CN101082571A - Method for measuring malic acid concentration and malic acid diagnose reagent kit - Google Patents
Method for measuring malic acid concentration and malic acid diagnose reagent kit Download PDFInfo
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- CN101082571A CN101082571A CN 200710024765 CN200710024765A CN101082571A CN 101082571 A CN101082571 A CN 101082571A CN 200710024765 CN200710024765 CN 200710024765 CN 200710024765 A CN200710024765 A CN 200710024765A CN 101082571 A CN101082571 A CN 101082571A
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Abstract
The invention relates to a kind of double enlarge method, enzyme colorimetric method and enzyme linked method and a diagnosis reagent box applying the malic acid dehydrogenase coupling pyruvate dehydrogenase enzymatic reaction ratio colorimetric method/end-point method. The reaction of the malic acid dehydrogenase enzymolysis malic acid generates pyruvate and at the same time makes the coenzyme reducing the reduction coenzyme. The pyruvate generated by acid dehydrogenase enzymolysis malic acid makes the coenzyme reducing the reduction coenzyme again by the coupling reaction of pyruvate dehydrogenase. Because of making the coenzyme reducing the reduction coenzyme two times the reaction sensitivity is enhanced double. So we can assay the degree/speed of the ascent absorbance at the place of 340nm. It can measure and calculate the density of citric acid by measuring the degree/speed of the fall-way absorbance at the place of 340nm. The method has high specificity and it is not polluted by the endogenous and exogenous object and the test result is precise and accurate. The invention can get the array result by the ultraviolet/visible light analytic instrument so it is convenience to extend and apply.
Description
Technical field
The present invention relates in the fields such as medicine, food, environment detection to malic acid concentration, relate in particular to double TRAP, enzymic colorimetric and enzyme-linked method and measure the method for malic acid concentration, and the malic acid diagnose reagent kit that makes thus, belong to the malic acid concentration technical field of analysis and detection.
Background technology
Traditional separation and the method for measuring malic acid are vapor-phase chromatographies, but must carry out derivatization earlier when analyzing malic acid, thus process and result that can impact analysis.The chromatography of ions (IC) and high performance liquid chromatography (HPLC) are to measure malic acid in recent years to adopt many methods, but sample preparation is comparatively complicated; The HPLC assay of malic acid adopts the Zorbax-sbC18 chromatographic column, and the range of linearity that HPLC analyzes malic acid is 0.280～9.020ug.It is a kind of fresh sample treatment technology that the matrix solid phase is disperseed, and has concentrated traditional sample homogenizing, and processes such as histocyte cracking, extraction, filtration, purification make The pretreatment become easy, has also avoided the loss of object simultaneously.
Enzymatic analysis is the analytical approach of the malic acid of generally acknowledging in the world, organized by a plurality of internal authorities or mechanism adopts: as IFU (international fruit juice association of producers), AIJN (European Union's Juice and beverage production person alliance), and MEBAK, OICC, VDLUFA etc. organize widely and praise highly, and are defined as standard detecting method.Be written into the food law of a plurality of countries such as Australia, Holland, Germany, Switzerland.
Summary of the invention
The purpose of this invention is to provide a kind of method of measuring malic acid concentration, and use the formulated malic acid diagnose reagent kit of this method.
For realizing purpose of the present invention, a kind of double TRAP, enzymic colorimetric and enzyme-linked method are measured the method for malic acid concentration, utilizing malic dehydrogenase to be the effect enzyme is again to develop the color enzyme, pyruvic dehydrogenase for the colour developing enzyme, and the malic acid in the testing sample is detected, and adopts following steps to carry out:
At first,, make it to take place following reaction with sample of measuring and the reagent mixing that contains malic dehydrogenase, coenzyme, pyruvic dehydrogenase, coacetylase,
Then, 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 size of malic acid concentration.
Above-mentioned double TRAP, enzymic colorimetric and enzyme-linked method are measured in the method for malic acid concentration, and described coenzyme is NADP
+, NAD
+Or thio-NAD
+In a kind of.
Realize that malic acid diagnose reagent kit of the present invention can be single agent, is grouped into by following one-tenth:
Damping fluid 20～500mmol/L,
Stabilizing agent 1～4000mmol/L,
DPN diphosphopyridine nucleotide～6mmol/L,
Coacetylase 1～50mmol/L,
Malic dehydrogenase 1000～80000U/L,
Pyruvic dehydrogenase 1000～80000U/L.
