CN107966556A - A kind of carbon dioxide kit using Enzymatic cycling - Google Patents

A kind of carbon dioxide kit using Enzymatic cycling Download PDF

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Publication number
CN107966556A
CN107966556A CN201711184134.3A CN201711184134A CN107966556A CN 107966556 A CN107966556 A CN 107966556A CN 201711184134 A CN201711184134 A CN 201711184134A CN 107966556 A CN107966556 A CN 107966556A
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kit
carbon dioxide
nadh
glucose
kit according
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CN201711184134.3A
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崔海林
黄小全
李冰
曾晓君
黄清媚
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Chuan Yi Biochemical Engineering Co Ltd Of Zhongshan City
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Chuan Yi Biochemical Engineering Co Ltd Of Zhongshan City
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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
    • G01N33/5002—Partitioning blood components

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  • Engineering & Computer Science (AREA)
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  • Hematology (AREA)
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  • Urology & Nephrology (AREA)
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  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a kind of carbon dioxide kit using Enzymatic cycling, kit is made of NADH analogs indirect regeneration and CO 2 measuring reagent, and wherein NADH analogs indirect regeneration is made of 6 glucose phosphate dehydrogenases, hexokinase, glucose, adenosine triphyosphate disodium salt.Carbon dioxide measurement kit preparation is simple, of low cost, wherein the NADH analogs indirect regeneration contained can extend the holding time of kit, effectively improves measuring stability, accuracy, precision.

