CN113588747B - Electrochemical test card for simultaneously measuring glutamic pyruvic transaminase and glutamic oxaloacetic transaminase - Google Patents
Electrochemical test card for simultaneously measuring glutamic pyruvic transaminase and glutamic oxaloacetic transaminase Download PDFInfo
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- 238000000840 electrochemical analysis Methods 0.000 title claims abstract description 25
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 title claims abstract description 16
- 108010082126 Alanine transaminase Proteins 0.000 title claims abstract description 16
- 108010003415 Aspartate Aminotransferases Proteins 0.000 title claims description 12
- 102000004625 Aspartate Aminotransferases Human genes 0.000 title claims description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 153
- 239000008280 blood Substances 0.000 claims abstract description 64
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims abstract description 63
- 210000004369 blood Anatomy 0.000 claims abstract description 55
- 229940107700 pyruvic acid Drugs 0.000 claims abstract description 32
- 239000000758 substrate Substances 0.000 claims abstract description 18
- 239000003292 glue Substances 0.000 claims abstract description 14
- 238000005070 sampling Methods 0.000 claims abstract description 11
- 108090000340 Transaminases Proteins 0.000 claims abstract description 4
- 102000014898 transaminase activity proteins Human genes 0.000 claims abstract description 3
- 239000012295 chemical reaction liquid Substances 0.000 claims description 19
- 108010042687 Pyruvate Oxidase Proteins 0.000 claims description 14
- 239000003814 drug Substances 0.000 claims description 14
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 claims description 12
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 12
- 229930006000 Sucrose Natural products 0.000 claims description 12
- 239000001913 cellulose Substances 0.000 claims description 12
- 229920002678 cellulose Polymers 0.000 claims description 12
- 229910052707 ruthenium Inorganic materials 0.000 claims description 12
- 239000005720 sucrose Substances 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 10
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 claims description 10
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 claims description 10
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 10
- 229960003767 alanine Drugs 0.000 claims description 10
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- KPGXRSRHYNQIFN-UHFFFAOYSA-L 2-oxoglutarate(2-) Chemical compound [O-]C(=O)CCC(=O)C([O-])=O KPGXRSRHYNQIFN-UHFFFAOYSA-L 0.000 claims description 5
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 5
- 108010017192 4-hydroxy-4-methyl-2-oxoglutarate aldolase Proteins 0.000 claims description 4
- 102100029589 Acylpyruvase FAHD1, mitochondrial Human genes 0.000 claims description 4
- 108010069823 Oxaloacetate decarboxylase Proteins 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 235000010299 hexamethylene tetramine Nutrition 0.000 claims description 3
- 239000004312 hexamethylene tetramine Substances 0.000 claims description 3
- VKYKSIONXSXAKP-UHFFFAOYSA-N hexamethylenetetramine Chemical compound C1N(C2)CN3CN1CN2C3 VKYKSIONXSXAKP-UHFFFAOYSA-N 0.000 claims description 3
- 238000007639 printing Methods 0.000 claims description 3
- 238000007650 screen-printing Methods 0.000 claims description 3
- 238000001514 detection method Methods 0.000 abstract description 11
- 238000005259 measurement Methods 0.000 abstract description 5
- 238000010241 blood sampling Methods 0.000 abstract description 4
- 238000012360 testing method Methods 0.000 description 22
- 238000001035 drying Methods 0.000 description 7
- 230000009471 action Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- YBCAZPLXEGKKFM-UHFFFAOYSA-K ruthenium(iii) chloride Chemical compound [Cl-].[Cl-].[Cl-].[Ru+3] YBCAZPLXEGKKFM-UHFFFAOYSA-K 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 2
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 2
- 206010067125 Liver injury Diseases 0.000 description 2
- 229960005261 aspartic acid Drugs 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229910052709 silver Inorganic materials 0.000 description 2
- 239000004332 silver Substances 0.000 description 2
- 230000001960 triggered effect Effects 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 231100000439 acute liver injury Toxicity 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
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- 238000009826 distribution Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 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
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
- G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- General Health & Medical Sciences (AREA)
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- Hematology (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses an electrochemical test card for simultaneously measuring glutamic pyruvic transaminase and glutamic oxalacetic transaminase, which comprises a PET substrate, a notching white glue layer, a hydrophilic film layer and a cover plate layer which are sequentially arranged, wherein an electrode layer is attached to the PET substrate, a pair of reaction electrodes and a full blood electrode are arranged on a reaction area on the electrode layer, the pair of reaction electrodes are positioned at one end close to a sampling port of the reaction area, the full blood electrode is positioned at one end far away from the sampling port of the reaction area, pins are arranged at one end, far away from the reaction area, of the PET substrate, and the pins are in one-to-one correspondence connection with the reaction electrodes and the full blood electrode. The electrochemical test card has small blood sampling amount and high detection speed, and eliminates the interference of endogenous pyruvic acid in fingertip blood on ALT and AST detection, so that the measurement result is more accurate.
