CN101173934A - Magnesium diagnosis/measuring reagent kit and method for measuring magnesium concentration - Google Patents
Magnesium diagnosis/measuring reagent kit and method for measuring magnesium concentration Download PDFInfo
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- CN101173934A CN101173934A CNA200610097311XA CN200610097311A CN101173934A CN 101173934 A CN101173934 A CN 101173934A CN A200610097311X A CNA200610097311X A CN A200610097311XA CN 200610097311 A CN200610097311 A CN 200610097311A CN 101173934 A CN101173934 A CN 101173934A
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- glycerol
- magnesium
- stabilizing agent
- coenzyme
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Abstract
The invention relates to a magnesium diagnostic/testing reagent kit using enzymatic colorimetric method and enzymic linkage technology as well as method for testing concentration of magnesium as well as composition and content of the reagent, belonging to technical field of medicine/food/environment test. The invention is characterized in that composition in the reagent kit comprises buffer solution, coenzyme, adenosine diphosphate, glycerol-3-phosphate, glycerol kinase, glycerol dehydrogenase and stabilizing agent; the sample is mixed with the reagent according to specified ratio to generate a series enzymatic reactions, then the reactant is arranged under a ultraviolet/visible light analyzer to test the increase speed of absorbance at wavelength of 340nm, thereby the magnesium concentration can be tested. The invention has the advantages that the test results can be easily obtained using ultraviolet/visible light analyzer.
Description
Technical field
The present invention relates to a kind of magnesium diagnosis/determination kit, the invention still further relates to the method for measuring magnesium density simultaneously, belong to medical science/food/environmental test determination techniques field.
Background technology
The magnesium determination method is a lot, summarizes and gets up to have colourimetry, fluorescence method, ion chromatography, ion-selective electrode method (ISE), enzyme process, atomic absorption spectrophotometry (AAS), isotope dilution mass spectrometry (ID-MS) etc.
The decisive method of magnesium determination is an isotope dilution mass spectrometry, and reference method is the atomic absorption spectrophotometry method.Though the result is accurate for these methods, the equipment complexity, the expense costliness is not suitable for Routine Test Lab and analysis automatically.
Colourimetry is most widely used method, comprises methyl thymol blue method (MTB), titan yellow method, OCPC method (OCPC), calmagite (Calmagite) method and 2-(8 '-hydroxyquinoline-5 '-sulfonic acid-7 '-azo)-chromotropic acid (8Q5SAC) method of reporting recently.Colourimetry is easy and simple to handle, and expense is low, is fit to Routine Test Lab and uses.But have reagent blank absorbance height, cholerythrin and other cationic interference contain in reagent stability difference and the reagent and are corrosive or shortcoming such as toxic component.
The ion-selective electrode method can be measured magnesium ion activity in the physiological solution.Adopt the accuracy of neutral carrier (ETH7025) liquid film ion selecting electrode determining higher.But also exist calcium to disturb (in the physiological range) Ca
2+Maximum interference 10% deficiency such as grade.
The total magnesium of ion-chromatographic determination serum needed sample is carried out pre-service before measuring, and comprised acidifying dilution and filtration.The definite value serum that this method of uses such as Thienpont is used atomic absorption spectrophotometry to five kinds of international standards and technology is analyzed, and mean deviation only is 0.35% as a result, thinks that the chromatography of ions can be used as total magnesium determination valuable reference method.
Mg
2+The enzymatic assays technology in recent years research very active, obtain bigger progress.Be applied to Mg
2+The Enzymology method of measuring has: 1. hexokinase and glucose-6-phosphate dehydrogenase coupling method; 2. glycerokinase, GPO and superoxide enzyme coupling method; 3. glucokinase and glucose-6-phosphate dehydrogenase coupling method; 4. enzyme joins chemoluminescence method; 5. phosphoglucomutase and glucose-6-phosphate dehydrogenase coupling method; 6. isocitric dehydrogenase method; 7. pyruvate kinase and lactic dehydrogenase enzyme coupling method.It is accurate that enzyme process detects the magnesium result, and disturbing effect is little, is suitable for automated analysis.Because enzyme reagent is brought in constant renewal in, make mensuration quick, sensitive, easy and simple to handle, thereby have application promise in clinical practice.
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, continuous monitoring reduced form nicotinamide coenzyme (reduced coenzyme) is in the variation of 340nm wavelength place absorbance, measured the method for magnesium density, simultaneously, the present invention also will provide in order to realize the magnesium diagnosis/determination kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carrying out magnesium density on the automatic clinical chemistry analyzer measures, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Magnesium density assay method principle of the present invention is as follows:
Adenosine diphosphate+glycerol-3-phosphate
Glycerokinase/Mg 2+ Glycerine+adenosine triphosphate
Glycerine+coenzyme
Glycerol dehydrogenaseGlyceric acid+reduced coenzyme
Glycerokinase (the Glycerokinase that this method application need magnesium ion activates; EC2.7.1.30) enzyme (idol) connection glycerol dehydrogenase (glycerol dehydrogenase; EC 1.1.1.6; EC 1.1.1.72; EC 1.1.1.156; EC 1.1.99.22) enzymatic reaction continuous monitoring method.Glycerokinase enzymolysis glycerol-3-phosphate produces glycerine, the effect of uniting glycerol dehydrogenase again by (idol), 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 the speed that reduced coenzyme rises in 340nm place absorbance, by measuring the speed that 340nm place absorbance rises, can calculate the concentration of magnesium.
