CN111189899A - Test strip for detecting creatinine by electrochemical method and preparation method thereof - Google Patents
Test strip for detecting creatinine by electrochemical method and preparation method thereof Download PDFInfo
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- CN111189899A CN111189899A CN202010026068.2A CN202010026068A CN111189899A CN 111189899 A CN111189899 A CN 111189899A CN 202010026068 A CN202010026068 A CN 202010026068A CN 111189899 A CN111189899 A CN 111189899A
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- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 title claims abstract description 74
- 238000012360 testing method Methods 0.000 title claims abstract description 54
- 229940109239 creatinine Drugs 0.000 title claims abstract description 37
- 238000002848 electrochemical method Methods 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims description 7
- 239000010410 layer Substances 0.000 claims abstract description 140
- 102000004190 Enzymes Human genes 0.000 claims abstract description 103
- 108090000790 Enzymes Proteins 0.000 claims abstract description 103
- 239000012790 adhesive layer Substances 0.000 claims abstract description 69
- 210000004369 blood Anatomy 0.000 claims abstract description 31
- 239000008280 blood Substances 0.000 claims abstract description 31
- CVSVTCORWBXHQV-UHFFFAOYSA-N anhydrous creatine Natural products NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 claims abstract description 29
- 229960003624 creatine Drugs 0.000 claims abstract description 27
- 239000006046 creatine Substances 0.000 claims abstract description 27
- 108090000604 Hydrolases Proteins 0.000 claims abstract description 25
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 25
- 108010060059 Sarcosine Oxidase Proteins 0.000 claims abstract description 25
- 102000008118 Sarcosine oxidase Human genes 0.000 claims abstract description 25
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 25
- 238000001914 filtration Methods 0.000 claims abstract description 23
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Natural products OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 20
- 102000004316 Oxidoreductases Human genes 0.000 claims abstract description 20
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 20
- 229960005070 ascorbic acid Drugs 0.000 claims abstract description 20
- 238000009792 diffusion process Methods 0.000 claims abstract description 20
- 239000004094 surface-active agent Substances 0.000 claims abstract description 17
- 239000003381 stabilizer Substances 0.000 claims abstract description 16
- 239000000758 substrate Substances 0.000 claims abstract description 14
- 108010066906 Creatininase Proteins 0.000 claims abstract description 13
- 239000002270 dispersing agent Substances 0.000 claims abstract description 7
- 238000009472 formulation Methods 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 38
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 21
- 229910052799 carbon Inorganic materials 0.000 claims description 21
- 238000001035 drying Methods 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 20
- 239000002211 L-ascorbic acid Substances 0.000 claims description 18
- 235000000069 L-ascorbic acid Nutrition 0.000 claims description 18
- 239000007853 buffer solution Substances 0.000 claims description 15
- 239000002002 slurry Substances 0.000 claims description 14
- 238000002791 soaking Methods 0.000 claims description 13
- 239000008363 phosphate buffer Substances 0.000 claims description 12
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 11
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 11
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 9
- 238000005520 cutting process Methods 0.000 claims description 7
- KTWOOEGAPBSYNW-UHFFFAOYSA-N ferrocene Chemical compound [Fe+2].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 KTWOOEGAPBSYNW-UHFFFAOYSA-N 0.000 claims description 7
- 239000002390 adhesive tape Substances 0.000 claims description 6
- 108010024957 Ascorbate Oxidase Proteins 0.000 claims description 5
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 claims description 3
- 239000007987 MES buffer Substances 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 229910021607 Silver chloride Inorganic materials 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 229920004896 Triton X-405 Polymers 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 3
- DCYOBGZUOMKFPA-UHFFFAOYSA-N iron(2+);iron(3+);octadecacyanide Chemical compound [Fe+2].[Fe+2].[Fe+2].[Fe+3].[Fe+3].[Fe+3].[Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] DCYOBGZUOMKFPA-UHFFFAOYSA-N 0.000 claims description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 239000005017 polysaccharide Substances 0.000 claims description 3
- 229920001282 polysaccharide Polymers 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- -1 potassium ferricyanide Chemical compound 0.000 claims description 3
- 229940124272 protein stabilizer Drugs 0.000 claims description 3
- 229960003351 prussian blue Drugs 0.000 claims description 3
- 239000013225 prussian blue Substances 0.000 claims description 3
- 229910052709 silver Inorganic materials 0.000 claims description 3
- 239000004332 silver Substances 0.000 claims description 3
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- 238000001514 detection method Methods 0.000 abstract description 20
- 239000012528 membrane Substances 0.000 abstract description 6
- 235000010323 ascorbic acid Nutrition 0.000 abstract 2
- 239000011668 ascorbic acid Substances 0.000 abstract 2
- 239000000872 buffer Substances 0.000 abstract 2
- 239000000047 product Substances 0.000 description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 239000000853 adhesive Substances 0.000 description 5
- 230000001070 adhesive effect Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 239000003365 glass fiber Substances 0.000 description 2
- 230000024924 glomerular filtration Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000012466 permeate Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
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- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
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Abstract
The invention discloses a test strip for detecting creatinine by an electrochemical method, which comprises: the device comprises a substrate, a working electrode, a reference electrode, a counter electrode, a starting electrode, an insulating layer, a first enzyme carrier layer, a second enzyme carrier layer, a blood filtering membrane, a diffusion layer, a double-sided adhesive layer, a siphon hole and a hydrophilic layer; the formulation of the first reaction solution on the first enzyme carrier layer comprises: creatininase, sarcosine oxidase, creatine hydrolase, ascorbic acid oxidase, peroxidase, dispersant, surfactant, buffer; the formula of the second reaction solution on the second enzyme carrier layer comprises: sarcosine oxidase, creatine hydrolase, ascorbic acid oxidase, peroxidase, stabilizer, surfactant, buffer; the invention has good matching performance between the detection result and the hospital detection result and is simple to use.