Description
Technical Field
The invention belongs to the technical field of electrochemical test paper, and particularly relates to an electrochemical test card for simultaneously measuring glutamic pyruvic transaminase and glutamic oxaloacetic transaminase.
Background
Glutamic pyruvic transaminase and glutamic oxaloacetic transaminase are two important enzymes of metabolism of human bodies, and the measurement of ALT and AST in blood mainly reflects sensitive indexes of liver injury, and various acute liver injuries can lead to elevation of ALT and AST. Thus, the concentration of ALT and AST in human body can be detected, and targeted treatment can be performed according to the damage degree of liver function of human body.
At present, two transaminases in a body need to be measured, and the operation is needed by a large-scale biochemical analyzer and a professional in a hospital, or a test card for testing ALT and AST by single electrochemical test is adopted, wherein the former increases the detection time and cost, the operation is inconvenient, and the latter can not measure ALT and AST simultaneously, so that inconvenience is brought to measurement. Therefore, it is imperative to invent a method for simultaneously measuring ALT and AST.
Disclosure of Invention
Based on the technical defects, the electrochemical test card for simultaneously measuring ALT and AST is small in blood sampling amount and high in detection speed, and interference on ALT and AST caused by the existence of endogenous pyruvic acid in fingertip blood is eliminated, so that a measurement result is more accurate.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
the utility model provides an electrochemical test card of simultaneous measurement glutamic pyruvic transaminase and glutamic oxaloacetic transaminase, includes PET substrate, the dashing white glue film layer that sets gradually, hydrophilic rete and cover sheet layer, it has the electrode layer to adhere to on the PET substrate, reaction zone on the electrode layer includes first reaction zone, second reaction zone and third reaction zone, be provided with a pair of reaction electrode and a full blood electrode on first reaction zone, second reaction zone and the third reaction zone respectively, a pair of reaction electrode is located the one end that is close to the reaction zone sampling mouth, full blood electrode is located the one end that is kept away from the reaction zone sampling mouth, a pair of reaction electrode in first reaction zone is used for detecting ALT and endogenous pyruvic acid, a pair of reaction electrode in second reaction zone is used for detecting AST and endogenous pyruvic acid, a pair of reaction electrode in third reaction zone is used for detecting endogenous pyruvic acid, full blood electrode is used for detecting the full condition of reaction zone blood sample, is provided with the pin one-to-one on the PET substrate that is kept away from the reaction zone, the pin is connected with reaction electrode one by one.
The electrode layer is also provided with a starting electrode, the starting electrode is formed by directly short-circuiting two electrodes, and when the detecting instrument is inserted, the two electrodes are in circuit connection with the detecting instrument, and the instrument is started automatically.
The electrode layer is formed by printing conductive silver paste, conductive carbon paste and insulating ink on a PET sheet in a screen printing mode.
