises a substrate, and the test strip is immobilized on the surface of the substrate. The substrate can be used for fixing the test paper, is convenient to use and is convenient for a user to observe a detection result. Typical substrates may be ABS, PP, PC, PVC, PE or PET. Wherein, the substrate and the detection test paper of the peroxidase can be fixed by using a double-sided adhesive tape.
According to a second aspect of the present invention, there is provided a method for preparing the above test paper for detecting peroxidase, wherein a base reagent and a core reagent are sequentially immobilized on the test paper, so as to obtain the test paper for detecting peroxidase.
The preparation method of the oxidase detection test paper provided by the invention is simple in process and convenient to operate, can be prepared by ordinary experimenters, and effectively saves labor cost. The basic reagent is fixed firstly, and then the core reagent is fixed, so that the fixation of the core reagent is more stable, and the sensitivity of a detection result is ensured.
The preparation method of the basic reagent and the core reagent is not limited, and the reagents with corresponding functions can be obtained by the conventional preparation method in the field, for example, the corresponding basic reagent or the core reagent can be obtained by taking the components with the formula amount, adding the components into the solvent and uniformly mixing the components.
In some preferred embodiments, the test strip is soaked in the basic reagent, then dried for the first time, and then soaked in the core reagent, and the test strip for detecting peroxidase is obtained after the second drying.
Different reagents are soaked and then are respectively dried, on one hand, the stability of fixing the basic reagent and the core reagent on the test paper can be increased through drying in batches, on the other hand, the reagents with different solubilities can be fully dissolved in respective solvents, different reagents are prevented from being mixed, and the detection sensitivity is guaranteed.
Preferably, the primary drying satisfies at least one of the following conditions:
the temperature is 70-100 deg.C, for example, but not limited to, 70 deg.C, 75 deg.C, 80 deg.C, 85 deg.C, 90 deg.C, 95 deg.C or 100 deg.C; the time is 30-60min, such as but not limited to 30min, 35min, 40min, 45min, 50min, 55min or 60 min;
the preferred conditions for primary drying are: the temperature is 80 deg.C, and the time is 50 min.
Preferably, the secondary drying satisfies at least one of the following conditions:
the temperature is 40-70 deg.C, such as but not limited to 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C or 70 deg.C; the time is 20-40min, for example, but not limited to, 20min, 25min, 30min, 35min or 40 min.
The preferred conditions for the secondary drying are: the temperature is 55 deg.C, and the time is 30 min.
Through the reasonable consideration of the characteristics of each component, when the drying conditions are selected to respectively dry the test paper soaking the basic reagent and the core reagent, the fixing effect of the reagent can be better, and the detection sensitivity is further ensured.
In addition, according to the third aspect of the invention, the application of the test paper for detecting peroxidase in identifying pleural effusion and pleural effusion is also provided.
Peroxidase enzymes decompose peroxide to release oxygen atoms, which oxidize the tetramethylbenzidine indicator, causing the indicator to change color. When a certain amount of peroxidase is contained in a pleural fluid or ascites sample of a human body, the color of the test paper changes from orange to yellow green or dark green, and the color of the test paper continues to turn dark blue due to the extremely high concentration of the peroxidase. Based on the detection result, the detection test paper for peroxidase can accurately and sensitively identify pleural effusion and pleural effusion.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1
The embodiment provides a test paper for detecting peroxidase, which comprises qualitative filter paper, and a basic reagent and a core reagent which are fixed on the qualitative filter paper;
the basic reagent comprises: 4g of monopotassium phosphate and 60g of disodium hydrogen phosphate, wherein the pH value is 7.5; 1g of hydrazine yellow; 20g of polyvinylpyrrolidone and 4g of polyoxyethylene lauryl ether; 3g of ethylene diamine tetraacetic acid disodium salt; 1000ml of purified water;
the core reagent comprises 3g of tetramethyl benzidine; cumene hydroperoxide 10 g; 60g of polyvinylpyrrolidone and 12g of polyoxyethylene lauryl ether; 1.5g of 4-hydroxyquinoline; 1000ml of ethanol.
And (3) immersing qualitative filter paper into a basic reagent, taking out, drying at 80 ℃ for 50min, then immersing into a core reagent, taking out, and drying at 55 ℃ for 30min to obtain the test paper for the peroxidase.
Example 2
The embodiment provides a test paper for detecting peroxidase, which comprises qualitative filter paper, and a basic reagent and a core reagent which are fixed on the qualitative filter paper;
the basic reagent comprises: 4g of monopotassium phosphate and 60g of disodium hydrogen phosphate, wherein the pH value is 7.5; 0.7g of hydrazine yellow; 25g of polyvinylpyrrolidone and 5g of polyoxyethylene lauryl ether; 3g of ethylene diamine tetraacetic acid disodium salt; 1000ml of purified water;
the core reagent comprises 2.5g of tetramethyl benzidine; cumene hydroperoxide 8 g; 50g of polyvinylpyrrolidone and 10g of polyoxyethylene lauryl ether; 1.2g of 4-hydroxyquinoline; 1000ml of ethanol.
