CN101762542A - Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol - Google Patents
Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol Download PDFInfo
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- CN101762542A CN101762542A CN200810244265A CN200810244265A CN101762542A CN 101762542 A CN101762542 A CN 101762542A CN 200810244265 A CN200810244265 A CN 200810244265A CN 200810244265 A CN200810244265 A CN 200810244265A CN 101762542 A CN101762542 A CN 101762542A
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- peroxidase
- stabilizing agent
- reduced coenzyme
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Abstract
The invention relates to a reagent (kit) for diagnosing/determining alcohol by using an enzyme cycling multiplication method, an enzyme colorimetric method and an enzyme link method, and also discloses a method for determining the concentration of the alcohol, a composition and components of the reagent, belonging to the technical field of medicine/food/environmental test and determination. The reagent (kit) comprises the main components: a buffer solution, a reduced coenzyme, an alcohol oxidase, a peroxidase, an NADH oxidase and a stabilizing agent. A sample and the reagent are mixed according to a certain volume ratio to generate a series of enzymatic reactions, and reactants are placed under an ultraviolet/visible light analyzer for detecting the decrease degree of absorbance at the dominant wavelength of 340 nm, thereby measuring and calculating the concentration of the alcohol.
Description
Technical field
The present invention relates to a kind of diagnosing/determining alcohol reagent (box), 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 enzyme circulation multiplication method (EnzymaticRecycling﹠amp that utilizes; Doubling Method), enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (Couple Reaction) technology, 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 reagent (box) 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 of the present invention is as follows:
Ethanol+oxygen
Alcohol oxidase enzymeAcetaldehyde+hydrogen peroxide
Acetaldehyde+water
Hydrogen peroxidaseEthanol+hydrogen peroxide
Hydrogen peroxide+reduced coenzyme
The NADH peroxidaseCoenzyme+2 water
This method is used alcohol oxidase enzyme (alcohol oxidase; EC 1.1.3.13) enzyme (idol) connection hydrogen peroxidase (Catalase; EC 1.11.1.6), NADH peroxidase (NADH peroxidase; EC 1.11.1.1; EC 1.11.1.2) enzymatic reaction continuous monitoring method.Alcohol oxidase enzyme is as the effect enzyme, and function is that the ethanol enzymolysis is produced acetaldehyde.Hydrogen peroxidase is as cyclophorase: hydrogen peroxidase becomes acetaldehyde again ethanol more once more, and it is recycling that ethanol constantly is repeated.The NADH peroxidase is as the colour developing enzyme, two hydrogen peroxide and reduced coenzyme (absorption peak being arranged at the 340nm place) are oxidized into oxidized coenzyme (not having absorption peak at the 340nm place), thereby measured the speed that reduced coenzyme descends in 340nm place absorbance, by measuring the 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 reagent of the present invention (box) of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Alcohol oxidase enzyme 6000U/L
Hydrogen peroxidase 8000U/L
NADH peroxidase 10000U/L
Diagnosing/determining alcohol reagent of the present invention (box) can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, alcohol oxidase enzyme, hydrogen peroxidase, NADH peroxidase.
Reagent (box) can be 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 oxidase enzyme, hydrogen peroxidase, NADH peroxidase.
Reduced coenzyme, alcohol oxidase enzyme, hydrogen peroxidase, the position of NADH peroxidase in reagent 1 or reagent 2 can not limit.Reagent (box) can be 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, hydrogen peroxidase, NADH peroxidase.
Reagent 3
Damping fluid, stabilizing agent, alcohol oxidase enzyme.
Reduced coenzyme, alcohol oxidase enzyme, hydrogen peroxidase, the position of NADH peroxidase in reagent 1, reagent 2 or reagent 3 can not limit.Reagent (box) can be 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 oxidase enzyme 6000U/L
Hydrogen peroxidase 8000U/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 1 minute of time delay is about 2 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 the degree 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 oxidase enzyme 6000U/L
Hydrogen peroxidase 8000U/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 1 minute of time delay is about 2 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 the degree 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
Hydrogen peroxidase 8000U/L
NADH peroxidase 10000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Alcohol oxidase enzyme 6000U/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 1 minute of time delay is about 2 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 the degree that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of ethanol size.
