CN101464328A - Selenium diagnosis/measuring reagent kit and method for measuring selenium concentration - Google Patents
Selenium diagnosis/measuring reagent kit and method for measuring selenium concentration Download PDFInfo
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- CN101464328A CN101464328A CNA2007101921210A CN200710192121A CN101464328A CN 101464328 A CN101464328 A CN 101464328A CN A2007101921210 A CNA2007101921210 A CN A2007101921210A CN 200710192121 A CN200710192121 A CN 200710192121A CN 101464328 A CN101464328 A CN 101464328A
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- glutathione
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- selenium
- stabilizing agent
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Abstract
The invention relates to a kit for diagnosing/measuring selenium by utilizing the selenium-dependent 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 measuring the concentration of selenium, and belongs to the technical field of medical/food/environmental inspection and measurement. The main components of the kit include a buffer solution, reduced coenzyme, glutathione disulfide, glutathione peroxidase, NADH peroxydase 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 340 nm of the dominant wavelength is detected, thereby measuring the concentration of selenium.
Description
Technical field
The present invention relates to a kind of selenium diagnosis/determination kit, the invention still further relates to the method for measuring selenium concentration simultaneously, belong to medical science/food/environmental test determination techniques field.
Background technology
Selenium is biological essential trace element, and plant selenium is the main source that the human and animal obtains selenium, and the too much absorption selenium of human body can cause selenosis.The selenium element can make the peroxide breakdown in the human body, protection cell membrane, the heart, kidney, lung, eye etc.; Can remove the free radical of human body, anti-ageing, strengthen immune function of human body, can suppress multiple carcinogen, human body lacks selenium can bring out angiocardiopathy and cancer.
In conjunction with both at home and abroad about the various detection methods of selenium. Chang Yong assay method wherein: as atomic absorption spectrography (AAS) (AAS), atomic fluorescence spectrometry (AFS) and ICP-AES (ICP-AES) etc.; Atomic absorption spectrum (AAS) has become the main method of trace element analysis as element special efficacy detecting device and GC coupling.Also have in addition, use liquid chromatography tandem mass spectrometry (LC-MS-MS) and identify selenium compound.
Summary of the invention
The technical problem to be solved in the present invention is: propose a kind of dependent enzymic colorimetric of selenium (Enzymatic Colorimetric 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 selenium concentration, simultaneously, the present invention also will provide in order to realize the selenium diagnosis/determination kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carry out measuring selenium concentration on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Method for measuring selenium concentration principle of the present invention is as follows:
Glutathione two sulphur+2 water
Glutathione peroxidase/Se
2 glutathione+hydrogen peroxide
Hydrogen peroxide+reduced coenzyme
The NADH peroxidaseCoenzyme+2 water
This method is utilized the dependent glutathione peroxidase of selenium (glutathione peroxidase; EC1.11.1.9) enzyme (idol) connection NADH peroxidase (NADH peroxidase; EC1.11.1.1; EC1.11.1.2) enzymatic reaction continuous monitoring method/speed ratio color method.Glutathione peroxidase enzymolysis glutathione two reaction of Salmon-Saxl produce hydrogen peroxide, effect by coupling NADH peroxidase 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 selenium.
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 selenium 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
Glutathione peroxidase 10000U/L
NADH peroxidase 12000U/L
Glutathione two sulphur 10mmol/L
Selenium diagnosis/determination kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, glutathione peroxidase, NADH peroxidase, glutathione two sulphur.
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, glutathione two sulphur.
Reagent 2
Damping fluid, stabilizing agent, glutathione peroxidase, NADH peroxidase.
Reduced coenzyme, glutathione peroxidase, NADH peroxidase, the position of glutathione two sulphur 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, glutathione two sulphur.
Reagent 2
Damping fluid, stabilizing agent, NADH peroxidase.
Reagent 3
Damping fluid, stabilizing agent, glutathione peroxidase.