The first reaction zone, the second reaction zone and the third reaction zone are isolated by a notching white glue layer, and isolated sampling ports are formed; the first reaction area, the second reaction area and the third reaction area are covered with enzyme, the notching white glue layer is covered with a hydrophilic film layer, the hydrophilic film layer is provided with an exhaust hole, the enzyme and the hydrophilic film layer form a siphon pool structure, and a blood sample is sucked into the three independent reaction areas from a sampling port through siphon action during blood sampling.
The hydrophilic film layer is covered with a cover plate, and the cover plate leaks out of the exhaust hole.
The reaction liquid stored in the first reaction zone comprises the following components in percentage by mass: 87% of buffer solution, 2% of alpha-ketoglutarate, 2% of L-alanine, 2% of sucrose, 2% of triton x-1001%, 1% of cellulose, 3% of ruthenium hexaammine chloride and 2% of pyruvate oxidase.
The reaction liquid stored in the second reaction zone comprises the following components in percentage by mass: 84% of buffer solution, 2% of L-aspartic acid, 2% of L-alanine, 2% of sucrose, 2% of triton x-1001%, 1% of cellulose, 3% of ruthenium hexamine chloride, 2% of oxaloacetate decarboxylase and 2% of pyruvate oxidase.
The reaction liquid stored in the third reaction zone comprises the following components in percentage by mass: 91% of buffer liquid medicine, 2% of sucrose, 1% of cellulose, 3% of ruthenium hexaammine chloride and 2% of pyruvate oxidase.
The beneficial effects of the invention are as follows:
1) According to the invention, through three independent reaction areas, the concentrations of ALT and AST are measured simultaneously, and the interference of endogenous pyruvic acid is eliminated, so that the test is more accurate, the blood sampling amount is small, and the test speed is high;
2) The invention is provided with the full blood electrode, when the reaction area is full of the blood sucking reaction area, the full blood electrode is conducted to start detection, if the blood is not full of the blood sucking reaction area, the full blood electrode is not conducted, the blood is prompted to be not full of the blood sucking reaction area, and the risk that the blood is misled by an inaccurate test result under the conditions that the blood is not full of the blood sucking reaction area and is not perceived in the test process can be completely avoided;
3) The silver electrode or the carbon electrode used in the invention is used for manufacturing the test card, and the used reagent is low in price, so that the production cost is greatly reduced.
Drawings
FIG. 1 is a schematic diagram of the layered structure of an electrochemical test card of the present invention;
FIG. 2 is a schematic diagram of the electrode distribution structure of the electrode layer of the electrochemical test card of the present invention;
FIG. 3 is a schematic view of the external structure of an electrochemical test card according to the present invention;
FIG. 4 is a standard curve for detecting ALT using the electrochemical test card of the present invention;
FIG. 5 is a standard curve of an electrochemical test card of the present invention for detecting AST;
wherein, 1, PET substrate, 2, notching white glue layer, 3, hydrophilic film layer, 4, cover plate layer; 5. a sampling port;
6. a first reaction zone, 6-1, a first full blood electrode, 6-2, a first reaction electrode a,6-3, a first reaction electrode b;
7. a second reaction zone, 7-1, a second full blood electrode, 7-2, a second reaction electrode a,7-3, a second reaction electrode b;
8. a third reaction zone, 8-1, a third full-blood electrode, 8-2, a third reaction electrode a,8-3, a third reaction electrode b;
9. and starting the electrode.
Detailed Description
The following description of the present invention will be made more complete and clear in view of the detailed description of the invention, which is to be taken in conjunction with the accompanying drawings that illustrate only some, but not all, of the embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides an electrochemical test card for simultaneously measuring glutamic pyruvic transaminase and glutamic oxalacetic transaminase, which is shown in figure 1 and sequentially comprises a PET substrate 1, a notching white glue layer 2, a hydrophilic film layer 3 and a cover plate 4 from bottom to top.
