CN1190664C - Blood beta-hydroxybutyric acid electrochemical sensor - Google Patents
Blood beta-hydroxybutyric acid electrochemical sensor Download PDFInfo
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- CN1190664C CN1190664C CNB031290957A CN03129095A CN1190664C CN 1190664 C CN1190664 C CN 1190664C CN B031290957 A CNB031290957 A CN B031290957A CN 03129095 A CN03129095 A CN 03129095A CN 1190664 C CN1190664 C CN 1190664C
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- blood
- hydroxybutyric acid
- hydroxybutyrate
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- WHBMMWSBFZVSSR-UHFFFAOYSA-N 3-hydroxybutyric acid Chemical compound CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 title claims abstract description 72
- 239000008280 blood Substances 0.000 title claims abstract description 32
- 210000004369 blood Anatomy 0.000 title claims abstract description 32
- 102000004190 Enzymes Human genes 0.000 claims abstract description 17
- 108090000790 Enzymes Proteins 0.000 claims abstract description 17
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 16
- 101710088194 Dehydrogenase Proteins 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- 239000003381 stabilizer Substances 0.000 claims abstract description 4
- 239000000758 substrate Substances 0.000 claims description 11
- 239000000463 material Substances 0.000 claims description 9
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 claims description 7
- 229950006238 nadide Drugs 0.000 claims description 7
- 238000009413 insulation Methods 0.000 claims description 6
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 5
- 239000004020 conductor Substances 0.000 claims description 3
- 230000002255 enzymatic effect Effects 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims 1
- YKHQSWIVNHQJSW-UHFFFAOYSA-N iron;oxalonitrile Chemical group [Fe].N#CC#N YKHQSWIVNHQJSW-UHFFFAOYSA-N 0.000 claims 1
- 238000005259 measurement Methods 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 2
- 230000027756 respiratory electron transport chain Effects 0.000 abstract 2
- 239000000126 substance Substances 0.000 abstract 2
- 230000007547 defect Effects 0.000 abstract 1
- 229920002521 macromolecule Polymers 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 10
- -1 carboxy methyl ethyl Chemical group 0.000 description 5
- 238000000034 method Methods 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 3
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 2
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 description 2
- 239000004354 Hydroxyethyl cellulose Substances 0.000 description 2
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 2
- 241001597008 Nomeidae Species 0.000 description 2
- 239000004372 Polyvinyl alcohol Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000007772 electrode material Substances 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 229940071826 hydroxyethyl cellulose Drugs 0.000 description 2
- 235000019447 hydroxyethyl cellulose Nutrition 0.000 description 2
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 2
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 2
- 229940071676 hydroxypropylcellulose Drugs 0.000 description 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 description 2
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 229940005561 1,4-benzoquinone Drugs 0.000 description 1
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- RXGJTUSBYWCRBK-UHFFFAOYSA-M 5-methylphenazinium methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2[N+](C)=C(C=CC=C3)C3=NC2=C1 RXGJTUSBYWCRBK-UHFFFAOYSA-M 0.000 description 1
- 229920000896 Ethulose Polymers 0.000 description 1
- 239000001856 Ethyl cellulose Substances 0.000 description 1
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 1
- 239000001859 Ethyl hydroxyethyl cellulose Substances 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 229910021607 Silver chloride Inorganic materials 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 229940105329 carboxymethylcellulose Drugs 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- CSVGEMRSDNSWRF-UHFFFAOYSA-L disodium;dihydrogen phosphate Chemical compound [Na+].[Na+].OP(O)([O-])=O.OP(O)([O-])=O CSVGEMRSDNSWRF-UHFFFAOYSA-L 0.000 description 1
- 238000003411 electrode reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 229920001249 ethyl cellulose Polymers 0.000 description 1
- 235000019325 ethyl cellulose Nutrition 0.000 description 1
- 235000019326 ethyl hydroxyethyl cellulose Nutrition 0.000 description 1
- 235000010944 ethyl methyl cellulose Nutrition 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 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 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 1
- 229920003087 methylethyl cellulose Polymers 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 229920001206 natural gum Polymers 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- 229920001308 poly(aminoacid) Polymers 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a sensor for the quantitative detection of beta-hydroxybutyrate in blood samples, which is used for the quantitative measurement of beta-hydroxybutyrate at a high speed. Traditional beta-hydroxybutyrate concentration detection methods are realized mainly through large blood biochemical analyzers and have the defects of large volume of required blood, long time and high price. The present invention comprises an insulating base board, and the base board is covered with a work electrode and a reference electrode; a work area for blood sample measurement is set on the base board, and the work electrode and the reference electrode stay within the range of the work area; the part outside the work area for blood sample measurement is covered with an insulating covering film with chemical inertia; the part inside the work area for blood sample measurement is covered with an enzyme reagent reaction film which can react with beta-hydroxybutyrate and generate electron transfer, wherein the enzyme reagent reaction film contains beta-hydroxybutyrate dehydrogenase, NAD, an electron transfer body, diaphorase, a stabilizing agent used for maintaining enzyme activity, and hydrophilic macromolecule substances. The present invention can be conveniently used and can measure the beta-hydroxybutyrate content in a short time with the need for only a trace volume of blood.