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 the various compositions in above single agent can be carried out formulated in combination and become two agent, such as:
| Reagent II | Damping fluid | 20～500mmol/L |
| Stabilizing agent | 1～4000mmol/L | |
| Malic dehydrogenase | 1000～80000U/L | |
| Pyruvic dehydrogenase | 1000～80000U/L |
The prescription of two agent is not limited only in the above-mentioned table listed, wherein the composition of reagent I: coenzyme, coacetylase can be placed on reagent II; Malic dehydrogenase among the reagent II, pyruvic dehydrogenase also can be put into reagent I, so can form multiple formulations, enumerate no longer one by one.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.
In addition, for reducing the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, more than add stabilizing agent usually in the middle of the reagent I/ reagent II of single agent, two agent, concentration is within 1～4000mmol/L or 0.1%～100% volume ratio scope.
Reagent can also be made into following three reagent:
Similar with two agent, three doses prescription also is not limited only to above-mentioned prescription, wherein the coenzyme among the reagent I, coacetylase can be placed among reagent II or the reagent III, pyruvic dehydrogenase among the reagent II can be placed among reagent I or the reagent III, malic dehydrogenase among the reagent III also can be put among reagent I or the reagent II, so can form multiple formulations, enumerate no longer one by one.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.
In addition, for reducing the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stabilizing agent in the middle of the reagent I/ reagent II of above single agent, two agent, three doses the reagent I/ reagent II/ reagent III, active concentration is within 1～4000mmol/L or 0.1%～100% volume ratio scope.
No matter be single agent, two agent or three doses, coenzyme can be: NADP
+, NAD
+Or thio-NAD
+In a kind of.
Material with used as stabilizers can be: at least a in ammonium sulfate (Ammonia Sulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), the ethylene glycol (Ethylene glycol).
No matter studies show that, take all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, be single agent or two agent, and the diagnosis/detection kit of following formula components relation is comparatively desirable, also is preferred version of the present invention:
Damping fluid 100mmol/L,
Stabilizing agent 500mmol/L,
Coenzyme 3mmol/L,
Coacetylase 6mmol/L,
Malic dehydrogenase 10000U/L,
Pyruvic dehydrogenase 12000U/L.
Utilize double TRAP (Double enchance), enzymic colorimetric (Enzymatic ColorimetricMethod) and enzyme-linked method (Couple Reaction) technology, metering/continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for malic acid concentration, simultaneously, the present invention gives in order to realize the malic acid diagnosis/determination reagent kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carrying out malic acid concentration on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
The outstanding substantive distinguishing features and the obvious improvement of technical solution of the present invention mainly shows:
(1) the present invention utilizes double TRAP, enzymic colorimetric and enzyme-linked method to measure malic acid concentration, and test result is accurate;
(2) composition of participation reaction all adds, and is not subjected to the pollution of inside and outside source material, test process degree of accuracy height;
(3) this method is easy, easy to operate, can obtain testing result fast, and the reaction be under buffer conditions, to carry out, do not pollute the environment;
(4) but this method just fast detecting on general ultraviolet/visible light analysis instrument or semi-automatic/automatic clinical chemistry analyzer does not need special or additional instruments, testing cost is cheap, is convenient to apply in the industry;
(5) use the reagent that assay method provided by the invention can be made various ways such as liquid reagent, powdered reagent, be used for measuring the size of various sample malic acid concentrations;
(6) liquid malic acid diagnosis/detection kit provided by the invention, good stability has guaranteed the application testing effect well.Be made into after two agent, can further reduce the cross influence between the various compositions, testing result is more credible, and reagent is more stable, can store for a long time.
Embodiment
A kind of double TRAP of the present invention, enzymic colorimetric and enzyme-linked method are measured the method and the diagnostic kit of malic acid concentration, utilization malic dehydrogenase (malate dehydrogenase; EC 1.1.1.83) coupling pyruvic dehydrogenase (pyruvate dehydrogenase; EC 1.2.1.51) enzyme ' s reaction speeding colourimetry/end-point method.The reaction of malic dehydrogenase enzymolysis malic acid produces pyruvic acid, simultaneously with coenzyme (not having absorption peak at the 340nm place) reduction becoming reduced coenzyme (absorption peak being arranged at the 340nm place).The pyruvic acid that malic dehydrogenase enzymolysis malic acid produces is the effect by the coupling pyruvic dehydrogenase again, once more with coenzyme (not having absorption peak) reduction becoming reduced coenzyme (absorption peak being arranged) at the 340nm place at the 340nm place, because therefore secondary can improve reaction sensitivity two times with coenzyme reduction becoming reduced coenzyme; Thereby measured degree/speed that reduced coenzyme rises in 340nm place absorbance,, can be calculated the concentration of malic acid by measuring degree/speed that 340nm place absorbance rises.