As shown in FIG. 2, an electrode layer is arranged on a PET substrate 1 of the invention, and the electrode layer consists of a first reaction electrode a 6-2, a first reaction electrode b 6-3, a second reaction electrode a 7-2, a second reaction electrode b 7-3, a third reaction electrode a 8-2, a third reaction electrode b 8-3, a first full blood electrode 6-1, a second full blood electrode 7-1, a third full blood electrode 8-1 and a starting electrode 9.
The first reaction electrode a 6-2, the first reaction electrode b 6-3 and the first full-blood electrode 6-1 are sequentially arranged on the first reaction zone 6 from top to bottom, the second reaction electrode b 7-3, the second reaction electrode a 7-2 and the second full-blood electrode 7-1 are arranged on the second reaction zone 7 from top to bottom, the third reaction electrode a 8-2, the third reaction electrode b 8-3 and the third full-blood electrode 8-1 are arranged on the third reaction zone 8 from top to bottom, the first full-blood electrode 6-1, the second full-blood electrode 7-1 and the third full-blood electrode 8-1 are respectively positioned at the bottoms of the first reaction zone 6, the second reaction zone 7 and the third reaction zone 8 for detecting the full degree of the blood sample, when the blood sample is not full, the full-blood sample is not conducted, the blood is not indicated to be full-blood, and the defect caused by the inaccurate test result due to the fact that the blood is not full and is not perceived in the test process can be completely avoided.
The first reaction electrode a 6-2 and the first reaction electrode b 6-3, the second reaction electrode b 7-3 and the second reaction electrode a 7-2, and the third reaction electrode a 8-2 and the third reaction electrode b 8-3 are respectively matched for use and are respectively used for detecting ALT and endogenous pyruvic acid, AST and endogenous pyruvic acid.
Pins corresponding to the first reaction electrode a 6-2, the first reaction electrode b 6-3, the second reaction electrode a 7-2, the second reaction electrode b 7-3, the third reaction electrode a 8-2, the third reaction electrode b 8-3, the first full blood electrode 6-1, the second full blood electrode 7-1 and the third full blood electrode 8-1 are arranged at the bottom of the PET substrate 1, are connected in a one-to-one correspondence manner and are used for being connected and conducted with a detection instrument.
The electrode layer is formed by conducting silver paste, conducting carbon paste and insulating ink in a screen printing mode. The conductive carbon paste is used for preparing each reaction electrode, the packed blood electrode and the pins, the conductive silver paste is used for preparing leads for connecting the reaction electrodes, the packed blood electrodes and the pins, and the insulating ink is attached to a circuit and leaks out of the reaction area and the pins.
The starting electrode 9 is arranged at the bottom of the PET substrate 1 and is formed by directly short-circuiting two electrodes, and when a detection instrument is inserted, the two electrodes are conducted with a starting circuit of the detection instrument in a cutting-off way, so that the instrument is automatically started.
The electrodes are covered with insulating ink for isolating interference among the electrodes, and the first reaction area 6, the second reaction area 7 and the third reaction area 8 at the end parts, the tail parts of the reaction electrodes and the full blood electrodes on the PET substrate 1 and the starting electrode 9 are used for being communicated with the detection circuits of the detection instrument.
Enzyme is respectively covered on the first reaction zone 6, the second reaction zone 7 and the third reaction zone 8, a notching white glue layer 2 is arranged on the enzyme, and the notching white glue layer is used for respectively and independently isolating the first reaction zone 6, the second reaction zone 7 and the third reaction zone 8 and forming three sampling ports 5, so that the mutual conduction interference is eliminated between the reaction zones.
A hydrophilic film layer 3 and a cover plate 4 are respectively and sequentially arranged on the notching white glue layer 2. The hydrophilic membrane layer 3 and the cover plate 4 are respectively provided with an exhaust hole corresponding to the first reaction zone 6, the second reaction zone 7 and the third reaction zone 8, the exhaust holes are as close to one side of each reaction zone, which is as close to the electrode, as possible, and the exhaust holes, the reaction zone enzymes and the hydrophilic membrane layer 3 form a siphon pool.
