CN101464270A - Carbon dioxide diagnosis/measuring reagent kit and carbon dioxide concentration determination method - Google Patents
Carbon dioxide diagnosis/measuring reagent kit and carbon dioxide concentration determination method Download PDFInfo
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- CN101464270A CN101464270A CNA2007101914236A CN200710191423A CN101464270A CN 101464270 A CN101464270 A CN 101464270A CN A2007101914236 A CNA2007101914236 A CN A2007101914236A CN 200710191423 A CN200710191423 A CN 200710191423A CN 101464270 A CN101464270 A CN 101464270A
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- reagent
- carbon dioxide
- acid
- stabilizing agent
- reduced coenzyme
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Abstract
The invention relates to a kit for diagnosing/measuring carbon dioxide 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 measuring the concentration of the carbon dioxide, 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, a phosphoenolpyruvic acid, phosphoenolpyruvate carboxykinase, 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 of the decrease in absorbance at 340 nm of the dominant wavelength is detected, thereby measuring the active concentration of the carbon dioxide.
Description
Technical field
The present invention relates to a kind of carbon dioxide diagnosis/determination reagent kit, the invention still further relates to the method for measuring gas concentration lwevel simultaneously, belong to medical science/food/environmental test determination techniques field.
Background technology
The content of carbon dioxide plays an important role to the adjusting of acid base equilibrium in the human body in the blood.The constant of blood pH is the activity essential condition that earns a bare living, and be the most important with bicarbonate buffer system in the multiple buffer system in the blood plasma, directly influences the pH of blood.
The content of carbon dioxide mainly reflects the metabolic disturbance of acid-base balance in the blood.During simple metabolic acid or alkali poisoning, blood bicarbonate radical [HCO
3 -] descend or rising, total CO 2 also descends thereupon or raises.And during simple respiratory acid or alkali poisoning, the blood bicarbonate radical raises or descends.
The assay method of total CO 2 and bicarbonate radical can be divided into direct mensuration and calculate two classes indirectly.Directly measure and mainly contain eudiometry, piezometric method, titrimetry, colourimetry, Conway microdiffusion, flow injection gaseous diffusion process etc.Calculate it is PH and the PCO that utilizes blood gas analyzer to record indirectly
2, according to the variation of H-H equation:
HCO
3 -(m mol/L)=0.03 * PCO
2(mmHg) * antilog (PH-p ka) or HCO
3 -(m mol/L)=0.225 * PCO
2(k Pa) * antilog (PH-p Ka) calculates acquisition.
In the formula: measured value when PH is 37 ℃, p ka is 6.10 at 37 ℃.
At present, more general with indirect predication method application, report that by little process computer of blood analyser and with the blood gas analysis result accuracy can meet clinical requirement substantially.
Directly determination method is most widely used general with titrimetry, but owing to be subjected to multiple factor affecting, error at measurment is bigger.
Enzymatic assays is fit to automated analysis, and reliable results should be up-and-coming assay method, but still remains at present to be furtherd investigate.
The retrieval Chinese patent finds, the application for a patent for invention that application number is 96190276.0, the applying date is 1996.02.06 discloses a kind of system and method that is used for deriving the Noninvasive of gas content in the patient blood.Gas concentration lwevel during this system exhales corresponding to cubing.Handle these data then and derive carbon dioxide level in the arterial blood.If data are time domain, processing can become the time domain data-switching volume territory.This method has also been assessed the conspicuousness of a plurality of variablees through iteration, the relation of gained can supply fast and exactly healthy people and trouble lunger be carried out the mensuration of gas content in the blood.In recent years, the utility model patent that application number is 02282386.7, the applying date is 2002.10.29 discloses a kind of medical blood bicarbonate radical analyzer, mainly by guidance panel, sample detection mechanism, decide dropping liquid mechanism, rabbling mechanism and be that the electrical control part of master element is formed with the single-chip microcomputer.Its guidance panel includes manual button, input keyboard and display screen; Sample detection mechanism is by being uniformly distributed along the circumference sample aperture and place the sample rotating disk of specimen bottle and vertically be assemblied in the drive motor below the rotating disk and form corresponding to the photoelectric detector of detecting position sample aperture setting in the hole; Decide dropping liquid mechanism by the micro pump of configuration motor and be arranged at sample aperture top decide water dropper and be configured in the drop recorder of deciding water dropper liquid droping port place form; Rabbling mechanism is made up of with the stirring rod that is assemblied in the disk on the stirring motor axle and be positioned in the specimen bottle the stirring motor that is arranged on relevant position, sample rotating disk below.
