CN101324628A - Method for determining ethanol concentration and ethanol diagnosis/determination reagent kit - Google Patents
Method for determining ethanol concentration and ethanol diagnosis/determination reagent kit Download PDFInfo
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- CN101324628A CN101324628A CNA2007100232070A CN200710023207A CN101324628A CN 101324628 A CN101324628 A CN 101324628A CN A2007100232070 A CNA2007100232070 A CN A2007100232070A CN 200710023207 A CN200710023207 A CN 200710023207A CN 101324628 A CN101324628 A CN 101324628A
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Abstract
The invention relates to a kit for diagnosing/mensurating ethanol by utilizing the technologies of the enzymic colorimetric method and the enzyme linked immunosorbent assay. The invention further relates to a method, a principle and the composition and the components of a reagent for mensurating the concentration of the ethanol, and belongs to the technology field of medical/food/environmental inspection and measurement. The main components of the kit include a buffer solution, reduced coenzyme, alcohol dehydrogenase, NADH peroxidase and a stabilizer. Through mixing a sample and the reagent by a certain volume ratio, a series of enzymatic reactions occur, then the reactant is placed under an ultraviolet/visible light analyzer, and the degree/velocity of the decrease in absorbance at 340nm of the dominant wavelength is detected, thereby mensurating the concentration of the ethanol. By adopting the kit, the required mensuration results can be completely obtained by the ultraviolet/visible light analytical instrument.
Description
Technical field
The present invention relates to a kind of diagnosing/determining alcohol kit, the invention still further relates to the method for measuring concentration of alcohol simultaneously, belong to medical science/food/environmental test determination techniques field.
Background technology
Ethanol tool habituation, alcohol abuse and alcohol dependence are one of serious social economic and public health problems in world today's scope.In western countries, alcohol correlativity hepatopathy has become the 6th dead reason.In China, over nearly 20 years, along with socio-economic development, the wine consumption rises significantly per capita, and the morbidity rate of alcohol dependence increases day by day.
The method for quantitatively determining of ethanol mainly contains chemical colorimetry in the biological sample, the enzyme terminal colorimetric analysis, and homogeneous EIA method (EIA), vapor-phase chromatography (GC) and expiratory air ethanol are analyzed.Serum infiltration capacity method can be made the ethanol semi-quantitative analysis in addition.
Have the number of chemical colourimetry once to be used for the ethanol quantitative measurement, microdiffusion is wherein more simple and practical a kind of, and the ethanol that trace overflows can make chromic acid be reduced into blue chromium oxide.
According to used reagent enzyme difference, the enzymatic assays of ethanol is divided into two kinds: oxidation of ethanol enzyme process and alcohol dehydrogenase enzyme process, to compare with the oxidation of ethanol enzyme process, and the selectivity height of alcohol dehydrogenase enzyme process can not play catalytic reaction with methyl alcohol and acetone.
It is generally acknowledged that vapor-phase chromatography can be used as reference method, especially most widely used in the high medical jurisprudence alcohol determining of accuracy and precision requirement.The distinct advantages of GC method is special, sensitive, and can analyze multiple volatility alcohols material simultaneously, as ethanol, methyl alcohol, acetone, acetaldehyde and isopropyl alcohol.
The assay method of other ethanol also has: homogeneous enzyme immunoassay analysis (Homogeneous enzymeimmunoassay), infiltration capacity method, expiratory air ethanol analytic approach; It is simple, quick to detect ethanol operation with expiratory air ethanol analyser, uses more in traffic administration department.A kind of according to the infrared Absorption principle design in this quasi-instrument, the blood concentration of alcohol correlativity that concentration of alcohol that this method is measured and enzyme process record is fine.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of enzymic colorimetric (EnzymaticColorimetric Method) and enzyme (even) united method (Couple Reaction) technology utilized, monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for concentration of alcohol, simultaneously, the present invention also will provide in order to realize the diagnosing/determining alcohol kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carry out determining concentration of alcohol on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Method for determining concentration of alcohol principle of the present invention is as follows:
Ethanol+oxygen
Alcohol dehydrogenaseAcetaldehyde+hydrogen peroxide
Hydrogen peroxide+reduced coenzyme
The NADH peroxidaseCoenzyme+2 water
This method is used alcohol dehydrogenase (Alcohol dehydrogenase; EC 1.1.3.13) enzyme (idol) connection NADH peroxidase (NADH peroxidase; EC 1.11.1.1; EC1.11.1.2) enzymatic reaction continuous monitoring method/speed ratio color method.Alcohol dehydrogenase enzymolysis ethanol synthesis produces hydrogen peroxide, the effect of uniting the NADH peroxidase again by (idol), reduced coenzyme (absorption peak being arranged at the 340nm place) is oxidized into coenzyme (not having absorption peak at the 340nm place) the most at last, thereby measured degree/speed that reduced coenzyme descends in 340nm place absorbance, by measuring degree/speed that 340nm place absorbance descends, can calculate the concentration of ethanol size.
Experiment shows, takes all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, no matter is single agent, two agent or three doses, and the diagnosing/determining alcohol kit of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Alcohol dehydrogenase 10000U/L
NADH peroxidase 10000U/L
Diagnosing/determining alcohol kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, alcohol dehydrogenase, NADH peroxidase.
Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Also above-mentioned single agent reagent can be made into following pair of agent reagent:
Reagent 1
Damping fluid, stabilizing agent, reduced coenzyme.
Reagent 2
Damping fluid, stabilizing agent, alcohol dehydrogenase, NADH peroxidase.