The first reaction zone 6 of the present invention stores the reaction liquid components as follows: 87% of buffer solution, 2% of alpha-ketoglutarate, 2% of L-alanine, 2% of sucrose, 2% of triton x-1001%, 1% of cellulose, 3% of ruthenium hexaammine chloride and 2% of pyruvate oxidase. The reaction zone is used for detecting ALT+endogenous pyruvic acid, L-alanine and alpha-ketoglutarate generate pyruvic acid under the action of ALT, and the pyruvic acid and electron mediator ruthenium hexammoniate generate oxidation-reduction reaction under the action of pyruvic acid oxidase to generate current.
The reaction liquid stored in the second reaction zone 7 comprises the following components: 84% of buffer solution, 2% of L-aspartic acid, 2% of L-alanine, 2% of sucrose, 2% of triton x-1001%, 1% of cellulose, 3% of ruthenium hexamine chloride, 2% of oxaloacetate decarboxylase and 2% of pyruvate oxidase. The reaction zone is used for detecting AST+endogenous pyruvic acid, L-aspartic acid and L-alanine generate pyruvic acid under the action of AST, and pyruvic acid reacts with electron mediator ruthenium hexammoniate under the action of pyruvic oxidase to generate current.
The third reaction zone 8 of the present invention stores the reaction liquid components as follows: 91% of buffer liquid medicine, 2% of sucrose, 1% of cellulose, 3% of ruthenium hexaammine chloride and 2% of pyruvate oxidase. The reaction zone is used for detecting endogenous pyruvic acid, and the endogenous pyruvic acid reacts with electron mediator ruthenium hexaammine chloride under the action of pyruvic oxidase and generates current.
The method comprises the steps of respectively differentiating the current generated by ALT+endogenous pyruvic acid and AST+endogenous pyruvic acid with the current generated by detecting endogenous pyruvic acid, eliminating interference of endogenous pyruvic acid, obtaining the current generated by ALT and AST in a reaction way, and obtaining the concentration of ALT and AST by utilizing a standard curve of the current and the concentration.
As shown in figure 3, the electrochemical test card for simultaneously measuring glutamic pyruvic transaminase and glutamic oxaloacetic transaminase has the advantages that when blood fills a reaction area, each full blood electrode and the reaction electrode are conducted, so that the blood sucking quantity is sufficient; when the reaction area is not full of blood, the reaction electrode generates current conduction, the full blood electrode is not triggered, or the reaction electrode is not triggered, the full blood electrode conduction indicates that the blood sucking amount of the reaction area is insufficient, and the detection instrument prompts that the blood sucking amount is insufficient. When the blood sucking amount of the three reaction areas is enough, the current generated by the respective reaction is detected, the current value generated by the pyruvic acid in the middle reaction area is subtracted from the current generated by the ALT reaction area and the AST reaction area, the interference of endogenous pyruvic acid can be eliminated, and the concentrations of ALT and AST are respectively obtained according to the fitted standard curve relation of the current value and the concentration.
In another embodiment of the present invention, the preparation of the electrochemical test card of the present invention comprises the steps of:
1) And printing conductive silver paste, conductive carbon paste and insulating ink on the PET substrate, wherein the electrodes consist of a reaction electrode, a blood filling electrode and a starting electrode. The three groups of reaction electrodes and the packed blood electrode are respectively in three independent reaction areas, ALT+endogenous pyruvic acid content, AST+endogenous pyruvic acid content and endogenous pyruvic acid content are respectively tested, the starting electrode is formed by directly shorting two electrodes, and when the instrument is inserted into a test card and then detects that conduction current exists between the two electrodes, the instrument is automatically started. The insulating ink covers the electrode to expose the reaction area and electrode pins to obtain the electrochemical test card substrate for testing ALT and AST.