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 magnesium diagnosis/determination kit of the present invention of following composition relation is comparatively desirable:
Damping fluid 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Glycerokinase 12000U/L
Glycerol dehydrogenase 20000U/L
Adenosine diphosphate 6mmol/L
Glycerol-3-phosphate 10mmol/L
Magnesium diagnosis/determination kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, coenzyme, adenosine diphosphate, glycerol-3-phosphate, glycerokinase, glycerol 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, coenzyme, adenosine diphosphate, glycerol-3-phosphate.
Reagent 2
Damping fluid, stabilizing agent, glycerokinase, glycerol dehydrogenase.
Coenzyme, adenosine diphosphate, glycerol-3-phosphate, glycerokinase, the position of glycerol 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, coenzyme.
Reagent 2
Damping fluid, stabilizing agent, adenosine diphosphate, glycerol-3-phosphate.
Reagent 3
Damping fluid, stabilizing agent, glycerokinase, glycerol dehydrogenase.
Coenzyme, adenosine diphosphate, glycerol-3-phosphate, glycerokinase, the position of glycerol 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 magnesium density, 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 magnesium diagnosing/determining reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Glycerokinase 12000U/L
Glycerol dehydrogenase 20000U/L
Adenosine diphosphate 6mmol/L
Glycerol-3-phosphate 10mmol/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 magnesium sample and reagent is 1/25, the Direction of Reaction is positive reaction (reaction of rising), 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 speed that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of magnesium.
Embodiment two
The magnesium diagnosing/determining reagent of present embodiment is double reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Adenosine diphosphate 8mmol/L
Glycerol-3-phosphate 12mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Glycerokinase 16000U/L
Glycerol dehydrogenase 30000U/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 magnesium sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is positive reaction (reaction of rising), 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 speed that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of magnesium.
Embodiment three
The magnesium diagnosing/determining reagent of present embodiment is three reagent, comprising:
Reagent 1
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Coenzyme 3mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Adenosine diphosphate 16mmol/L
Glycerol-3-phosphate 30mmol/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 50mmol/L
Glycerokinase 24000U/L
Glycerol dehydrogenase 36000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring magnesium density, 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 magnesium 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 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 speed that predominant wavelength 340nm absorbance rises, thereby calculates the concentration of magnesium.
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 magnesium density assay method of an enzymic colorimetric, its method principle is as follows:
Adenosine diphosphate+glycerol-3-phosphate glycerokinase/Mg
2+Glycerine+
Adenosine triphosphate
Glycerine+coenzyme
Glycerol dehydrogenaseGlyceric acid+reduced coenzyme
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the speed that predominant wavelength 340nm absorbance rises, calculate the concentration measurement result of magnesium.
2. magnesium diagnosis/determination kit, principal ingredient comprises:
Damping fluid 20-500mmol/L
Stabilizing agent 1-50mmol/L
DPN diphosphopyridine nucleotide-6mmol/L
Glycerokinase 1000-80000U/L
Glycerol dehydrogenase 1000-80000U/L
Adenosine diphosphate 1-50mmol/L
Glycerol-3-phosphate 1-50mmol/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 magnesium diagnosis/determination kit of claim 2, it is characterized in that: form single agent reagent by damping fluid, stabilizing agent, coenzyme, adenosine diphosphate, glycerol-3-phosphate, glycerokinase, glycerol dehydrogenase.
4. according to the described magnesium diagnosis/determination kit of claim 2, it is characterized in that: form two agent reagent by damping fluid, stabilizing agent, coenzyme, adenosine diphosphate, glycerol-3-phosphate, glycerokinase, glycerol dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme, adenosine diphosphate, glycerol-3-phosphate; Reagent 2 is made up of damping fluid, stabilizing agent, glycerokinase, glycerol dehydrogenase.Coenzyme, adenosine diphosphate, glycerol-3-phosphate, glycerokinase, the position of glycerol dehydrogenase in reagent 1 or reagent 2 can not limit.
5. according to the described magnesium diagnosis/determination kit of claim 2, it is characterized in that: form multi-agent reagent by damping fluid, stabilizing agent, coenzyme, adenosine diphosphate, glycerol-3-phosphate, glycerokinase, glycerol dehydrogenase; Reagent 1 is made up of damping fluid, stabilizing agent, coenzyme; Reagent 2 is made up of damping fluid, stabilizing agent, adenosine diphosphate, glycerol-3-phosphate; Reagent 3 is made up of damping fluid, stabilizing agent, glycerokinase, glycerol dehydrogenase.Coenzyme, adenosine diphosphate, glycerol-3-phosphate, glycerokinase, the position of glycerol dehydrogenase in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described magnesium 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 |
|---|---|---|---|
| CNA200610097311XA CN101173934A (en) | 2006-10-30 | 2006-10-30 | Magnesium diagnosis/measuring reagent kit and method for measuring magnesium concentration |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200610097311XA CN101173934A (en) | 2006-10-30 | 2006-10-30 | Magnesium diagnosis/measuring reagent kit and method for measuring magnesium concentration |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101173934A true CN101173934A (en) | 2008-05-07 |
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ID=39422568
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|---|---|---|---|
| CNA200610097311XA Pending CN101173934A (en) | 2006-10-30 | 2006-10-30 | Magnesium diagnosis/measuring reagent kit and method for measuring magnesium concentration |
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|---|---|
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2006
- 2006-10-30 CN CNA200610097311XA patent/CN101173934A/en active Pending
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Application publication date: 20080507 |