Reduced coenzyme, alcohol dehydrogenase, the position of NADH peroxidase in reagent 1 or reagent 2 can not limit.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
Above-mentioned single agent reagent can also be made into following three doses of reagent:
Reagent 1
Damping fluid, stabilizing agent, reduced coenzyme.
Reagent 2
Damping fluid, stabilizing agent, NADH peroxidase.
Reagent 3
Damping fluid, stabilizing agent, alcohol dehydrogenase.
Reduced coenzyme, alcohol dehydrogenase, the position of NADH peroxidase in reagent 1, reagent 2 or reagent 3 can not limit.Kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
No matter be single agent, two agent or three doses, the present invention measures the method for concentration of alcohol, and its reduced coenzyme can be a kind of among NADPH, NADH or the thio-NADH.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
The diagnosing/determining alcohol reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Alcohol dehydrogenase 10000U/L
NADH peroxidase 10000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and carries out freeze drying, makes powdered reagent; Before the use, add pure water, use after redissolving.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested ethanol sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), about about 0 minute of time delay is about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of ethanol size.
Embodiment two
The diagnosing/determining alcohol reagent of present embodiment is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Alcohol dehydrogenase 10000U/L
NADH peroxidase 10000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid double reagent, can directly use.
On automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested ethanol sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), about about 0 minute of time delay is about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of ethanol size.
Embodiment three
The diagnosing/determining alcohol reagent of present embodiment is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Reduced coenzyme 0.25mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
NADH peroxidase 10000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Alcohol dehydrogenase 10000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring concentration of alcohol, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested ethanol sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is negative reaction (reaction descends), and about about 0 minute of time delay is about 5 minutes detection times.
After adding sample and reagent, make them mixed and have reaction, reactant places under the Biochemical Analyzer the most at last, detects degree/speed that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of ethanol size.
The applicant adopts other various reduced form chromogens combinations of putting down in writing in the above summary of the invention all can reach purpose of the present invention through experimental verification, in view of situation such as determination step and above embodiment roughly the same, do not separately enumerate.
In a word, experiment showed, and adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully, and highly sensitive, degree of accuracy good, and is easy to utilize.
Claims (6)
1. method for determining concentration of alcohol that utilizes enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:
Ethanol+oxygen
Alcohol dehydrogenaseAcetaldehyde+hydrogen peroxide
Hydrogen peroxide+reduced coenzyme
The NADH peroxidaseCoenzyme+2 water
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect degree/speed that predominant wavelength 340nm absorbance descends, calculate concentration of ethanol size measurement result.
2. diagnosing/determining alcohol kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
Reduced coenzyme 0.1---0.35mmol/L
Alcohol dehydrogenase 1000---80000U/L
NADH peroxidase 1000---80000U/L
It is characterized in that: kit can be a dry powder, and use the back that is dissolved in water before use; Also can be mixed with liquid reagent, directly use.
3. according to the described diagnosing/determining alcohol kit of claim 2, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, alcohol dehydrogenase, NADH peroxidase.
4. according to the described diagnosing/determining alcohol kit of claim 2, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, alcohol dehydrogenase, NADH peroxidase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme; Reagent 2 is made up of damping fluid, stabilizing agent, alcohol dehydrogenase, NADH peroxidase.Reduced coenzyme, alcohol dehydrogenase, the position of NADH peroxidase in reagent 1 or reagent 2 can not limit.
5. according to the described diagnosing/determining alcohol kit of claim 2, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, alcohol dehydrogenase, NADH peroxidase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme; Reagent 2 is made up of damping fluid, stabilizing agent, NADH peroxidase; Reagent 3 is made up of damping fluid, stabilizing agent, alcohol dehydrogenase.Reduced coenzyme, alcohol dehydrogenase, the position of NADH peroxidase in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described diagnosing/determining alcohol kit of claim 2, it is characterized in that: also comprise stabilizing agent 1---4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: ammonium sulfate (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (PropyleneGlycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100232070A CN101324628A (en) | 2007-06-13 | 2007-06-13 | Method for determining ethanol concentration and ethanol diagnosis/determination reagent kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100232070A CN101324628A (en) | 2007-06-13 | 2007-06-13 | Method for determining ethanol concentration and ethanol diagnosis/determination reagent kit |
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| CN101324628A true CN101324628A (en) | 2008-12-17 |
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| CNA2007100232070A Pending CN101324628A (en) | 2007-06-13 | 2007-06-13 | Method for determining ethanol concentration and ethanol diagnosis/determination reagent kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102495220A (en) * | 2011-11-07 | 2012-06-13 | 宁波美康生物科技股份有限公司 | Stable reagent kit of ethanol determination liquid |
| CN106885803A (en) * | 2015-12-16 | 2017-06-23 | 山东博科生物产业有限公司 | A kind of strong antijamming capability and degree of accuracy enzyme process ethanol detection reagent high |
-
2007
- 2007-06-13 CN CNA2007100232070A patent/CN101324628A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102495220A (en) * | 2011-11-07 | 2012-06-13 | 宁波美康生物科技股份有限公司 | Stable reagent kit of ethanol determination liquid |
| CN102495220B (en) * | 2011-11-07 | 2013-11-06 | 宁波美康生物科技股份有限公司 | Stable reagent kit of ethanol determination liquid |
| CN106885803A (en) * | 2015-12-16 | 2017-06-23 | 山东博科生物产业有限公司 | A kind of strong antijamming capability and degree of accuracy enzyme process ethanol detection reagent high |
| CN106885803B (en) * | 2015-12-16 | 2019-06-28 | 山东博科生物产业有限公司 | A kind of strong antijamming capability and the high enzyme process ethyl alcohol detection reagent of accuracy |
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Open date: 20081217 |