Description

A kind of carbon dioxide kit using Enzymatic cycling
Technical field
The present invention relates to in-vitro diagnosis field, more particularly to a kind of carbon dioxide kit using Enzymatic cycling.
Background technology
Total carbon dioxide capacity (T-CO2) refer to various forms of CO in blood plasma2Summation, including HCO3 -(95%), a small amount of thing Manage the CO of dissolving2And CO existing for minimal amount of other forms2.Therefore influenced in vivo by breathing and being metabolized two factors, it is main If the influence of metabolic factor.T-CO2Enzyme process, electrode method, titration or amount area method etc. can be used.Wherein enzymatic assays T-CO2, Reagent, which opens and uses the rear carbon dioxide easily absorbed in air, causes the NADH classes material in reagent by oxidation deactivation, electrode method one As combined with blomelicalbloodgasandelectrolrteanalyzers, CO2Electrode film needs to regularly replace.NADH classes material is used in enzyme process as reaction Thing is demarcated, since the presence of Carbon Dioxide in Air easily causes carbon dioxide measurement kit deficient in stability, the holding time is short, Directly affect Clinical practice.
Report at present on CO 2 measuring reagent NADH analog indirect regenerations is very limited.Patent CN 2653492Y provides a kind of carbon dioxide combining power analyzer；The test method used is amount area method, not to suitable for biochemistry The stability of the kit of analyzer is improved；Patent CN200610097207.0 provides a kind of carbon dioxide kit Preparation method, but do not make the relevant discussion of NADH analog circular regenerations, the stability of kit is poor in practical applications；CN 103698500B provides a kind of method for strengthening stability of carbon dioxide detection reagent, but the means mainly taken are addition oiliness Material and surfactant are to reduce contact of the reagent with Carbon Dioxide in Air, but the oily matter introduced may be to biochemistry point Each mechanical module of analyzer has other influences, and the NADH stability of kit is not improved；Patent CN103558370 A Energy supply material when having made some discussion on the circular regeneration of reducing substances, but being not directed to circular regeneration, fails effectively Ensure the stability requirement in reagent kit use.Therefore, a kind of preparation of research is simple, of low cost, is surveyed for carbon dioxide Determining kit has the NADH analog indirect regenerations of stablizing effect, has suitable market value.
The content of the invention
It is an object of the invention to disclose a kind of carbon dioxide kit using Enzymatic cycling.
The technical solution used in the present invention is：
A kind of carbon dioxide kit using Enzymatic cycling, kit is by NADH analogs indirect regeneration and dioxy Change carbon measure reagent composition.
Preferably, in kit NADH analogs indirect regeneration by 6- phosphate-dextroses dehydrogenase, hexokinase, Glucose, adenosine triphyosphate disodium salt composition.
Preferably, in kit NADH analogs indirect regeneration by 6- phosphate-dextrose dehydrogenases 5-50U/L, oneself Sugared kinases 50-500U/L, glucose 1-5g/L, adenosine triphyosphate disodium salt 0.5-2.5g/L compositions.
Preferably, in kit NADH analogs indirect regeneration by 6- phosphate-dextrose dehydrogenase 2s 0U/L, hexose Kinases 200U/L, glucose 2g/L, adenosine triphyosphate disodium salt 1g/L compositions.
Preferably, in kit CO 2 measuring reagent by Trisbase, hydrochloric acid, stabilizer, magnesium acetate, oxamic acid Sodium, phosphoric acid enol pyruvic acid carboxylase, phosphoenolpyruvate potassium, APADH, malic dehydrogenase, ethylenediamine tetra-acetic acid Disodium, Proclin300 compositions.
Preferably, stabilizer is at least one of gelatin, sorbic alcohol, glycerine, ethylene glycol, bovine serum albumin(BSA).
Preferably, in kit CO 2 measuring reagent by Trisbase 6g/L, hydrochloric acid 1.8g/L, stabilizer 1g/L, Magnesium acetate 1g/L, Sodium oxamate 0.3g/L, phosphoric acid enol pyruvic acid carboxylase 0.1g/L, phosphoenolpyruvate potassium 0.6g/L, APADH 0.5g/L, malic dehydrogenase 0.05g/L, disodium ethylene diamine tetraacetate 1g/L, Proclin300 1g/L Composition.
The beneficial effects of the invention are as follows：Carbon dioxide measurement kit preparation is simple, of low cost, wherein contain NADH analogs indirect regeneration can extend the holding time of kit, effectively improve measuring stability, accuracy, precision Degree.
Embodiment
Embodiment 1
NADH analog indirect regenerations are formed by following components：6- phosphate-dextrose dehydrogenase 2s 0U/L, hexose swash Enzyme 200U/L, glucose 2g/L, adenosine triphyosphate disodium salt 1g/L.
Above-mentioned each component is fully dissolved, is mixed in former carbon dioxide measurement kit.Former CO 2 measuring reagent Box, is made of liquid single-reagent：Trisbase 6g/L, hydrochloric acid 1.8g/L, stabilizer 1g/L, magnesium acetate 1g/L, Sodium oxamate 0.3g/L, phosphoric acid enol pyruvic acid carboxylase 0.1g/L, phosphoenolpyruvate potassium 0.6g/L, APADH 0.5g/L, apple Tartaric acid dehydrogenase 0.05g/L, disodium ethylene diamine tetraacetate 1g/L, Proclin300 1g/L.Wherein, stabilizer is gelatin, three pears At least one of alcohol, glycerine, ethylene glycol, bovine serum albumin(BSA).