The applicant adopts other assay methods 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, experimental results show that: adopt assay method of the present invention can draw required measurement result by general biochemical analyzer fully---the blank reagent absorbance changes (Δ A/min)≤0.0005; Absorbance time response curve should be decline curve until terminal point; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 20mmol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 3%; The coefficient of variation (CV)≤1% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.030 ± 0.015 Δ A/mmol/L; Reagent is preserved down at 2-8 ℃, and activity can be stablized 1 year;---the present invention is highly sensitive, degree of accuracy good, the linear range broadness, and stationary phase is long, is enough to easy to utilize.
Claims (6)
1. method for determining concentration of alcohol that utilizes enzyme circulation multiplication method, enzymic colorimetric and enzyme-linked method technology, its method is as follows:
Ethanol+oxygen
Alcohol oxidase enzymeAcetaldehyde+hydrogen peroxide
Acetaldehyde+water
Hydrogen peroxidaseEthanol+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 the degree that predominant wavelength 340nm absorbance descends, calculate concentration of ethanol size measurement result.
2. a diagnosing/determining alcohol reagent (box), principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
Reduced coenzyme 0.1---0.35mmol/L
Alcohol oxidase enzyme 1000---80000U/L
Hydrogen peroxidase 1000---80000U/L
NADH peroxidase 1000---80000U/L
The concentration of reagent composition not necessarily is only limited to above-mentioned scope; Effect is better in this scope, and outside this scope, reagent still can reagentia.
It is characterized in that: reagent (box) can be 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 reagent of claim 2 (box), it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, alcohol oxidase enzyme, hydrogen peroxidase, NADH peroxidase.
4. according to the described diagnosing/determining alcohol reagent of claim 2 (box), it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, alcohol oxidase enzyme, hydrogen peroxidase, 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 oxidase enzyme, hydrogen peroxidase, NADH peroxidase.Reduced coenzyme, alcohol oxidase enzyme, hydrogen peroxidase, the position of NADH peroxidase in reagent 1 or reagent 2 can not limit.
5. according to the described diagnosing/determining alcohol reagent of claim 2 (box), it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, alcohol oxidase enzyme, hydrogen peroxidase, NADH peroxidase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme; Reagent 2 is made up of damping fluid, stabilizing agent, hydrogen peroxidase, NADH peroxidase; Reagent 3 is made up of damping fluid, stabilizing agent, alcohol oxidase enzyme.Reduced coenzyme, alcohol oxidase enzyme, hydrogen peroxidase, the position of NADH peroxidase in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described diagnosing/determining alcohol reagent of claim 2 (box), it is characterized in that: also comprise stabilizing agent 1-4000mmol/L or 0.1%-100% volume ratio.Described stabilizing agent is: ammonium sulfate (AmmoniaSulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810244265A CN101762542A (en) | 2008-12-10 | 2008-12-10 | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810244265A CN101762542A (en) | 2008-12-10 | 2008-12-10 | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol |
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| CN101762542A true CN101762542A (en) | 2010-06-30 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN200810244265A Pending CN101762542A (en) | 2008-12-10 | 2008-12-10 | Reagent (kit) for diagnosing/determining alcohol and method for determining concentration of alcohol |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104198420A (en) * | 2014-08-14 | 2014-12-10 | 上海睿康生物科技有限公司 | Stable detection kit of total bile acid |
| CN105319316A (en) * | 2014-07-16 | 2016-02-10 | 中国科学院天津工业生物技术研究所 | Method for rapidly measuring content of threonine in fermentation broth |
-
2008
- 2008-12-10 CN CN200810244265A patent/CN101762542A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105319316A (en) * | 2014-07-16 | 2016-02-10 | 中国科学院天津工业生物技术研究所 | Method for rapidly measuring content of threonine in fermentation broth |
| CN104198420A (en) * | 2014-08-14 | 2014-12-10 | 上海睿康生物科技有限公司 | Stable detection kit of total bile acid |
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Addressee: Aijie Biological Science & Technology Co., Ltd., Suzhou City Wang Erzhong Document name: Notification that Application Deemed to be Withdrawn |
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Application publication date: 20100630 |