And (3) immersing qualitative filter paper into a basic reagent, taking out, drying at 80 ℃ for 50min, then immersing into a core reagent, taking out, and drying at 55 ℃ for 30min to obtain the test paper for the peroxidase.
Example 3
The embodiment provides a test paper for detecting peroxidase, which comprises qualitative filter paper, and a basic reagent and a core reagent which are fixed on the qualitative filter paper;
the basic reagent comprises: 4g of monopotassium phosphate and 60g of disodium hydrogen phosphate, wherein the pH value is 7.5; 1.2g of hydrazine yellow; 30g of polyvinylpyrrolidone and 6g of polyoxyethylene lauryl ether; 3g of ethylene diamine tetraacetic acid disodium salt; 1000ml of purified water;
the core reagent comprises 2g of tetramethyl benzidine; 6g of cumene hydroperoxide; 60g of polyvinylpyrrolidone and 12g of polyoxyethylene lauryl ether; 1.5g of 4-hydroxyquinoline; 1000ml of ethanol.
And (3) immersing qualitative filter paper into a basic reagent, taking out, drying at 80 ℃ for 50min, then immersing into a core reagent, taking out, and drying at 55 ℃ for 30min to obtain the test paper for the peroxidase.
Example 4
The embodiment provides a test paper for detecting peroxidase, which comprises qualitative filter paper, and a basic reagent and a core reagent which are fixed on the qualitative filter paper;
the basic reagent comprises: 3g of monopotassium phosphate and 47g of disodium hydrogen phosphate, wherein the pH value is 7.5; 1.5g of hydrazine yellow; 42g of polyvinylpyrrolidone and 8g of polyoxyethylene lauryl ether; 2g of ethylene diamine tetraacetic acid disodium salt; 1000ml of purified water;
the core reagent comprises 4g of tetramethyl benzidine; cumene hydroperoxide 3.5 g; 67g of polyvinylpyrrolidone and 13g of polyoxyethylene lauryl ether; 0.6g of 4-hydroxyquinoline; 1000ml of ethanol.
And (3) immersing qualitative filter paper into a basic reagent, taking out, drying at 80 ℃ for 50min, then immersing into a core reagent, taking out, and drying at 55 ℃ for 30min to obtain the test paper for the peroxidase.
Example 5
The embodiment provides a test paper for detecting peroxidase, which comprises qualitative filter paper, and a basic reagent and a core reagent which are fixed on the qualitative filter paper;
the basic reagent comprises: 6.25g of monopotassium phosphate and 93.75g of disodium hydrogen phosphate, wherein the pH is 7.5; 1.5g of hydrazine yellow; 17g of polyvinylpyrrolidone and 3g of polyoxyethylene lauryl ether; 4g of ethylene diamine tetraacetic acid disodium salt; 1000ml of purified water;
the core reagent comprises 2g of tetramethyl benzidine; cumene hydroperoxide 15 g; 17g of polyvinylpyrrolidone and 3g of polyoxyethylene lauryl ether; 3g of 4-hydroxyquinoline; 1000ml of ethanol.
And (3) immersing qualitative filter paper into a basic reagent, taking out, drying at 80 ℃ for 50min, then immersing into a core reagent, taking out, and drying at 55 ℃ for 30min to obtain the test paper for the peroxidase.
Example 6
The embodiment provides a test paper for detecting peroxidase, which comprises qualitative filter paper, and a basic reagent and a core reagent which are fixed on the qualitative filter paper;
the basic reagent comprises: 2g of monopotassium phosphate and 40g of disodium hydrogen phosphate, wherein the pH value is 7.5; 0.5g of hydrazine yellow; 45g of polyvinylpyrrolidone and 10g of polyoxyethylene lauryl ether; 5g of ethylene diamine tetraacetic acid disodium salt; 1000ml of purified water;
the core reagent comprises 1g of tetramethyl benzidine; cumene hydroperoxide (18 g); 70g of polyvinylpyrrolidone and 20g of polyoxyethylene lauryl ether; 0.5g of 4-hydroxyquinoline; 1000ml of ethanol.
And (3) immersing qualitative filter paper into a basic reagent, taking out, drying at 80 ℃ for 50min, then immersing into a core reagent, taking out, and drying at 55 ℃ for 30min to obtain the test paper for the peroxidase.