Description
Technical Field
The invention relates to the field of detection, in particular to a test strip for detecting creatinine by an electrochemical method and a preparation method thereof.
Background
Creatinine (CRE) is a product of human muscle metabolism, including both exogenous and endogenous. Exogenous creatinine is a product of meat metabolism in vivo; endogenous creatinine is a product of in vivo muscle tissue metabolism. Under normal conditions, the creatinine content in the human body is substantially stable. If the volume of the body muscle is not changed obviously, the generation amount of the endogenous creatinine is relatively constant and is mainly discharged out of the body through glomerular filtration. Therefore, under the condition that exogenous creatinine is stable, the concentration of creatinine in blood can be used as one of the indexes for detecting the glomerular filtration function. In general: male: 44-133 mu mol/L female: 70-106 mu mol/L.
The creatinine is mainly detected 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 performed by professionals. Causing some inconvenience to the user or patient. The dry test strip detection is free of products in China, international and international creatinine dry sheets of ISATA exist, but the dry sheet detection of other manufacturers is basically inaccurate due to the fact that price is too high and limitation of storage conditions is large, and the dry sheet detection mainly cannot remove interference of creatine in a sample, so that the detection result is uneven, and few people use the dry method to detect creatinine.
The test strip is required to be capable of removing creatine interference in a sample and good in matching of creatinine content detection and hospital detection results, and the problem is solved.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide the test strip for detecting the creatinine by the electrochemical method and the preparation method thereof, and the test strip can be matched with a dry biochemical analyzer for use to quickly, accurately and quantitatively detect the content of the creatinine in whole blood, serum/plasma and other body fluids in a human body or an animal body; the detection result is well matched with the hospital detection result, the use is simple, and the operation of professional personnel is not needed.
In order to achieve the above object, the present invention adopts the following technical solutions:
a test strip for detecting creatinine by an electrochemical method comprises: the electrode comprises a substrate, a working electrode, a comparison electrode, a counter electrode, a starting electrode, an insulating layer, a first enzyme carrier layer, a second enzyme carrier layer, a blood filtering membrane, a diffusion layer, a double-sided adhesive layer, a siphon hole and a hydrophilic layer, wherein the working electrode, the comparison electrode, the counter electrode and the starting electrode are arranged on the substrate;
the formulation of the first reaction solution on the first enzyme carrier layer comprises: 5-100KU/L creatininase, 5-200KU/L sarcosine oxidase, 5-200KU/L creatine hydrolase, 1-100KU/L ascorbic acid oxidase, 1-100KU/L peroxidase, 10-200g/L dispersant, 0.1-10g/L surfactant, 0.05-1M buffer solution;
the formula of the second reaction solution on the second enzyme carrier layer comprises: 5-200KU/L sarcosine oxidase, 5-200KU/L creatine hydrolase, 1-100KU/L ascorbic acid oxidase, 1-100KU/L peroxidase, 10-200g/L stabilizer, 0.1-10g/L surfactant, and 0.05-1M buffer solution.
The test strip for detecting creatinine by an electrochemical method comprises a double-sided adhesive layer: the enzyme carrier layer is placed on the insulating layer, and the first double-sided adhesive layer, the second double-sided adhesive layer, the third double-sided adhesive layer and the fourth double-sided adhesive layer are arranged at the upper end and two sides of the diffusion layer; the first double-sided adhesive layer is provided with mounting holes for placing the first enzyme carrier layer and the second enzyme carrier layer.