Below in conjunction with specific embodiment technical solution of the present invention is described further.These examples only are some exemplary applications, can not be interpreted as a kind of restriction to claim protection domain of the present invention.
Embodiment one (single agent)
Prepare malic acid diagnose reagent kit by following composition and consumption:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ethylene glycol 500mmol/L,
thio-NAD
+ 3mmol/L，
Coacetylase 6mmol/L,
Malic dehydrogenase 10000U/L,
Pyruvic 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 malic acid sample and reagent is 1: 25, and the Direction of Reaction is positive reaction (reaction of rising), 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 malic acid.
Embodiment two (two agent)
Prepare malic acid diagnose reagent kit by following composition and consumption:
Reagent I---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Propylene glycol 50mmol/L,
NAD
+ 3mmol/L，
Coacetylase 6mmol/L;
Reagent II---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Glycerine 500mmol/L,
Malic dehydrogenase 10000U/L,
Pyruvic 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 malic acid sample and reagent I, reagent II is 2: 20: 5, and the Direction of Reaction is positive reaction (reaction of rising), 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 malic acid.
Embodiment three (three doses)
Prepare malic acid diagnose reagent kit by following composition and consumption:
Reagent I---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Ammonium sulfate 50mmol/L,
NADP
+ 3mmol/L，
Coacetylase 6mmol/L;
Reagent II---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Glycerine 500mmol/L,
Pyruvic dehydrogenase 12000U/L;
Reagent III---
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L,
Propylene glycol 500mmol/L,
Malic 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 malic acid 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 malic acid sample and reagent I, reagent II, reagent III is 4: 40: 5: 5, the Direction of Reaction is positive reaction (reaction of rising), 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 malic acid.
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, be not subjected in the pollution of allogenic material, easy to utilize.
Claims (6)
1. the assay method of malic acid concentration is characterized in that may further comprise the steps:
3) 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 size of malic acid concentration.
2. malic acid diagnose reagent kit, reagent is grouped into by following one-tenth in the box:
Damping fluid 20～500mmol/L,
Stabilizing agent 1～4000mmol/L,
DPN diphosphopyridine nucleotide～6mmol/L,
Coacetylase 1～50mmol/L,
Malic dehydrogenase 1000～80000U/L,
Pyruvic dehydrogenase 1000～80000U/L.
3. malic acid diagnose reagent kit according to claim 2 is characterized in that: described stabilizing agent is at least a in ammonium sulfate, glycerine, propylene glycol, the ethylene glycol.
4. malic acid diagnose reagent kit according to claim 2 is characterized in that: described coenzyme is NADP
+, NAD
+Or thio-NAD
+In a kind of.
5. any one malic acid diagnose reagent kit in the claim 2～4 is characterized in that: described reagent is made into single agent or two agent or three doses.
6. any one malic acid diagnose reagent kit in the claim 2～4, it is characterized in that: described kit is powdered reagent box or liquid reagent box.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710024765 CN101082571A (en) | 2007-06-28 | 2007-06-28 | Method for measuring malic acid concentration and malic acid diagnose reagent kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710024765 CN101082571A (en) | 2007-06-28 | 2007-06-28 | Method for measuring malic acid concentration and malic acid diagnose reagent kit |
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| Publication Number | Publication Date |
|---|---|
| CN101082571A true CN101082571A (en) | 2007-12-05 |
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ID=38912255
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|---|---|---|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107664619A (en) * | 2017-08-28 | 2018-02-06 | 青岛贝美生物技术有限公司 | A kind of free fatty acid determination reagent kit |
-
2007
- 2007-06-28 CN CN 200710024765 patent/CN101082571A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107664619A (en) * | 2017-08-28 | 2018-02-06 | 青岛贝美生物技术有限公司 | A kind of free fatty acid determination reagent kit |
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Open date: 20071205 |