Description
Technical field
The present invention relates to a kind of beta-hydroxybutyric acid in the blood sample be carried out the sensor of detection by quantitative, be used for quantitatively measuring fast blood ketoboidies level.
Background technology
In recent years, in diagnosis, measure the content of beta-hydroxybutyric acid concentration in the blood and more and more paid attention to.At present, the beta-hydroxybutyric acid detection method mainly is to utilize the beta-hydroxybutyric acid kit to carry out by large-scale seralyzer.But the problem that this method exists is: need to use special device, measure that required blood volume is more, the time is longer, cost an arm and a leg, only be adapted at carrying out in the large hospital, can not use in mini clinic or patient family.
Summary of the invention
The object of the present invention is to provide a kind of sensor that can conveniently measure beta-hydroxybutyric acid content in the blood in various places fast, and the present invention only needs micro blood just can measure the content of beta-hydroxybutyric acid at short notice.
The present invention is achieved in that blood beta-hydroxybutyric acid electrochemical sensor comprises the insulativity substrate, is covered with working electrode and contrast electrode on the substrate.Set a blood sample surveying work district on the substrate, comprised working electrode and contrast electrode in this workspace scope.Scope outside, blood sample surveying work district is covered with chemically inert insulation coverlay.Be covered with on blood sample surveying work district scope internal work electrode and the contrast electrode and can and produce the enzyme reagent reacting film of electron transport with the beta-hydroxybutyric acid reaction, enzyme reagent reacting film links to each other with contrast electrode with working electrode respectively.Working electrode and contrast electrode are respectively by outside the contact conductor pass-out insulation coverlay.
Described enzyme reagent reacting film contains beta-hydroxybutyric dehydrogenase, diaphorase, nicotinamide adenine dinucleotide (being called for short NAD), electron transit mediator, is used for stabilizing agent and hydrophilic high mol material that enzymatic activity keeps.Beta-hydroxybutyric dehydrogenase is every square centimeter of 0.01-200 μ active unit.The beta-hydroxybutyric dehydrogenase that surpasses 100 units in the responding layer not only can increase cost of products, also can cause the instability of response current.Diaphorase is every square centimeter of 0.01-600 μ active unit.
The hydrophilic high mol material can make sample solution penetrate into enzyme reaction layer apace to react, and the hydrophilic high mol material can be: carboxy methyl cellulose (CMC), hydroxy ethyl cellulose (HEC), hydroxy propyl cellulose (HPC), methylcellulose and ethyl cellulose, the ethyl hydroxy ethyl cellulose, carboxy methyl ethyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol (PVA), polylysine and polyaminoacid, the polystyrene acid iodide, latex, acrylic acid or its salt, methacrylic acid or its salt, the starch or derivatives thereof, maleic anhydride or its esters, gelatin, Ago-Gel, natural gum etc.