In the test strip for detecting creatinine by the electrochemical method, the working electrode and the reference electrode are provided with the electron mediator; the electron mediator includes: potassium ferricyanide, ferrocene and its derivatives, benzoquinone and its derivatives, prussian blue.
In the foregoing test strip for detecting creatinine by an electrochemical method, the counter electrode includes: a first pair of electrodes corresponding to the position of the working electrode, a second pair of electrodes corresponding to the position of the contrast electrode, and the first pair of electrodes and the second pair of electrodes are silver + silver chloride electrodes.
The test strip for detecting creatinine by an electrochemical method comprises the following stabilizers: protein stabilizer, polysaccharide stabilizer, and polyethylene glycol stabilizer.
In the foregoing test strip for detecting creatinine by an electrochemical method, the surfactant includes: tween-20, tween-80, triton X-100, triton X-405, Emulgen B66, OP-10.
The test strip for detecting creatinine by an electrochemical method comprises a buffer solution: phosphate buffer, TRIS buffer, MES buffer.
In the foregoing test strip for detecting creatinine by an electrochemical method, the formula of the first reaction solution includes: 50KU/L creatininase, 100KU/L sarcosine oxidase, 100KU/L creatine hydrolase, 20KU/L ascorbic acid oxidase, 50KU/L peroxidase, 20g/L polyvinylpyrrolidone, 0.1g/L Emulgen B66, 0.05MPH ═ 6 phosphate buffer.
In the test strip for detecting creatinine by an electrochemical method, the formula of the second reaction solution includes: 100KU/L sarcosine oxidase, 100KU/L creatine hydrolase, 20KU/L ascorbate oxidase, 50KU/L peroxidase, 20g/L polyvinylpyrrolidone, 0.1g/L Emulgen B66, and 0.05MPH 6.
A preparation method of a test strip for detecting creatinine by an electrochemical method comprises the following steps:
the method comprises the following steps: preparing a first reaction solution according to the following formula, wherein the formula comprises: 5-100KU/L creatininase, 5-200KU/L sarcosine oxidase, 5-200KU/L creatine hydrolase, 1-100KU/L ascorbic acid oxidase, 1-100KU/L peroxidase, 10-200g/L dispersant, 0.1-10g/L surfactant, 0.05-1M buffer solution; stirring for one hour for later use;
step two: preparing a second reaction solution according to the following formula, wherein the formula comprises: 5-200KU/L sarcosine oxidase, 5-200KU/L creatine hydrolase, 1-100KU/L ascorbic acid oxidase, 1-100KU/L peroxidase, 10-200g/L stabilizer, 0.1-10g/L surfactant, 0.05-1M buffer solution; stirring for one hour for later use;
step three: a first enzyme carrier layer is prepared and,
soaking the enzyme carrier layer in the first reaction solution for 1-10min, taking the enzyme carrier layer, and drying at 25-50 deg.C for 10-60min to obtain a first enzyme carrier layer;
step four: a second enzyme carrier layer is prepared and,
soaking the enzyme carrier layer in the second reaction solution for 1-10min, taking the enzyme carrier layer, and drying at 25-50 deg.C for 10-60min to obtain a second enzyme carrier layer;
step five: adding an electron mediator into the carbon slurry, and uniformly stirring to obtain mixed carbon slurry for later use;
step six: taking a substrate, printing a counter electrode, then taking the mixed carbon paste to print a working electrode and a reference electrode, then printing an insulating layer to obtain an electrode plate, and drying for later use;
step seven: taking an electric pole plate, sticking a double-sided adhesive tape, placing a first enzyme carrier layer and a second enzyme carrier layer on a first double-sided adhesive layer, sticking a blood filtration film on the first enzyme carrier layer and the second enzyme carrier layer, sticking a diffusion film on the blood filtration film, finally sticking a second double-sided adhesive layer, a third double-sided adhesive layer and a fourth double-sided adhesive layer, sticking a hydrophilic film on the second double-sided adhesive layer, the third double-sided adhesive layer and the fourth double-sided adhesive layer to form a siphon channel, and cutting to obtain a finished test strip product.
The invention has the advantages that:
the invention improves the test strip from formulation and structure, make the test strip and analyzer use and can be fast, accurate, quantitative determination human or animal internal whole blood, serum/plasma, other body fluid have creatinine content, the invention uses simply, does not need professional person's operation, suitable for each pharmacy, clinic, institute, community hospital, pet hospital, testing center or other government to check generally;
compared with the hospital value of the detection result of the test card, the R square value is more than 0.9, which shows that the test card has good matching with the hospital detection result.