Example 7
This example provides a peroxidase test strip, which is different from example 1 only in that a phosphate buffer solution in a basic reagent is replaced with a Tris buffer solution, including 60g, to adjust the pH to 9.0.
Example 8
This example provides a peroxidase test strip, which is different from example 1 only in that a phosphate buffer solution in a basic reagent is replaced with a citric acid buffer solution, which includes 3g of citric acid and 60g of sodium citrate, and the pH is adjusted to 7.0.
Example 9
This example provides a test strip for peroxidase, which is different from example 1 only in that hydrazine yellow in the base reagent is replaced with methyl orange.
Example 10
This example provides a test strip for peroxidase, which is different from example 1 only in that hydrazine yellow in the base reagent is replaced with sunset yellow.
Example 11
This example provides a test strip for detecting peroxidase, which is different from example 1 only in that polyvinylpyrrolidone and laureth in the basic reagent are replaced with 36g of polyethylene glycol.
Example 12
This example provides a test strip for detecting peroxidase, which is different from example 1 only in that polyvinyl pyrrolidone and laureth in the base reagent are replaced with 36g of polyvinyl alcohol.
Example 13
This example provides a peroxidase test strip, which is different from example 1 only in that disodium salt of ethylenediaminetetraacetic acid in the base reagent is replaced with dipotassium salt of ethylenediaminetetraacetic acid.
Example 14
This example provides a peroxidase test strip, which is different from example 1 only in that disodium ethylenediaminetetraacetate contained in the basic reagent is replaced with diethylenetriaminepentaacetic acid.
Example 15
This example provides a test strip for peroxidase, which is different from example 1 only in that cumene hydroperoxide in the core reagent is replaced with di-tert-butyl cumene hydroperoxide.
Example 16
This example provides a test strip for detecting peroxidase, which is different from example 1 only in that polyvinylpyrrolidone and laureth in the core reagent are replaced with 72g of polyethylene glycol.
Example 17
This example provides a test strip for detecting peroxidase, which is different from example 1 only in that polyvinylpyrrolidone and laureth in the core reagent are replaced with 72g of polyvinyl alcohol.
Example 18
This example provides a test strip for peroxidase, which is different from example 1 only in that 4-hydroxyquinoline in the core reagent is replaced with quinoline.
Example 19
This example provides a test strip for detecting peroxidase, which is different from example 1 only in that the solvent ethanol in the core reagent is replaced by methanol.
Example 20
This example provides a test strip for peroxidase, which is different from example 1 only in that qualitative filter paper is replaced with chromatographic filter paper.
Example 21
This example provides a test strip for detecting peroxidase, which is different from example 1 only in that: and (3) immersing qualitative filter paper into a basic reagent, taking out, drying at 70 ℃ for 60min, then immersing into a core reagent, taking out, and drying at 40 ℃ for 40min to obtain the test paper for the peroxidase.
Example 22
This example provides a test strip for detecting peroxidase, which is different from example 1 only in that: and (3) immersing qualitative filter paper into a basic reagent, taking out, drying at 100 ℃ for 30min, then immersing into a core reagent, taking out, and drying at 70 ℃ for 20min to obtain the test paper for the peroxidase.
Example 23
This example provides a test strip for detecting peroxidase, which is different from example 1 only in that: and (3) immersing qualitative filter paper into a basic reagent, taking out, drying at 65 ℃ for 25min, then immersing into a core reagent, taking out, and drying at 75 ℃ for 45min to obtain the test paper for the peroxidase.
Comparative example 1
This comparative example provides a test strip for peroxidase, which is different from example 1 only in that it does not contain a basic reagent.
Comparative example 2
This comparative example provides a test strip for peroxidase, which is different from example 1 only in that the base reagent does not contain a metal ion chelating agent.
Comparative example 3
This comparative example provides a test strip for peroxidase, which is different from example 1 only in that: and (3) immersing qualitative filter paper into the core reagent, taking out, drying at 55 ℃ for 30min, then immersing into the basic reagent, taking out, and drying at 80 ℃ for 50min to obtain the test paper for the peroxidase.
Experimental example 1
The peroxidase test strips provided in examples 1 to 23 and comparative examples 1 to 3 were used to test positive reference samples, and hemoglobin was used as a positive reference reagent, and was diluted in a gradient of 10. mu.g/ml, 20. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml, 200. mu.g/ml and 500. mu.g/ml. The detection sensitivity is shown in the following table:
according to the experimental results, the detection sensitivity of the peroxidase detection test paper provided by the embodiment of the invention is higher than that of the peroxidase detection test paper in the comparative example, which indicates that the detection test paper obtained by limiting the reagent composition and the preparation method of the detection test paper has higher sensitivity and can obtain a more accurate detection result.
