CN101324516A - Method for determining formic acid concentration and formic acid diagnosis/determination reagent kit - Google Patents
Method for determining formic acid concentration and formic acid diagnosis/determination reagent kit Download PDFInfo
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- CN101324516A CN101324516A CNA2007100233887A CN200710023388A CN101324516A CN 101324516 A CN101324516 A CN 101324516A CN A2007100233887 A CNA2007100233887 A CN A2007100233887A CN 200710023388 A CN200710023388 A CN 200710023388A CN 101324516 A CN101324516 A CN 101324516A
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- reagent
- formic acid
- ferricytochrome
- stabilizing agent
- hydrogenlyase
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Abstract
The invention relates to a kit for diagnosing/mensurating formic acid by utilizing the technologies of the enzymic colorimetry 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 formic acid, and belongs to the technology field of medicine/food/environment/industry inspection and measurement. The main components of the kit include a buffer solution, reduced coenzyme, ferricytochrome, pyruvic acid, formate dehydrogenlyase, malic dehydrogenase 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 formic acid.
Description
Technical field
The present invention relates to a kind of formic acid diagnosis/determination kit, the invention still further relates to the method for measuring formic acid concn simultaneously, belong to medical science/food/environment/industrial inspection determination techniques field.
Background technology
Formic acid (HCOOH) at room temperature has the characteristic of natural decomposition, simultaneously, in the test process inevitably some moisture sneak into formic acid, formic acid is the water white transparency and the liquid of being fuming. strong pungency tart flavour is arranged.Generally in industry and environmental monitoring, use vapor-phase chromatography; On medicine and agricultural, use enzymatic analysis, enzymatic analysis is 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.
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 formic acid concn, simultaneously, the present invention also will provide in order to realize the formic acid diagnosis/determination kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carrying out formic acid concn on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Formic acid concn assay method principle of the present invention is as follows:
Formic acid+ferricytochrome
HydrogenlyaseCarbon dioxide+ferricytochrome
Carbon dioxide+pyruvic acid+reduced coenzyme
Malic dehydrogenase (decarboxylation)
L MALIC ACID+coenzyme
This method is used hydrogenlyase (formate dehydrogenase; EC 1.2.2.1; EC1.2.2.3) coupling malic dehydrogenase (malate dehydrogenase; EC 1.1.1.38; EC 1.1.1.39; EC 1.1.1.40; EC 1.1.1.83) enzymatic reaction continuous monitoring method/terminal colorimetric analysis.The reaction of hydrogenlyase enzymolysis formic acid produces carbon dioxide, effect by the coupling malic dehydrogenase again, 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 formic acid.
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 formic acid diagnosis/determination 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
Ferricytochrome 10mmol/L
Pyruvic acid 30mmol/L
Hydrogenlyase 10000U/L
Malic dehydrogenase 16000U/L
Formic acid diagnosis/determination kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, ferricytochrome, pyruvic acid, hydrogenlyase, malic dehydrogenase.
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, ferricytochrome, pyruvic acid.
Reagent 2
Damping fluid, stabilizing agent, hydrogenlyase, malic dehydrogenase.
Reduced coenzyme, ferricytochrome, pyruvic acid, hydrogenlyase, the position of malic dehydrogenase 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, ferricytochrome, pyruvic acid.
Reagent 2
Damping fluid, stabilizing agent, malic dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, hydrogenlyase.
Reduced coenzyme, ferricytochrome, pyruvic acid, hydrogenlyase, the position of malic dehydrogenase 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 formic acid concn, 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 formic acid diagnosing/determining reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Ferricytochrome 10mmol/L
Pyruvic acid 30mmol/L
Hydrogenlyase 10000U/L
Malic dehydrogenase 16000U/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 formic acid 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 formic acid.
Embodiment two
The formic acid diagnosing/determining 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
Ferricytochrome 10mmol/L
Pyruvic acid 30mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Hydrogenlyase 10000U/L
Malic dehydrogenase 16000U/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 formic acid 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 formic acid.
Embodiment three
The formic acid diagnosing/determining 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
Ferricytochrome 10mmol/L
Pyruvic acid 30mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Malic dehydrogenase 16000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Hydrogenlyase 10000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring formic acid concn, 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 formic acid 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 formic 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, and is easy to utilize.
Claims (6)
1. formic acid concn assay method that utilizes enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:
Formic acid+ferricytochrome
HydrogenlyaseCarbon dioxide+ferricytochrome
Carbon dioxide+pyruvic acid+reduced coenzyme
Malic dehydrogenase (decarboxylation)
L MALIC ACID+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 descends, calculate the concentration measurement result of formic acid.
2. formic acid diagnosis/determination kit, principal ingredient comprises:
Damping fluid 20-500mmol/L
Stabilizing agent 14000mmol/L
Reduced coenzyme 0.1-0.35mmol/L
Ferricytochrome 1-50mmol/L
Pyruvic acid 1-50mmol/L
Hydrogenlyase 1000-80000U/L
Malic 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 formic acid diagnosis/determination kit of claim 2, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, ferricytochrome, pyruvic acid, hydrogenlyase, malic dehydrogenase.
4. according to the described formic acid diagnosis/determination kit of claim 2, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, ferricytochrome, pyruvic acid, hydrogenlyase, malic dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, ferricytochrome, pyruvic acid; Reagent 2 is made up of damping fluid, stabilizing agent, hydrogenlyase, malic dehydrogenase.Reduced coenzyme, ferricytochrome, pyruvic acid, hydrogenlyase, the position of malic dehydrogenase in reagent 1 or reagent 2 can not limit.
5. according to the described formic acid diagnosis/determination kit of claim 2, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, ferricytochrome, pyruvic acid, hydrogenlyase, malic dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, ferricytochrome, pyruvic acid; Reagent 2 is made up of damping fluid, stabilizing agent, malic dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, hydrogenlyase.Reduced coenzyme, ferricytochrome, pyruvic acid, hydrogenlyase, the position of malic dehydrogenase in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described formic acid diagnosis/determination 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 (Propylene Glycol), ethylene glycol (Ethylene glycol) and at least one of the preservatives.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100233887A CN101324516A (en) | 2007-06-13 | 2007-06-13 | Method for determining formic acid concentration and formic acid diagnosis/determination reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100233887A CN101324516A (en) | 2007-06-13 | 2007-06-13 | Method for determining formic acid concentration and formic acid diagnosis/determination reagent kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101324516A true CN101324516A (en) | 2008-12-17 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100233887A Pending CN101324516A (en) | 2007-06-13 | 2007-06-13 | Method for determining formic acid concentration and formic acid diagnosis/determination reagent kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101324516A (en) |
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2007
- 2007-06-13 CN CNA2007100233887A patent/CN101324516A/en active Pending
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Open date: 20081217 |