Drawings
FIG. 1 is a schematic structural diagram of an embodiment of the present invention;
FIG. 2 is a cross-sectional view of one embodiment of the present invention;
FIG. 3 is a graph showing the experimental results of example 1 of the present invention;
FIG. 4 is a graph showing the experimental results of example 2 of the present invention;
FIG. 5 is a graph showing the results of an experiment in example 3 of the present invention;
FIG. 6 is a graph showing the experimental results of example 4 of the present invention.
The meaning of the reference symbols in the figures:
100 substrates, 101 working electrodes, 102 comparison electrodes, 111 first pair electrodes, 112 second pair electrodes, 121, 122, 123 starting electrodes, 201 insulating layers, 301 first double-sided adhesive layers, 311 and 312 mounting holes, 321 first enzyme carrier layers, 322 second enzyme carrier layers, 401 blood filtering films, 501 diffusion films, 601 second double-sided adhesive layers, 602 third double-sided adhesive layers, 603 fourth double-sided adhesive layers, 701 hydrophilic films and 801 siphon holes.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
A test strip for detecting creatinine by an electrochemical method comprises: the electrode comprises a substrate 100, a working electrode 101, a comparison electrode 102, a counter electrode, a starting electrode 121, 122, 123, an insulating layer 201 arranged on the electrode layer, a first enzyme carrier layer 321 arranged on the insulating layer 201 and corresponding to the position of the working electrode 101, a second enzyme carrier layer 322 arranged on the insulating layer 201 and corresponding to the position of the comparison electrode 102, a blood filtering film arranged on the enzyme carrier layer, a diffusion layer arranged on the blood filtering film, a double-sided adhesive layer covering the enzyme carrier layer, the blood filtering film 401 and two sides of the diffusion layer, a siphon hole 801 formed on the double-sided adhesive layer and communicated with the diffusion layer, and a hydrophilic layer arranged on the double-sided adhesive layer and forming a siphon structure with the double-sided adhesive layer.
After passing through the siphon hole 801, the sample reaches the diffusion layer through the siphon channel and then permeates into the blood filtering membrane 401 at a constant speed, after passing through the blood filtering membrane 401, red blood cells are retained above the blood filtering membrane 401, the serum permeates through the blood filtering membrane 401 and reaches the first enzyme carrier layer 321 and the second enzyme carrier layer 322 to react, electrons generated by the reaction are transmitted through the electronic mediators on the working electrode 101 and the contrast electrode 102, then current is generated, and the electrons are converted into concentration through the analyzer.
The formulation of the first reaction solution on the first enzyme support layer 321 includes: 5-100KU/L creatininase, 5-200KU/L sarcosine oxidase, 5-200KU/L creatine hydrolase, 1-100KU/L ascorbic acid oxidase, 1-100KU/L peroxidase, 10-200g/L dispersant, 0.1-10g/L surfactant, and 0.05-1M buffer solution.
The formulation of the second reaction solution on the second enzyme carrier layer 322 includes: 5-200KU/L sarcosine oxidase, 5-200KU/L creatine hydrolase, 1-100KU/L ascorbic acid oxidase, 1-100KU/L peroxidase, 10-200g/L stabilizer, 0.1-10g/L surfactant, and 0.05-1M buffer solution.
As an example, the stabilizer includes: protein stabilizers, polysaccharide stabilizers, polyethylene glycol stabilizers; the surfactant includes: tween-20, tween-80, triton X-100, triton X-405, Emulgen B66, OP-10; the buffer solution comprises: phosphate buffer, TRIS buffer, MES buffer.
The double-sided adhesive layer includes: a first double-sided adhesive layer 301 which is used for placing an enzyme carrier layer and is arranged on the insulating layer 201, and a second double-sided adhesive layer 601, a third double-sided adhesive layer 602 and a fourth double-sided adhesive layer 603 which are arranged at the upper end and two sides of the diffusion layer; the first double-sided adhesive layer 301 is provided with mounting holes 311, 312 for placing a first enzyme carrier layer 321 and a second enzyme carrier layer 322.
The working electrode 101 and the reference electrode 101 are provided with electron mediators; as an example, the electron mediator includes: potassium ferricyanide, ferrocene and its derivatives, benzoquinone and its derivatives, prussian blue.
The counter electrode includes: a first pair of electrodes 111 corresponding to the position of the working electrode 101, and a second pair of electrodes 112 corresponding to the position of the reference electrode 102, wherein the first pair of electrodes 111 and the second pair of electrodes 112 are silver + silver chloride electrodes as an example.