Wherein, the effect of the embodiment 1 is better than that of the embodiments 4-6, which shows that the test paper with the component content in the preferable range of the invention has higher detection sensitivity; the effect of example 1 is superior to or equal to
Examples 7-20, which illustrate that test strips with raw material components selected within the preferred ranges of the present invention, have higher detection sensitivity; example 1 is superior to examples 21 to 23 in the effect, and shows that the test paper prepared by applying the preparation conditions within the preferable range of the present invention is also important in the preparation conditions of the test paper, and the sensitivity is higher.
Experimental example 2
Clinically, 53 cases of pleural effusion and 67 cases of pleural effusion are selected, and a bubble method and the test paper provided by the embodiment 1 of the invention are respectively used for differential diagnosis, and the detection results are as follows:
compared with the prior art that the thoracic cavity effusion and the thoracic cavity effusion are identified by the existing bubble method, the detection test paper provided by the invention has higher accuracy of the detection result, and the positive rate of the detection on the thoracic cavity effusion reaches 94.3 percent and is far higher than 60.4 percent of the detection by the existing bubble method; the negative rate of detection on pleural effusion is more 100%.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The test paper for detecting the peroxidase is characterized by comprising test paper and a peroxidase detection reagent fixed on the test paper, wherein the peroxidase detection reagent comprises a basic reagent and a core reagent;
the base reagent comprises a buffer system, a dye, a metal ion chelating agent and an optional surfactant;
the core reagent comprises tetramethylbenzidine, peroxide, an optional surfactant, and an optional catalyst.
2. The test strip of claim 1, wherein the solvent of the base reagent comprises water;
preferably, in the basic agent, every 1000ml of water contains 50-100g of buffer system, 0.7-1.5g of dye, 2-4g of metal ion chelating agent and 20-50g of surfactant;
preferably, in the base reagent, 64g of the buffer system, 1.0g of the dye, 3g of the metal ion chelating agent and 24g of the surfactant are contained per 1000ml of water.
3. The test strip according to claim 1, wherein the buffer system comprises tris buffer, phosphate buffer or citrate buffer, and the pH of the buffer system is preferably 7 to 10;
preferably, the dye comprises one or more of hydrazine yellow, methyl orange or sunset yellow;
preferably, the surfactant comprises a non-ionic surfactant, preferably comprising one or more of polyvinylpyrrolidone, polyethylene glycol, polyoxyethylene lauryl ether or polyvinyl alcohol;
preferably, the metal ion chelating agent comprises an organometallic ion chelating agent, preferably comprising one or more of ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid dipotassium salt or diethylenetriaminepentaacetic acid.
4. The test strip of claim 1, wherein the solvent of the core reagent comprises an organic solvent;
preferably, in the core reagent, every 1000ml of organic solvent contains 2-4g of tetramethyl benzidine, 3.5-15g of peroxide, 20-80g of surfactant and 0.6-3g of catalyst;
preferably, in the core reagent, every 1000ml of organic solvent contains 3g of tetramethyl benzidine, 10g of peroxide, 72g of surfactant and 1.5g of catalyst;
preferably, the organic solvent comprises a lower alcohol solvent or a lower alcohol derivative solvent, preferably comprising methanol, ethanol, a methanol derivative or an ethanol derivative.
5. The detection reagent of claim 1, wherein the peroxide comprises cumene hydroperoxide and/or a derivative of cumene hydroperoxide;
preferably, the surfactant comprises one or more of polyvinylpyrrolidone, polyethylene glycol, polyoxyethylene lauryl ether or polyvinyl alcohol;
preferably, the catalyst comprises quinoline and/or a derivative of quinoline.
6. The peroxidase detection test strip according to claim 1, wherein the test strip comprises a filter paper, preferably a qualitative filter paper, a quantitative filter paper or a chromatographic filter paper.
7. The peroxidase test strip according to any one of claims 1 to 6, further comprising a substrate, wherein the test strip is immobilized on a surface of the substrate.
8. The method of producing a test strip for detecting peroxidase according to any one of claims 1 to 7, wherein a base reagent and a core reagent are immobilized on the test strip in this order to obtain the test strip for detecting peroxidase.
9. The preparation method of claim 8, wherein the test paper is soaked in a basic reagent, then dried for the first time, and then soaked in the core reagent, and the test paper for peroxidase is obtained after secondary drying;
preferably, the primary drying satisfies at least one of the following conditions:
the temperature is 70-100 ℃, and preferably 80 ℃; the time is 30-60min, preferably 50 min;
preferably, the secondary drying satisfies at least one of the following conditions:
the temperature is 40-70 ℃, and the preferred temperature is 55 ℃; the time is 20-40min, preferably 30 min.
10. Use of a peroxidase test strip according to any one of claims 1 to 7 in the manufacture of a product for the identification of pleural effusion and pleural effusion.
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