In the present invention, contain electron accepter in the responding layer, when reduced form nicotinamide adenine dinucleotide (being called for short NADH) was oxidized in electrode, electron accepter was reduced.Electron transit mediator can be: the potassium ferricyanide, 1,4-benzoquinone, phenazine methosulfate, indophenols and derivant thereof, β-Nai Kun-4-Huang Suanjia, methylene blue, ferrocene and derivant thereof etc.Electron transit mediator is preferably fewrricyanic acid radical compound especially.
During measurement, the sample solution that contains beta-hydroxybutyric acid is applied on the enzyme reagent reacting film of this biology sensor, beta-hydroxybutyric acid in the sample under certain voltage by the catalysis of beta-hydroxybutyric dehydrogenase, redox reaction takes place with NAD, the NADH of generation under the effect of diaphorase with the Fe in the fewrricyanic acid radical compound
3+Reduction, the response current that is produced in the oxidation-reduction process is proportional to the concentration of the beta-hydroxybutyric acid substrate in the sample.Therefore, as long as measure the content that the sensing response electric current is promptly known beta-hydroxybutyric acid, measure convenient, time spent weak point.
This blood beta-hydroxybutyric acid electrochemical sensor method for making comprises following steps:
1, at first utilize thick film silk-screen or film evaporation technology on insulating substrate, to form the electrode system that comprises working electrode and contrast electrode.The working electrode material therefor is selected a kind of of the stable inert material of electrochemical properties such as gold, platinum, carbon; The contrast electrode material is good with Ag/AgCl, also can adopt a kind of in the stable inert material of electrochemical properties such as gold, platinum, carbon.When adopting aforementioned inert material,, select for use usually and the working electrode identical materials for reducing process complexity.
2, adopt thick film silk-screen or other technology to form reaction operation interval with certain working electrode area and contrast electrode area.By suitable geometry designs, make the working electrode in the reaction workspace have constant area, to guarantee the measuring accuracy of this invention.
3, the preparation contain have certain effective dose, can with the enzyme of beta-hydroxybutyric acid reagentia and the solution of auxiliary reagent and electron transit mediator.
4, adopt accurate some model machine or other point sample equipment will contain fixedly enzyme and the auxiliary reagent and the electron transport liquid solution of effective dose, spray drips on the electrode of reaction workspace.
5, the drying and forming-film of biochemical reagents is handled.
6, the beta-hydroxybutyric acid electrochemica biological sensor cuts apart and encapsulates.
Adopt beta-hydroxybutyric acid electrochemical sensor of the present invention to carry, can comprise the medium various places use of mini clinic or patient family, and only need micro blood just can in one minute, measure the content of beta-hydroxybutyric acid fast.
Description of drawings
Fig. 1 is a STRUCTURE DECOMPOSITION synoptic diagram of the present invention;
Fig. 2 is the local longitudinal sectional drawing of Fig. 1;
Beta-hydroxybutyric acid concentration/sensor response current graph of a relation that Fig. 3 measures for one embodiment of the invention.
Embodiment
As depicted in figs. 1 and 2, blood beta-hydroxybutyric acid electrochemical sensor comprises insulativity substrate 1, is covered with working electrode 2 and contrast electrode 3 on the substrate 1.Set a blood sample surveying work district 6 on the substrate 1, comprised working electrode 2 and contrast electrode 3 in these workspace 6 scopes.6 scope outsides, blood sample surveying work district are covered with chemically inert insulation coverlay 5.Be covered with on blood sample surveying work district 6 scope internal work electrodes 2 and the contrast electrode 3 and can and produce the enzyme reagent reacting film 4 of electron transport with the beta-hydroxybutyric acid reaction.Enzyme reagent reacting film 4 links to each other with contrast electrode 3 with working electrode 2 respectively, and working electrode 2 and contrast electrode 3 are respectively by outside contact conductor 7, the 8 pass-outs insulation coverlay 5.