Reduced coenzyme, glutathione peroxidase, NADH peroxidase, the position of glutathione two sulphur 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 selenium concentration, 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 selenium 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
Glutathione peroxidase 10000U/L
NADH peroxidase 12000U/L
Glutathione two sulphur 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 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested selenium sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), about about 1 minute of time delay is about 3 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 selenium.
Embodiment two
The selenium 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
Glutathione two sulphur 10mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glutathione peroxidase 10000U/L
NADH peroxidase 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 1.8 ± 0.7, test predominant wavelength 340nm, test commplementary wave length 405nm, the volume ratio of tested selenium 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 3 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 selenium.
Embodiment three
The selenium 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
Glutathione two sulphur 10mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
NADH peroxidase 12000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Glutathione peroxidase 10000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring selenium concentration, 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 selenium 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 3 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 selenium.
The applicant adopts the assay method 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)≤0.0012; Absorbance time response curve should linearly descend; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 0.6mmol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 6%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach that 0.056 ± 0.028 Δ A/mmol/L---the present invention is highly sensitive, degree of accuracy good, and the linear range broadness is enough to easy to utilize.
Claims (6)
1. method for measuring selenium concentration that utilizes dependent enzymic colorimetric of selenium and enzyme-linked method technology, its method principle is as follows:
Glutathione two sulphur+2 water
Glutathione peroxidase/Se
2 glutathione+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 degree/speed that predominant wavelength 340nm absorbance descends, calculate the concentration measurement result of selenium.
2. selenium diagnosis/determination kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
Reduced coenzyme 0.1---0.35mmol/L
Glutathione peroxidase 1000---80000U/L
NADH peroxidase 1000---80000U/L
Glutathione two sulphur 1---50mmol/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: 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 selenium diagnosis/determination kit of claim 2, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, glutathione peroxidase, NADH peroxidase, glutathione two sulphur.
4. according to the described selenium diagnosis/determination kit of claim 2, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, glutathione peroxidase, NADH peroxidase, glutathione two sulphur; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, glutathione two sulphur; Reagent 2 is made up of damping fluid, stabilizing agent, glutathione peroxidase, NADH peroxidase.Reduced coenzyme, glutathione peroxidase, NADH peroxidase, the position of glutathione two sulphur in reagent 1 or reagent 2 can not limit.
5. according to the described selenium diagnosis/determination kit of claim 2, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, glutathione peroxidase, NADH peroxidase, glutathione two sulphur; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, glutathione two sulphur; Reagent 2 is made up of damping fluid, stabilizing agent, NADH peroxidase; Reagent 3 is made up of damping fluid, stabilizing agent, glutathione peroxidase.Reduced coenzyme, glutathione peroxidase, NADH peroxidase, the position of glutathione two sulphur in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described selenium 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 (AmmoniaSulfate), glycerine (Glycerol), propylene glycol (Propylene Glycol), ethylene glycol (Ethyleneglycol) and at least one of the preservatives.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101921210A CN101464328A (en) | 2007-12-19 | 2007-12-19 | Selenium diagnosis/measuring reagent kit and method for measuring selenium concentration |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101921210A CN101464328A (en) | 2007-12-19 | 2007-12-19 | Selenium diagnosis/measuring reagent kit and method for measuring selenium concentration |
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| CN101464328A true CN101464328A (en) | 2009-06-24 |
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| CNA2007101921210A Pending CN101464328A (en) | 2007-12-19 | 2007-12-19 | Selenium diagnosis/measuring reagent kit and method for measuring selenium concentration |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105092562A (en) * | 2014-05-07 | 2015-11-25 | 河南蓝环医疗设备有限公司 | Kit for detecting selenium ion and detection method thereof |
-
2007
- 2007-12-19 CN CNA2007101921210A patent/CN101464328A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105092562A (en) * | 2014-05-07 | 2015-11-25 | 河南蓝环医疗设备有限公司 | Kit for detecting selenium ion and detection method thereof |
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Open date: 20090624 |