Above patent is all irrelevant with enzymatic assays, and this is from a side illustration, and domestic experimental study in this respect extremely lacks.
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 gas concentration lwevel, simultaneously, the present invention also will provide in order to realize the carbon dioxide diagnosis/determination reagent kit of this method, adopt this reagent not only can be ultraviolet analyser or half, carry out carbon dioxide concentration determination on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.
Carbon dioxide concentration determination method principle of the present invention is as follows:
Carbon dioxide+phosphoenolpyruvic acid+water
Phosphoric acid enol pyruvic acid carboxylase
Oxaloacetic acid+phosphate radical
Oxaloacetic acid+reduced coenzyme
Malic dehydrogenaseMalic acid+coenzyme
This method is used phosphoric acid enol pyruvic acid carboxylase (phosphoenolpyruvate carboxylase; EC 4.1.1.31) enzyme (idol) connection malic dehydrogenase (Malate dehydrogenase; EC 1.1.1.37) enzyme ' s reaction speeding colourimetry.Phosphoric acid enol pyruvic acid carboxylase enzymolysis carbon dioxide reaction produces oxaloacetic acid, the effect of uniting malic dehydrogenase again by (idol), 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 the degree that reduced coenzyme descends in 340nm place absorbance, by measuring the degree that 340nm place absorbance descends, can calculate the concentration of carbon dioxide size.
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 carbon dioxide diagnosis/determination reagent 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
Phosphoric acid enol pyruvic acid carboxylase 6000U/L
Malic dehydrogenase 10000U/L
Phosphoenolpyruvic acid 6mmol/L
Carbon dioxide diagnosis/determination reagent kit of the present invention can be single agent, comprising:
Damping fluid, stabilizing agent, reduced coenzyme, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, phosphoenolpyruvic acid.
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, phosphoenolpyruvic acid.
Reagent 2
Damping fluid, stabilizing agent, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase.
Reduced coenzyme, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, the position of phosphoenolpyruvic acid 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, phosphoenolpyruvic acid.
Reagent 2
Damping fluid, stabilizing agent, malic dehydrogenase.
Reagent 3
Damping fluid, stabilizing agent, phosphoric acid enol pyruvic acid carboxylase.
Reduced coenzyme, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, the position of phosphoenolpyruvic acid 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 gas concentration lwevel, 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 carbon dioxide diagnosis/determination reagent of present embodiment is single reagent, comprising:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Phosphoric acid enol pyruvic acid carboxylase 6000U/L
Malic dehydrogenase 10000U/L
Phosphoenolpyruvic acid 6mmol/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 carbon dioxide sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), 0 minute 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 the degree that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of carbon dioxide size.
Embodiment two
The carbon dioxide diagnosis/determination 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
Phosphoenolpyruvic acid 6mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Phosphoric acid enol pyruvic acid carboxylase 6000U/L
Malic dehydrogenase 10000U/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 carbon dioxide sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), 0 minute 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 the degree that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of carbon dioxide size.
Embodiment three
The carbon dioxide diagnosis/determination 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
Phosphoenolpyruvic acid 6mmol/L
Reagent 2
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Malic dehydrogenase 10000U/L
Reagent 3
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Stabilizing agent 500mmol/L
Phosphoric acid enol pyruvic acid carboxylase 6000U/L
Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.