Enzyme reagent reacting film 4 contains beta-hydroxybutyric dehydrogenase, NAD, electron transit mediator, diaphorase, is used for stabilizing agent and hydrophilic high mol material that enzymatic activity keeps.Concrete is is made in the pH value is 0.2M sodium hydrogen phosphate-sodium dihydrogen phosphate cocktail buffer of 8.0 and contains beta-hydroxybutyric dehydrogenase 500 active units/ml, diaphorase 1500 active units/ml, NAD 8mg/ml, potassium ferricyanide 50mg/ml, the enzyme reagent solution of carboxymethyl cellulose 4mg/ml, getting the above-mentioned enzyme drop of 2 microlitres is added in the electrode reaction district of Fig. 1, with its in 45 degrees centigrade thermostatic drying chamber dry five minutes, form enzyme reaction layer.
After tested, with concentration be respectively 0,10,20,35, beta-hydroxybutyric acid solution 2 microlitres of 70mg/dL drip respectively in the reaction zone of the beta-hydroxybutyric acid sensor that said method is made, test its kinetic current after 60 seconds under 300mV voltage, the result is as shown in the table:
| Beta-hydroxybutyric acid concentration (mg/dL) | 0 | 10 | 20 | 35 | 70 |
| Sensor response current (μ A) | 1.05 | 2.11 | 3.09 | 3.68 | 5.02 |
Beta-hydroxybutyric acid concentration and sensor response current concern that as shown in Figure 3 therefore visible beta-hydroxybutyric acid concentration and the linear proportional relationship of sensor response current only need measure the content that the sensing response size of current can be measured beta-hydroxybutyric acid quickly and easily.
Claims (4)
1, blood beta-hydroxybutyric acid electrochemical sensor comprises insulativity substrate (1), it is characterized in that being covered with on the substrate (1) working electrode (2) and contrast electrode (3); Substrate (1) is gone up and is set a blood sample surveying work district (6), has comprised working electrode (2) and contrast electrode (3) in the scope of this workspace (6); Scope outside, blood sample surveying work district (6) is covered with chemically inert insulation coverlay (5); Scope inside, blood sample surveying work district (6) is covered with and can and produces the enzyme reagent reacting film (4) of electron transport with beta-hydroxybutyric acid reaction, enzyme reagent reacting film (4) respectively with working electrode (2) link to each other with contrast electrode (3); Working electrode (2) and contrast electrode (3) are respectively by outside contact conductor (7,8) the pass-out insulation coverlay (5).
2, blood beta-hydroxybutyric acid electrochemical sensor as claimed in claim 1 is characterized in that described enzyme reagent reacting film (4) contains beta-hydroxybutyric dehydrogenase, diaphorase, nicotinamide adenine dinucleotide, electron transit mediator, is used for stabilizing agent and hydrophilic high mol material that enzymatic activity keeps.
3, blood beta-hydroxybutyric acid electrochemical sensor as claimed in claim 2 is characterized in that described electron transit mediator is an iron cyanogen radical compound.
4, blood beta-hydroxybutyric acid electrochemical sensor as claimed in claim 2 is characterized in that described beta-hydroxybutyric dehydrogenase is that every square centimeter of 0.01-200 μ active unit, diaphorase are every square centimeter of 0.01-600 μ active unit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031290957A CN1190664C (en) | 2003-06-06 | 2003-06-06 | Blood beta-hydroxybutyric acid electrochemical sensor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031290957A CN1190664C (en) | 2003-06-06 | 2003-06-06 | Blood beta-hydroxybutyric acid electrochemical sensor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1456880A CN1456880A (en) | 2003-11-19 |
| CN1190664C true CN1190664C (en) | 2005-02-23 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB031290957A Expired - Fee Related CN1190664C (en) | 2003-06-06 | 2003-06-06 | Blood beta-hydroxybutyric acid electrochemical sensor |
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| Country | Link |
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| CN (1) | CN1190664C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101900734B (en) * | 2010-02-03 | 2013-03-27 | 上海高丰医疗电器有限公司 | Diagnosis test paper for diabetic ketosis and other symptoms of relatively high ketone body |
| CN108896635A (en) * | 2018-09-20 | 2018-11-27 | 江苏鱼跃医疗设备股份有限公司 | A kind of beta-hydroxybutyric acid electrochemical sensor |
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- 2003-06-06 CN CNB031290957A patent/CN1190664C/en not_active Expired - Fee Related
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| Publication number | Publication date |
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| CN1456880A (en) | 2003-11-19 |
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