2) Preparing a treatment solution containing polyvinyl alcohol, carrying out hydrophilic treatment on the reaction area by using the treatment solution, and drying in a drying tunnel. The drying temperature of the treatment fluid is 50 ℃ and the time is 4min.
3) Three groups of reaction liquid medicine are prepared respectively, wherein the first group is the reaction liquid medicine for testing ALT+endogenous pyruvic acid, the second group is the reaction liquid medicine for testing AST+endogenous pyruvic acid, and the third group is the reaction liquid medicine for testing endogenous pyruvic acid. And (3) respectively putting the prepared three groups of reaction liquid medicines in corresponding reaction areas, and putting the three groups of reaction liquid medicines into a drying channel for drying after the sample application is finished, wherein the drying temperature is 50 ℃ and the time is 20-25min.
The buffer solution of the three reaction liquid medicines is Tris buffer solution with pH=7.3 and 0.5 mmol/L.
In the first group of reaction liquid medicine, the buffer solution accounts for 87 percent, the alpha-ketoglutarate accounts for 2 percent, the L-alanine accounts for 2 percent, the sucrose accounts for 2 percent, the surfactant is triton x-100 accounts for 1 percent, the cellulose accounts for 1 percent, the electron mediator is hexaammine ruthenium chloride accounts for 3 percent, and the pyruvate oxidase accounts for 2 percent.
In the second group of reaction liquid medicine, the buffer solution accounts for 84 percent, the L-aspartic acid accounts for 2 percent, the L-alanine accounts for 2 percent, the sucrose accounts for 2 percent, the surfactant is triton x-100 accounts for 1 percent, the cellulose accounts for 1 percent, the electron mediator is hexaammine ruthenium chloride accounts for 3 percent, the oxaloacetate decarboxylase accounts for 2 percent and the pyruvate oxidase accounts for 2 percent.
In the third group of reaction liquid medicine, the buffer liquid medicine accounts for 91 percent, the sucrose accounts for 2 percent, the surfactant triton x-100 accounts for 1 percent, the cellulose accounts for 1 percent, the electron mediator hexaammine ruthenium chloride accounts for 3 percent, and the pyruvate oxidase accounts for 2 percent.
4) After the drying is finished, the notching white glue layer, the hydrophilic film layer and the cover plate are respectively covered. The material of the notching white glue layer is Soweis WH234343 type double faced glue, the hydrophilic film is subjected to single-sided hydrophilic treatment, 3M 9901P type is used, and the material of the cover plate is synthetic paper.
Example 1
5 samples of fresh whole blood were prepared, each of which had a gradient of ALT and AST, and each of the concentrations was divided into 3 parts, the first was measured for ALT and AST respectively using a biochemical analyzer, the second was used for the test card to confirm the standard curves of ALT and AST, and the third was used for the test card to compare the concentrations of ALT and AST with the biochemical concentrations after confirming the standard curves.
In the standard curve confirmation process, 10 repeated tests are respectively carried out on samples with each concentration, the CV of the tested current value is within 3%, and the average value of the tested current with each concentration is shown in the following table 1:
TABLE 1 test currents at different concentrations ALT and AST
| Glutamic pyruvic transaminase concentration (g/L) | 20 | 50 | 100 | 300 | 500 |
| Glutamic-oxaloacetic transaminase concentration (g/L) | 20 | 50 | 100 | 300 | 500 |
| First reaction zone Current (nA) | 236 | 287 | 364 | 636 | 962 |
| Second reaction zone Current (nA) | 225 | 268 | 358 | 645 | 958 |
| Third reaction zone Current (nA) | 203 | 197 | 201 | 198 | 200 |
| ALT corresponding current (nA) | 33 | 90 | 163 | 438 | 762 |
| AST corresponding current (nA) | 22 | 71 | 157 | 447 | 758 |
The biochemical concentrations of ALT and AST are respectively subjected to linear fitting with the corresponding current values, and standard curves of ALT and AST can be obtained (figures 4 and 5). From the standard curve, the biochemical concentrations of ALT and AST are better in linearity with the corresponding current values, which can reach 0.9987 and 0.9997 respectively.
