CN101329335A - Glycerol diagnosis / determination reagent kit and method for determining glycerol concentration - Google Patents
Glycerol diagnosis / determination reagent kit and method for determining glycerol concentration Download PDFInfo
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- CN101329335A CN101329335A CNA2007100247305A CN200710024730A CN101329335A CN 101329335 A CN101329335 A CN 101329335A CN A2007100247305 A CNA2007100247305 A CN A2007100247305A CN 200710024730 A CN200710024730 A CN 200710024730A CN 101329335 A CN101329335 A CN 101329335A
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- glycerol
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- coenzyme
- glycerokinase
- stabilizing agent
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Abstract
The invention relates to a glycerine diagnosis/determination kit which uses the techniques of an enzyme colorimetry method and an enzyme-linked method; simultaneously, the invention also relates to a method principle used for determining glycerine consistency, reagent composition and components, belonging to the field of medical/food/environment detection determination technique. The main compositions of the kit of the invention comprise buffer solution, coenzyme, adenosine triphosphate, glycerokinase, glycerinee-3-phosphate dehydrogenase and stabilizer; samples and reagents are mixed according to a certain volume proportion so as to generate a series of enzymatic reactions; subsequently, reaction matters are arranged under an ultraviolet/visible light analyser so as to detect the degree/speed of the absorbency increment of the main wavelength at the 340nm position, thus calculating the consistency of the glycerine.
Description
Technical field
The present invention relates to a kind of glycerol diagnosis/mensuration kit, the invention still further relates to the method for measuring glycerol concentration simultaneously, belong to medical science/food/environmental test determination techniques field.
Background technology
Glycerine has another name called glycerine, be a kind of colourless, do not have a sweet thick liquid of smelling, distinguish the flavor of.Glycerine is in a large number as industrial chemicals, be used to make synthetic resin, plastics, paint, monobel, grease and beeswax etc., also be used for industry such as pharmacy, spices, cosmetics, amenities, glycerine is normally used sweetener and NMF in the food processing industry, appears at mostly in motion food and the milk replacer.
A standard that detects free glycerol content of European Union is EN 14106, and this standard at first extracts biodiesel, through the laggard promoting the circulation of qi analysis of hplc of extraction, up to the present, has had several gas chromatography analysis methods to be in the news.Lozano et al. (1996) detects the glycerite that extracts with the high performance liquid chromatography that has the pulse ampere detector from methyl esters or ethyl ester.
The chromatogram of other detection free glycerol content and non-chromatogram method of testing have: Greenhill has advised a kind of alternative method of enzyme process test, glycerine is used as the conversion product that forms the quinone imines pigment through the H2O2 that generates after the enzymatic conversion, and the concentration of pigment can detect by spectroscopic methodology.
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, metering/continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for glycerol concentration, simultaneously, the present invention also will provide in order to the glycerol diagnosis of realizing this method/mensuration kit, adopt this reagent not only can be ultraviolet analyser or half, carrying out glycerol concentration on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Glycerol concentration assay method principle of the present invention is as follows:
Glycerine+adenosine triphosphate
GlycerokinaseGlycerol-3-phosphate+adenosine diphosphate
Glycerol-3-phosphate+coenzyme
Glycerol-3-phosphate dehydrogenaseGlyceric acid phosphoric acid+reduced coenzyme
This method is used glycerokinase (Glycerokinase; EC 2.7.1.30) coupling glycerol-3-phosphate dehydrogenase (glycerol 3-phosphate dehydrogenase; EC 1.1.1.8; EC 1.1.1.94; EC1.1.1.177) enzyme ' s reaction speeding colourimetry/end-point method.Glycerokinase enzymolysis glycerine reaction produces glycerol-3-phosphate, effect by the coupling glycerol-3-phosphate dehydrogenase again, coenzyme (not having absorption peak at the 340nm place) reduces the most at last becomes reduced coenzyme (absorption peak being arranged at the 340nm place), thereby measured degree/speed that reduced coenzyme rises in 340nm place absorbance, by measuring degree/speed that 340nm place absorbance rises, can calculate the concentration of glycerine.
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 glycerol diagnosis of the present invention/the mensurations kit of following composition relation is ideal comparatively:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Adenosine triphosphate 8mmol/L
Glycerokinase 10000U/L
Glycerol-3-phosphate dehydrogenase 12000U/L
Glycerol diagnosis of the present invention/mensuration kit can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, glycerokinase, glycerol-3-phosphate dehydrogenase, adenosine triphosphate.
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, coenzyme, adenosine triphosphate.
Reagent 2
Damping fluid, stabilizing agent, glycerokinase, glycerol-3-phosphate dehydrogenase.
Coenzyme, glycerokinase, glycerol-3-phosphate dehydrogenase, the position of adenosine triphosphate 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, coenzyme, adenosine triphosphate.
Reagent 2
Damping fluid, stabilizing agent, glycerol-3-phosphate dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, glycerokinase.
Coenzyme, glycerokinase, glycerol-3-phosphate dehydrogenase, the position of adenosine triphosphate 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 glycerol concentration, and its coenzyme can be NADP
+, NAD
+Or thio-NAD
+In a kind of.