A preparation method of a test strip for detecting creatinine by an electrochemical method comprises the following steps:
the method comprises the following steps: preparing a first reaction solution according to the following formula, wherein the formula comprises: 5-100KU/L creatininase, 5-200KU/L sarcosine oxidase, 5-200KU/L creatine hydrolase, 1-100KU/L ascorbic acid oxidase, 1-100KU/L peroxidase, 10-200g/L dispersant, 0.1-10g/L surfactant, 0.05-1M buffer solution; stirring for one hour for later use;
step two: preparing a second reaction solution according to the following formula, wherein the formula comprises: 5-200KU/L sarcosine oxidase, 5-200KU/L creatine hydrolase, 1-100KU/L ascorbic acid oxidase, 1-100KU/L peroxidase, 10-200g/L stabilizer, 0.1-10g/L surfactant, 0.05-1M buffer solution; stirring for one hour for later use;
step three: a first enzyme carrier layer 321 is prepared,
soaking the enzyme carrier layer in the first reaction solution for 1-10min, taking the enzyme carrier layer, and drying at 25-50 ℃ for 10-60min to obtain a first enzyme carrier layer 321 for later use; as an example, the enzyme carrier layer comprises: glass fiber film, filter paper and asymmetric film;
step four: a second enzyme carrier layer 322 is prepared,
soaking the enzyme carrier layer in the second reaction solution for 1-10min, taking the enzyme carrier layer, and drying at 25-50 deg.C for 10-60min to obtain a second enzyme carrier layer 322 for use; as an example, the enzyme carrier layer comprises: glass fiber film, filter paper and asymmetric film;
step five: adding an electron mediator into the carbon slurry, and uniformly stirring to obtain mixed carbon slurry for later use;
step six: taking a substrate 100, printing a counter electrode, then taking mixed carbon paste to print a working electrode 101 and a reference electrode 102, then printing an insulating layer 201 to obtain an electrode plate, and drying for later use;
step seven: taking an electrode plate, sticking a double-sided adhesive tape, placing a first enzyme carrier layer 321 and a second enzyme carrier layer 322 on a first double-sided adhesive layer 301, sticking a blood filtering film 401 on the first enzyme carrier layer 321 and the second enzyme carrier layer 322, sticking a diffusion film 501 on the blood filtering film 401, finally sticking a second double-sided adhesive layer 601, a third double-sided adhesive layer 602 and a fourth double-sided adhesive layer 603, sticking a hydrophilic film 701 on the second double-sided adhesive layer 601, the third double-sided adhesive layer 602 and the fourth double-sided adhesive layer 603 to construct a siphon channel, and cutting to obtain a finished test strip.
The following tests verify that the detection result has good matching with the hospital test result:
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 a first reaction solution according to the following formula, wherein the formula comprises: 50KU/L creatininase, 100KU/L sarcosine oxidase, 100KU/L creatine hydrolase, 20KU/L ascorbic acid oxidase, 50KU/L peroxidase, 20g/L polyvinylpyrrolidone, 0.1g/L Emulgen B66, 0.05MPH ═ 6 phosphate buffer; stirring for one hour for later use;
step two: enzyme carrier layer 321: soaking the enzyme carrier layer in the reaction solution for 5min, taking the enzyme carrier layer out, and drying at 45 ℃ for 20min for later use;
step three: preparing working carbon slurry: adding electron mediator ferrocene methanol into carbon slurry CH-10, and uniformly stirring for later use
Step four: taking the substrate 100, printing the counter electrodes 111 and 112, then taking the prepared working carbon paste to print the working electrodes 101 and 102, then printing the insulating layer 201, and drying for later use;
step five: taking an electrode plate, then attaching a first double-sided adhesive layer 301, then placing an enzyme carrier layer 321 on the first double-sided adhesive layer 301, then attaching a blood filtering film 401 on the first double-sided adhesive layer, then attaching a diffusion film 501, finally attaching a second double-sided adhesive layer 601, a third double-sided adhesive layer 602, a fourth double-sided adhesive layer 603 and a hydrophilic film 701 to construct a siphon channel, and cutting to obtain a test strip finished product 1.
The test paper strips are tested with a self-made analyzer developed by the company, 10 venous blood samples with gradient are randomly drawn for testing, and then compared with hospital values for evaluation.
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 matched with an analyzer developed by the company for testing, 20 venous blood samples with gradient are randomly drawn for testing, and then compared with a hospital value, the R square value is 0.884, which indicates that the matching of the detection result and the hospital inspection result is general, and the normal value and the abnormal value are distinguished.