A third blood sample was taken and tested for ALT and AST using a different electrochemical test card of the present invention and the results are shown in tables 2 and 3.
TABLE 2 ALT test results
TABLE 3 AST test results
As can be seen from tables 2 and 3, the concentrations of ALT and AST tested by the test card have small deviation from biochemical concentrations, and the interference of endogenous pyruvic acid is eliminated by utilizing the differential principle, so that the ALT and AST can be measured simultaneously by one drop of blood by the test card, and the result has high accuracy.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. The electrochemical test card for simultaneously measuring glutamic pyruvic transaminase and glutamic oxalacetic transaminase comprises a PET substrate, a notching white glue layer, a hydrophilic film layer and a cover plate layer which are sequentially arranged, and is characterized in that an electrode layer is attached to the PET substrate, a reaction area on the electrode layer comprises a first reaction area, a second reaction area and a third reaction area, a pair of reaction electrodes and a full blood electrode are respectively arranged on the first reaction area, the second reaction area and the third reaction area, the pair of reaction electrodes are positioned at one end close to a sampling port of the reaction area, the full blood electrode is positioned at one end far from the sampling port of the reaction area, the pair of reaction electrodes of the first reaction area are used for detecting glutamic pyruvic transaminase and endogenous pyruvic acid, the pair of reaction electrodes of the second reaction area are used for detecting endogenous pyruvic acid, the full blood electrode of the third reaction area is used for detecting full blood sample of the reaction area, and pins are arranged at one end, far from the reaction area, of the PET substrate, and the pins are in one-to-one correspondence with the reaction electrodes;
according to mass fraction:
the reaction liquid stored in the first reaction zone comprises the following components: 87% of buffer solution, 2% of alpha-ketoglutarate, 2% of L-alanine, 2% of sucrose, 1% of triton x-100%, 1% of cellulose, 3% of ruthenium hexammoniate chloride and 2% of pyruvate oxidase;
the reaction liquid stored in the second reaction zone comprises the following components: 84% of buffer solution, 2% of L-aspartic acid, 2% of L-alanine, 2% of sucrose, 1% of triton x-100%, 1% of cellulose, 3% of ruthenium hexamine chloride, 2% of oxaloacetate decarboxylase and 2% of pyruvate oxidase;
the reaction liquid stored in the third reaction zone comprises the following components: 91% of buffer liquid medicine, 2% of sucrose, 1% of triton x-100%, 1% of cellulose, 3% of ruthenium hexaammine chloride and 2% of pyruvate oxidase.
2. The electrochemical test card for simultaneously measuring glutamic pyruvic transaminase and glutamic oxaloacetic transaminase according to claim 1, wherein the electrode layer is provided with a starting electrode, and the starting electrode is formed by directly short-circuiting two electrodes.
3. The electrochemical test card for simultaneously measuring glutamic pyruvic transaminase and glutamic oxaloacetic transaminase according to claim 2, wherein the electrode layer is formed by printing conductive silver paste, conductive carbon paste and insulating ink on a PET substrate in a screen printing mode.
4. The electrochemical test card for simultaneously measuring glutamic pyruvic transaminase and glutamic oxaloacetic transaminase of claim 1, wherein the first reaction zone, the second reaction zone and the third reaction zone are isolated by a notching white glue layer and form isolated sampling ports.
5. The electrochemical test card for simultaneously measuring glutamic pyruvic transaminase and glutamic oxaloacetic transaminase according to claim 1, wherein the hydrophilic membrane layer is provided with an exhaust hole, and the exhaust hole, enzyme and hydrophilic membrane layer form a siphon pool structure.
6. An electrochemical test card for simultaneously measuring glutamic pyruvic transaminase and glutamic oxaloacetic transaminase according to claim 5, wherein the hydrophilic film layer is covered with a cover sheet, and the cover sheet leaks out of the vent hole.
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