Embodiment
The present invention is further illustrated below in conjunction with examples of implementation.
Embodiment one
The glycerol diagnosis of present embodiment/mensuration reagent is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Coenzyme 3mmol/L
Adenosine triphosphate 8mmol/L
Glycerokinase 10000U/L
Glycerol-3-phosphate dehydrogenase 12000U/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≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested glycerine sample and reagent is 1/25, the Direction of Reaction is positive reaction (reaction of rising), 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 rises, thereby calculates the concentration of glycerine.
Embodiment two
The glycerol diagnosis of present embodiment/mensuration reagent is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Adenosine triphosphate 8mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glycerokinase 10000U/L
Glycerol-3-phosphate dehydrogenase 12000U/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≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested glycerine sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is positive reaction (reaction of rising), 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 rises, thereby calculates the concentration of glycerine.
Embodiment three
The glycerol diagnosis of present embodiment/mensuration reagent is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Adenosine triphosphate 8mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glycerol-3-phosphate dehydrogenase 12000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glycerokinase 10000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring glycerol concentration, on automatic clinical chemistry analyzer, set: 37 ℃ of temperature, 10 minutes reaction time, initial absorbance≤0.1, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested glycerine sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is positive reaction (reaction of rising), 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 rises, thereby calculates the concentration of glycerine.
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. the method for measurement of concentration of the glycerine of enzymic colorimetric and enzyme-linked method, its method principle is as follows:
Glycerine+adenosine triphosphate
GlycerokinaseGlycerol-3-phosphate+adenosine diphosphate
Glycerol-3-phosphate+coenzyme
Glycerol-3-phosphate dehydrogenaseGlyceric acid phosphoric acid+
Reduced coenzyme
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect degree/speed that predominant wavelength 340nm absorbance rises, calculate the concentration measurement result of glycerine.
2. glycerol diagnosis/mensuration kit, principal ingredient comprises:
Damping fluid 20-500mmol/L
Stabilizing agent 1-4000mmol/L
DPN diphosphopyridine nucleotide-6mmol/L
Adenosine triphosphate 1-50mmol/L
Glycerokinase 1000-80000U/L
Glycerol-3-phosphate dehydrogenase 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 glycerol diagnosis of claim 2/mensuration kit, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, coenzyme, glycerokinase, glycerol-3-phosphate dehydrogenase, adenosine triphosphate.
4. according to the described glycerol diagnosis of claim 2/mensuration kit, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, coenzyme, glycerokinase, glycerol-3-phosphate dehydrogenase, adenosine triphosphate; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, adenosine triphosphate; Reagent 2 is made up of damping fluid, stabilizing agent, glycerokinase, glycerol-3-phosphate dehydrogenase.Coenzyme, glycerokinase, glycerol-3-phosphate dehydrogenase, the position of adenosine triphosphate in reagent 1 or reagent 2 can not limit.
5. according to the described glycerol diagnosis of claim 2/mensuration kit, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, glycerokinase, glycerol-3-phosphate dehydrogenase, adenosine triphosphate; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, adenosine triphosphate; Reagent 2 is made up of damping fluid, stabilizing agent, glycerol-3-phosphate dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, glycerokinase.Coenzyme, glycerokinase, glycerol-3-phosphate dehydrogenase, the position of adenosine triphosphate in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described glycerol diagnosis of claim 2/mensuration kit, 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 (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100247305A CN101329335A (en) | 2007-06-21 | 2007-06-21 | Glycerol diagnosis / determination reagent kit and method for determining glycerol concentration |
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| CNA2007100247305A CN101329335A (en) | 2007-06-21 | 2007-06-21 | Glycerol diagnosis / determination reagent kit and method for determining glycerol concentration |
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|---|---|
| CN101329335A true CN101329335A (en) | 2008-12-24 |
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| CNA2007100247305A Pending CN101329335A (en) | 2007-06-21 | 2007-06-21 | Glycerol diagnosis / determination reagent kit and method for determining glycerol concentration |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112011592A (en) * | 2020-07-28 | 2020-12-01 | 苏州市药品检验检测研究中心 | Method for measuring content of glycerin in medium/long-chain fat emulsion injection |
| CN113804872A (en) * | 2020-02-13 | 2021-12-17 | 浙江东方基因生物制品股份有限公司 | Device and method for distinguishing electronic cigarette smoking from tobacco cigarette smoking |
-
2007
- 2007-06-21 CN CNA2007100247305A patent/CN101329335A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113804872A (en) * | 2020-02-13 | 2021-12-17 | 浙江东方基因生物制品股份有限公司 | Device and method for distinguishing electronic cigarette smoking from tobacco cigarette smoking |
| CN112011592A (en) * | 2020-07-28 | 2020-12-01 | 苏州市药品检验检测研究中心 | Method for measuring content of glycerin in medium/long-chain fat emulsion injection |
| CN112011592B (en) * | 2020-07-28 | 2023-09-08 | 苏州市药品检验检测研究中心 | Method for measuring glycerol content in medium/long-chain fat emulsion injection |
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Open date: 20081224 |