With Britain's Landau routine biochemistry Quality Control level 2 (Quality Control 2) and routine biochemistry Quality Control level 3 (Quality Control 3) for sample, mark Quasi- product are Britain's Landau routine biochemistry calibrated horizontal 2, are detected using 7020 type automatic clinical chemistry analyzer of Hitachi, if one Skip test, two test samples (skip test replaces sample with physiological saline).The ratio of sample and reagent is 2 μ l：200μ L, the dominant wavelength set is 405nm, a length of 700nm of complementary wave, after sample is mixed with reagent 1, after 37 DEG C are incubated 2min, read and counts Calculate 120 seconds absorbance change rate Λ/min, deduct reagent blank after calculate sample, standard items absorbance change rate Λ/ Min, then root calculate carbon dioxide content in sample automatically.Carbon dioxide target value is 12.9mmol/L in Quality Control 2, Quality Control 3 The target value of middle carbon dioxide is 16.4mmol/L, it is allowed to which deviation range is ± 20%.
According to above assay method, using same batch with bottled Quality Control 2, Quality Control 3 as detection sample, respectively with addition The carbon dioxide measurement kit of NADH analog indirect regenerations and do not add the two of NADH analog indirect regenerations Carbonoxide assay kit carry out stability trace detection, obtain Self-made reagent and contrast agents corkage stability, 37 DEG C plus Fast stability, long-term stable experiment result.
With contrast agents performance for stability index results of comparison (table 1)
Embodiment 2
NADH analog indirect regenerations are formed by following components：6- phosphate-dextrose dehydrogenases 5U/L, hexokinase 50U/L, glucose 1g/L, adenosine triphyosphate disodium salt 0.5g/L.
Above-mentioned each component is fully dissolved, is mixed in former carbon dioxide measurement kit.Former CO 2 measuring reagent Box, is made of liquid single-reagent：Trisbase 6g/L, hydrochloric acid 1.8g/L, stabilizer, magnesium acetate 1g/L, Sodium oxamate 0.3g/ L, phosphoric acid enol pyruvic acid carboxylase 0.1g/L, phosphoenolpyruvate potassium 0.6g/L, APADH 0.5g/L, malic acid take off Hydrogen enzyme 0.05g/L, disodium ethylene diamine tetraacetate 1g/L, Proclin300 1g/L.
Method according to embodiment 1 is detected.
With contrast agents performance for stability index results of comparison (table 2)
Embodiment 3
NADH analog indirect regenerations are formed by following components：6- phosphate-dextrose dehydrogenases 50U/L, hexose swash Enzyme 500U/L, glucose 5g/L, adenosine triphyosphate disodium salt 2.5g/L.
Above-mentioned each component is fully dissolved, is mixed in former carbon dioxide measurement kit.Former CO 2 measuring reagent Box, is made of liquid single-reagent：Trisbase 6g/L, hydrochloric acid 1.8g/L, stabilizer, magnesium acetate 1g/L, Sodium oxamate 0.3g/ L, phosphoric acid enol pyruvic acid carboxylase 0.1g/L, phosphoenolpyruvate potassium 0.6g/L, APADH 0.5g/L, malic acid take off Hydrogen enzyme 0.05g/L, disodium ethylene diamine tetraacetate 1g/L, Proclin300 1g/L.
Method according to embodiment 1 is detected.
With contrast agents performance for stability index results of comparison (table 3)
The data of 3 embodiments more than, do not add the CO 2 measuring of NADH analog indirect regenerations For kit compared with adding the kit of NADH analog indirect regenerations, its testing result reagent blank is relatively low, marks relatively Quasi- deviation is substantially higher.Illustrate very unstable without the carbon dioxide measurement kit for adding NADH analog indirect regenerations It is fixed.The carbon dioxide measurement kit of NADH analog indirect regenerations is added in whole test period, reagent blank, phase Standard deviation is showed it is good, illustrate 37 DEG C remain silent and place 4 weeks after, testing inspection data are normal, and reagent blank absorbance A exists 1.600 left and right；After 4 DEG C of corkages are placed 30 days, testing inspection data are normal, and reagent blank absorbance A is 1.600 or so；4 DEG C are closed After mouth is placed 1.5 years, testing inspection data are normal, and reagent blank absorbance A is 1.600 or so.It can be seen from the above that it with the addition of NADH The carbon dioxide measurement kit of analog indirect regeneration shows more in terms of measuring stability, accuracy, precision Excellent, NADH analogs indirect regeneration has it obvious stablizing effect, can effectively extend carbon dioxide measurement kit Pot-life.
It can be obtained from data above and analysis result, it is similar that carbon dioxide measurement kit adds NADH of the present invention After thing indirect regeneration, the holding time of kit can be extended, effectively improve measuring stability, accuracy, precision.Should NADH analog indirect regenerations are of low cost, prepare simple, worth further genralrlization use.