Example 2
The manufacturing method of the finished product 2 comprises the following steps:
the method comprises the following steps: preparing a first reaction solution according to the following formula, wherein the formula comprises: 50KU/L creatininase, 100KU/L sarcosine oxidase, 100KU/L creatine hydrolase, 20KU/L ascorbic acid oxidase, 50KU/L peroxidase, 20g/L polyvinylpyrrolidone, 0.1g/L Emulgen B66, 0.05MPH ═ 6 phosphate buffer; stirring for one hour for later use;
step two: preparing a second reaction solution according to the following formula, wherein the formula comprises: 100KU/L sarcosine oxidase, 100KU/L creatine hydrolase, 20KU/L ascorbate oxidase, 50KU/L peroxidase, 20g/L polyvinylpyrrolidone, 0.1g/LEmulgen B66, 0.05MPH ═ 6 phosphate buffer; stirring for one hour for later use;
step three: first enzyme carrier layer 321: soaking the enzyme carrier layer in the first reaction solution for 5min, taking the enzyme carrier layer out, and drying at 45 ℃ for 20min for later use;
step four: second enzyme carrier layer 322: soaking the enzyme carrier layer in the second reaction solution for 5min, taking the enzyme carrier layer out, and drying the enzyme carrier layer for 20min at the temperature of 45 ℃ for later use;
step five: preparing working carbon slurry: adding electron mediator ferrocene methanol into carbon slurry CH-10, and uniformly stirring for later use
Step six: taking a substrate 100, printing a counter electrode, then taking the prepared working carbon paste to print a working electrode 101 and a counter electrode 102, then printing an insulating layer 201, and drying for later use;
step seven: taking an electrode plate, then pasting a first double-sided adhesive layer 301, then placing a first enzyme carrier layer 321 and a second enzyme carrier layer 322 on a double-sided adhesive, then pasting a blood filtering film 401 on the double-sided adhesive, then pasting a diffusion film 501, finally pasting a second double-sided adhesive layer 601, a third double-sided adhesive layer 602, a fourth double-sided adhesive layer 603 and a hydrophilic film 701 to construct a siphon channel, and cutting to obtain a test strip finished product 2.
The results of the data for finished product 2 are shown in table 2 below and fig. 4;
TABLE 2
And (4) analyzing results: the finished product 2 is matched with an analyzer developed by the company for testing, 20 venous blood samples with gradient are randomly drawn for testing, and then compared with hospital values, the R square value is 0.989, which shows that the matching of the detection result and the hospital test result is excellent, particularly the low concentration is accurate, and the normal value and the abnormal value can be distinguished.
Example 3
The manufacturing method of the finished product 3 comprises the following steps:
the method comprises the following steps: preparing a first reaction solution according to the following formula, wherein the formula comprises: phosphate buffer of 10KU/L creatininase, 10KU/L sarcosine oxidase, 10KU/L creatine hydrolase, 1KU/L ascorbic acid oxidase, 10KU/L peroxidase, 10g/L polyvinylpyrrolidone, 0.1g/L Emulgen B66, 0.05MPH ═ 6; stirring for one hour for later use;
step two: preparing a second reaction solution according to the following formula, wherein the formula comprises: phosphate buffer of 10KU/L sarcosine oxidase, 10KU/L creatine hydrolase, 1KU/L ascorbate oxidase, 10KU/L peroxidase, 10g/L polyvinylpyrrolidone, 0.1g/LEmulgen B66, 0.05MPH ═ 6; stirring for one hour for later use;
step three: first enzyme carrier layer 321: soaking the enzyme carrier layer in the first reaction solution for 2min, taking the enzyme carrier layer out, and drying at 50 ℃ for 20min for later use;
step four: second enzyme carrier layer 322: soaking the enzyme carrier layer in the second reaction solution for 2min, taking the enzyme carrier layer out, and drying the enzyme carrier layer for 20min at the temperature of 50 ℃ for later use;
step five: preparing working carbon slurry: adding electron mediator ferrocene methanol into carbon slurry CH-10, and uniformly stirring for later use
Step six: taking a substrate 100, printing a counter electrode, then taking the prepared working carbon paste to print a working electrode 101 and a counter electrode 102, then printing an insulating layer 201, and drying for later use;
step seven: taking an electrode plate, then pasting a first double-sided adhesive tape, placing a first enzyme carrier layer 321 and a second enzyme carrier layer on the double-sided adhesive tape, pasting a blood filtering film 401 on the double-sided adhesive tape, pasting a diffusion film 501, finally pasting a second double-sided adhesive layer 601, a third double-sided adhesive layer 602 and a fourth double-sided adhesive layer 603, pasting a hydrophilic film 701 to construct a siphon channel, and cutting to obtain a test strip finished product 3.