When measuring gas concentration lwevel, 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 carbon dioxide sample and reagent 1, reagent 2, reagent 3 is 4/40/5/5, the Direction of Reaction is negative reaction (reaction descends), and 0 minute 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 the degree that predominant wavelength 340nm absorbance descends, thereby calculates the concentration of carbon dioxide size.
The applicant adopts other assay methods 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)≤0.05; Absorbance time response curve should be decline curve until terminal point; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 25mmol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 5%; The coefficient of variation (CV)≤2% of the precision of reagent test (repeatability); The sensitivity of reagent can reach that 0.068 ± 0.034 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. carbon dioxide concentration determination method that utilizes enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:
Carbon dioxide+phosphoenolpyruvic acid+water
Phosphoric acid enol pyruvic acid carboxylase
Oxaloacetic acid+phosphate radical
Oxaloacetic acid+reduced coenzyme
Malic dehydrogenaseMalic acid+coenzyme
The end reaction thing is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the degree that predominant wavelength 340nm absorbance descends, calculate concentration of carbon dioxide size measurement result.
2. carbon dioxide diagnosis/determination reagent kit, principal ingredient comprises:
Damping fluid 20---500mmol/L
Stabilizing agent 1---4000mmol/L
Reduced coenzyme 0.1---0.35mmol/L
Phosphoric acid enol pyruvic acid carboxylase 1000---80000U/L
Malic dehydrogenase 1000---80000U/L
Phosphoenolpyruvic acid 1---50mmol/L
The concentration of reagent composition not necessarily is only limited to above-mentioned scope; Concentration effect in above-mentioned scope is better, 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 carbon dioxide diagnosis/determination reagent kit of claim 2, it is characterized in that:
Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, phosphoenolpyruvic acid.
4. according to the described carbon dioxide diagnosis/determination reagent kit of claim 2, it is characterized in that:
Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, phosphoenolpyruvic acid; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, phosphoenolpyruvic acid; Reagent 2 is made up of damping fluid, stabilizing agent, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase.Reduced coenzyme, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, the position of phosphoenolpyruvic acid in reagent 1 or reagent 2 can not limit.
5. according to the described carbon dioxide diagnosis/determination reagent kit of claim 2, it is characterized in that:
Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, phosphoenolpyruvic acid; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, phosphoenolpyruvic acid; Reagent 2 is made up of damping fluid, stabilizing agent, malic dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, phosphoric acid enol pyruvic acid carboxylase.Reduced coenzyme, phosphoric acid enol pyruvic acid carboxylase, malic dehydrogenase, the position of phosphoenolpyruvic acid in reagent 1, reagent 2 or reagent 3 can not limit.
6. according to the described carbon dioxide diagnosis/determination reagent 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 |
|---|---|---|---|
| CNA2007101914236A CN101464270A (en) | 2007-12-19 | 2007-12-19 | Carbon dioxide diagnosis/measuring reagent kit and carbon dioxide concentration determination method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101914236A CN101464270A (en) | 2007-12-19 | 2007-12-19 | Carbon dioxide diagnosis/measuring reagent kit and carbon dioxide concentration determination method |
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| CN101464270A true CN101464270A (en) | 2009-06-24 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109613225A (en) * | 2018-12-28 | 2019-04-12 | 上海高踪医疗器械科技有限公司 | A kind of carbon dioxide detection reagent box |
| CN110082344A (en) * | 2019-05-20 | 2019-08-02 | 吉林瑞特生物科技有限公司 | Carbon dioxide measurement kit and preparation method thereof |
-
2007
- 2007-12-19 CN CNA2007101914236A patent/CN101464270A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109613225A (en) * | 2018-12-28 | 2019-04-12 | 上海高踪医疗器械科技有限公司 | A kind of carbon dioxide detection reagent box |
| CN110082344A (en) * | 2019-05-20 | 2019-08-02 | 吉林瑞特生物科技有限公司 | Carbon dioxide measurement kit and preparation method thereof |
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Open date: 20090624 |