Claims (7)

1. a kind of carbon dioxide kit using Enzymatic cycling, it is characterised in that the kit is circulated by NADH analogs Regenerative system and CO 2 measuring reagent composition.
2. carbon dioxide kit according to claim 1, it is characterised in that NADH analogs circular regeneration in kit System is made of 6- phosphate-dextroses dehydrogenase, hexokinase, glucose, adenosine triphyosphate disodium salt.
3. carbon dioxide kit according to claim 1, it is characterised in that NADH analogs circular regeneration in kit System is by 6- phosphate-dextrose dehydrogenases 5-50U/L, hexokinase 50-500U/L, glucose 1-5g/L, adenosine three Disodic alkaliine 0.5-2.5g/L is formed.
4. carbon dioxide kit according to claim 1, it is characterised in that NADH analogs circular regeneration in kit System is by 6- phosphate-dextrose dehydrogenase 2s 0U/L, Hexokinase 2 00U/L, glucose 2g/L, adenosine triphyosphate disodium Salt 1g/L is formed.
5. carbon dioxide kit according to claim 1, it is characterised in that in kit CO 2 measuring reagent by Trisbase, hydrochloric acid, stabilizer, magnesium acetate, Sodium oxamate, phosphoric acid enol pyruvic acid carboxylase, phosphoenolpyruvate Potassium, APADH, malic dehydrogenase, disodium ethylene diamine tetraacetate, Proclin300 compositions.
6. carbon dioxide kit according to claim 5, it is characterised in that stabilizer is gelatin, sorbic alcohol, the third three At least one of alcohol, ethylene glycol, bovine serum albumin(BSA).
7. carbon dioxide kit according to claim 1, it is characterised in that in kit CO 2 measuring reagent by Trisbase 6g/L, hydrochloric acid 1.8g/L, stabilizer 1g/L, magnesium acetate 1g/L, Sodium oxamate 0.3g/L, phosphoric acid enol form propanone Sour carboxylase 0.1g/L, phosphoenolpyruvate potassium 0.6g/L, APADH 0.5g/L, malic dehydrogenase 0.05g/L, second two Amine tetraacethyl disodium 1g/L, Proclin300 1g/L are formed.
CN201711184134.3A 2017-11-23 2017-11-23 A kind of carbon dioxide kit using Enzymatic cycling Pending CN107966556A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108998501A (en) * 2018-08-08 2018-12-14 大连大学 A kind of efficient cryogenic uses the carbon dioxide kit of Enzymatic cycling
CN109613225A (en) * 2018-12-28 2019-04-12 上海高踪医疗器械科技有限公司 A kind of carbon dioxide detection reagent box
CN111893162A (en) * 2020-06-16 2020-11-06 武汉生之源生物科技股份有限公司 Carbon dioxide enzyme method determination kit
CN111996234A (en) * 2020-08-20 2020-11-27 安徽伊普诺康生物技术股份有限公司 Serum carbon dioxide detection kit and preparation method thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1133070A (en) * 1993-09-17 1996-10-09 微量科学有限公司 Reagent
CN1179792A (en) * 1995-03-28 1998-04-22 微量科学有限公司 Reagent stabilized using coenzyme reduction system
CN1344330A (en) * 1999-03-18 2002-04-10 扶桑药品工业株式会社 Enzymatic fluorimetric assay of cAMP and adenylate cyclase
CN1500884A (en) * 2002-11-15 2004-06-02 江西特康科技有限公司 Nicotinamide agent and preparation method thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1133070A (en) * 1993-09-17 1996-10-09 微量科学有限公司 Reagent
CN1179792A (en) * 1995-03-28 1998-04-22 微量科学有限公司 Reagent stabilized using coenzyme reduction system
CN1344330A (en) * 1999-03-18 2002-04-10 扶桑药品工业株式会社 Enzymatic fluorimetric assay of cAMP and adenylate cyclase
CN1500884A (en) * 2002-11-15 2004-06-02 江西特康科技有限公司 Nicotinamide agent and preparation method thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108998501A (en) * 2018-08-08 2018-12-14 大连大学 A kind of efficient cryogenic uses the carbon dioxide kit of Enzymatic cycling
CN108998501B (en) * 2018-08-08 2022-04-01 大连大学 Carbon dioxide kit for efficiently using cycloenzyme method at low temperature
CN109613225A (en) * 2018-12-28 2019-04-12 上海高踪医疗器械科技有限公司 A kind of carbon dioxide detection reagent box
CN111893162A (en) * 2020-06-16 2020-11-06 武汉生之源生物科技股份有限公司 Carbon dioxide enzyme method determination kit
CN111996234A (en) * 2020-08-20 2020-11-27 安徽伊普诺康生物技术股份有限公司 Serum carbon dioxide detection kit and preparation method thereof

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Application publication date: 20180427