The results for the data for finished product 3 are shown in table 3 below and fig. 5;
TABLE 3
And (4) analyzing results: the finished product 3 is matched with an analyzer developed by the company for testing, 10 venous blood samples with gradient are randomly drawn for testing, and then compared with a hospital value, the R square value is 0.9, which shows that the matching of the detection result and the hospital test result is good, but the linearity is poor compared with the finished product 2.
Example 4
The manufacturing method of the finished product 4 comprises the following steps:
the method comprises the following steps: preparing a first reaction solution according to the following formula, wherein the formula comprises: 100KU/L creatininase, 200KU/L sarcosine oxidase, 200KU/L creatine hydrolase, 80KU/L ascorbic acid oxidase, 80KU/L peroxidase, 200g/L polyvinylpyrrolidone, 10g/L Emulgen B66, 0.05MPH ═ 6 phosphate buffer; stirring for one hour for later use;
step two: preparing a second reaction solution according to the following formula, wherein the formula comprises: 200KU/L sarcosine oxidase, 200KU/L creatine hydrolase, 80KU/L ascorbate oxidase, 80KU/L peroxidase, 200g/L polyvinylpyrrolidone, 10g/LEmulgen B66, 0.05MPH 6 phosphate buffer; stirring for one hour for later use;
step three: first enzyme carrier layer 321: soaking the enzyme carrier layer in the first reaction solution for 10min, taking the enzyme carrier layer out, and drying the enzyme carrier layer for 20min at 25 ℃ for later use;
step four: second enzyme carrier layer 322: soaking the enzyme carrier layer in the second reaction solution for 10min, taking the enzyme carrier layer out, and drying the enzyme carrier layer for 20min at 25 ℃ for later use;
step five: preparing working carbon slurry: adding electron mediator ferrocene methanol into carbon slurry CH-10, and uniformly stirring for later use
Step six: taking the substrate 100, printing the counter electrodes 111 and 112, then taking the prepared working carbon paste to print the working electrode 101 and the counter electrode 102, then printing the insulating layer 201, and drying for later use;
step seven: taking an electrode plate, then pasting a first double-sided adhesive layer 301, then placing a first enzyme carrier layer 321 and a second enzyme carrier layer 322 on the first double-sided adhesive, then pasting a blood filtering film 401 on the first double-sided adhesive, then pasting a diffusion film 501, finally pasting a second double-sided adhesive layer 601, a third double-sided adhesive 602, a fourth double-sided adhesive layer 603 and a hydrophilic film 701 to construct a siphon channel, and cutting to obtain a test strip finished product 4.
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 matched with an analyzer developed by the company for testing, 10 venous blood samples with gradient are randomly drawn for testing, and then compared with a hospital value, the R square value is 0.952, which shows that the matching of the detection result and the hospital inspection result is good, but the linear relation is poorer than that of the finished product 2.
And (3) comprehensive analysis: the results of the finished products 2-4 and the finished product 1 are compared, and the results of the finished products 2-4 are compared, so that the test paper prepared according to the formula and the structure of the test paper strip disclosed by the invention has better matching performance with the hospital test results, and the formula in the example 2 is the optimal formula.
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 strip for detecting creatinine by an electrochemical method is characterized by comprising: the electrode comprises a substrate, a working electrode, a comparison electrode, a counter electrode and a starting electrode which are arranged on the substrate, an insulating layer arranged on the electrode layer, a first enzyme carrier layer arranged on the insulating layer and corresponding to the position of the working electrode, a second enzyme carrier layer arranged on the insulating layer and corresponding to the position of the comparison electrode, a blood filtering film arranged on the enzyme carrier layer, a diffusion layer arranged on the blood filtering film, a double-sided adhesive layer covering the enzyme carrier layer, the blood filtering film and two sides of the diffusion layer, a siphon hole formed on the double-sided adhesive layer and communicated with the diffusion layer, and a hydrophilic layer arranged on the double-sided adhesive layer and forming a siphon structure with the double-sided adhesive layer;
the formula of the first reaction solution on the first enzyme carrier layer comprises: 5-100KU/L creatininase, 5-200KU/L sarcosine oxidase, 5-200KU/L creatine hydrolase, 1-100KU/L ascorbic acid oxidase, 1-100KU/L peroxidase, 10-200g/L dispersant, 0.1-10g/L surfactant, 0.05-1M buffer solution;
the formula of the second reaction solution on the second enzyme carrier layer comprises: 5-200KU/L sarcosine oxidase, 5-200KU/L creatine hydrolase, 1-100KU/L ascorbic acid oxidase, 1-100KU/L peroxidase, 10-200g/L stabilizer, 0.1-10g/L surfactant, and 0.05-1M buffer solution.
2. The test strip for detecting creatinine by an electrochemical method according to claim 1, wherein the double-sided adhesive layer comprises: the enzyme carrier layer is placed on the insulating layer, and the first double-sided adhesive layer, the second double-sided adhesive layer, the third double-sided adhesive layer and the fourth double-sided adhesive layer are arranged at the upper end and two sides of the diffusion layer; the first double-sided adhesive layer is provided with mounting holes for placing a first enzyme carrier layer and a second enzyme carrier layer.
3. The strip of claim 1, wherein the working electrode and the control electrode have electron mediators; the electron mediator includes: potassium ferricyanide, ferrocene and its derivatives, benzoquinone and its derivatives, prussian blue.
4. The test strip for detecting creatinine by an electrochemical method according to claim 1, wherein the counter electrode comprises: a first pair of electrodes corresponding to the position of the working electrode, and a second pair of electrodes corresponding to the position of the contrast electrode, wherein the first pair of electrodes and the second pair of electrodes are silver + silver chloride electrodes.
5. The strip of claim 1, wherein the stabilizer comprises: protein stabilizer, polysaccharide stabilizer, and polyethylene glycol stabilizer.
6. The strip of claim 1, wherein the surfactant comprises: tween-20, tween-80, triton X-100, triton X-405, Emulgen B66, OP-10.
7. The strip of claim 1, wherein the buffer solution comprises: phosphate buffer, TRIS buffer, MES buffer.
8. The test strip for detecting creatinine by an electrochemical method according to claim 1, wherein the formulation of the first reaction solution comprises: 50KU/L creatininase, 100KU/L sarcosine oxidase, 100KU/L creatine hydrolase, 20KU/L ascorbic acid oxidase, 50KU/L peroxidase, 20g/L polyvinylpyrrolidone, 0.1g/L Emulgen B66, 0.05MPH ═ 6 phosphate buffer.
9. The test strip for detecting creatinine by an electrochemical method according to claim 1, wherein the formulation of the second reaction solution comprises: 100KU/L sarcosine oxidase, 100KU/L creatine hydrolase, 20KU/L ascorbate oxidase, 50KU/L peroxidase, 20g/L polyvinylpyrrolidone, 0.1g/L Emulgen B66, and 0.05MPH 6.
10. A preparation method of a test strip for detecting creatinine by an electrochemical method is characterized by comprising the following steps:
the method comprises the following steps: preparing a first reaction solution according to the following formula, wherein the formula comprises: 5-100KU/L creatininase, 5-200KU/L sarcosine oxidase, 5-200KU/L creatine hydrolase, 1-100KU/L ascorbic acid oxidase, 1-100KU/L peroxidase, 10-200g/L dispersant, 0.1-10g/L surfactant, 0.05-1M buffer solution; stirring for one hour for later use;
step two: preparing a second reaction solution according to the following formula, wherein the formula comprises: 5-200KU/L sarcosine oxidase, 5-200KU/L creatine hydrolase, 1-100KU/L ascorbic acid oxidase, 1-100KU/L peroxidase, 10-200g/L stabilizer, 0.1-10g/L surfactant, 0.05-1M buffer solution; stirring for one hour for later use;
step three: a first enzyme carrier layer is prepared and,
soaking the enzyme carrier layer in the first reaction solution for 1-10min, taking the enzyme carrier layer, and drying at 25-50 deg.C for 10-60min to obtain a first enzyme carrier layer;
step four: a second enzyme carrier layer is prepared and,
soaking the enzyme carrier layer in the second reaction solution for 1-10min, taking the enzyme carrier layer, and drying at 25-50 deg.C for 10-60min to obtain a second enzyme carrier layer;
step five: adding an electron mediator into the carbon slurry, and uniformly stirring to obtain mixed carbon slurry for later use;
step six: taking a substrate, printing a counter electrode, then taking the mixed carbon paste to print a working electrode and a reference electrode, then printing an insulating layer to obtain an electrode plate, and drying for later use;
step seven: taking an electric pole plate, sticking a double-sided adhesive tape, placing a first enzyme carrier layer and a second enzyme carrier layer on a first double-sided adhesive layer, sticking a blood filtration film on the first enzyme carrier layer and the second enzyme carrier layer, sticking a diffusion film on the blood filtration film, finally sticking a second double-sided adhesive layer, a third double-sided adhesive layer and a fourth double-sided adhesive layer, sticking a hydrophilic film on the second double-sided adhesive layer, the third double-sided adhesive layer and the fourth double-sided adhesive layer to form a siphon channel, and cutting to obtain a finished test strip product.
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Application publication date: 20200522 |