CN101464405A - Kalium ion diagnosis/measuring reagent kit and kalium ion concentration determination method - Google Patents

Abstract

The invention relates to a kit for diagnosing/measuring kalium (ions) 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 kalium (ions), 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, adenosine diphosphate, enolphosphopyruvate, ferricytochrome b1, oxoglutarate, pyruvate kinase, pyruvate dehydrogenase, isocitrate 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 velocity of the decrease in absorbance at 340 nm of the dominant wavelength is detected, thereby measuring the concentration of kalium (ions).

Description

The method for measurement of concentration of potassium (ion) diagnosis/determination kit and potassium (ion)

Technical field

The present invention relates to a kind of potassium (ion) diagnosis/determination kit, the invention still further relates to the method for measuring potassium (ion) concentration simultaneously, belong to medical science/food/environmental test determination techniques field.

Background technology

Renal glomerular disease, renal function are weak, the poisoning of diabetes ketosis, hypoadrenocorticism etc.When blood potassium up to 7.5mmol/L or when above patient arrhythmia cordis will appear, should consider to determine suitable therapeutic scheme.When serum potassium surpasses 10mmol/L, ventricular fibrillation can take place, cardiac arrest and death.High potassium makes cardiac arrest diastole.

Potassium is measured method commonly used at present has: isotope dilution mass spectrometry, neutron activation method, flare photometer, chemical assay, ion selective electrode method, atom spectrophotometric method, enzyme kinetics method.

Flame photometry nineteen fifty is brought into use and the flame photometry always used till today detects the Na of serum, urine, cerebrospinal fluid and ascites pleural fluid ⁺And K ⁺, be a kind of emission spectrometry method.Assay method is divided into internal reference method and two kinds of outer standard laws.Outer standard law operate miss is bigger, does not generally adopt.The main now internal standard method of using, promptly sample and titer adopt internal standard element lithium or the caesium of adding same concentrations to measure.During operation, the solution that will contain lithium is measured the concentration of K, Na and Li simultaneously as dilution, with the Na/Li and the K/Li ratio of sample and titer, calculates Na, K concentration.Because serum diluting multiple is big, the influence of serum proteins viscosity almost can be ignored.

Chemical assay mainly utilizes multiple ring crown compound such as cave crown ether or spherical crown ether, also be called crown ether, being ionophore measures, owing in the macrocyclic structure hole is arranged, the intramolecule oxygen atom has not, and share electron pair can combine with metallic ion, according to the hole size, alternative metallic ion in conjunction with different-diameter, thus can reach the purpose of measuring ion concentration.Measure serum K, generally adopt common crown ether anionic dye to carry out colorimetric assay.

Ion selective electrode method ISE method is to adopt sensitive specific electrode special, at the K of body fluid such as the cleer and peaceful urine of the enterprising promoting circulation of blood of instrumentation ⁺, Na ⁺Mensuration, because of the sample consumption is few, quick and precisely, the trend that replaces additive method is arranged almost.Its Instrument measuring principle is that ion-selective electrode and contrast electrode combination are immersed in the sample solution to be measured, detects.Present existing type of electrodes is: 1. glass-membrane electrode, inductive material be glass film pH electrode, K arranged ⁺Electrode and Na ⁺Electrode; 2. immobilon-p electrode by the extrusion forming of slightly solubility metallics, has Cl with solid film or odd-numbered day film as the electrode of sense film ^-Electrode and F ^-Electrode; 3. liquid film electrode is with epoxy resin or the interior dress polyvinyl chloride Ca that is sense film ²⁺Electrode; 4. the K that makes with the valinomycins film ⁺Electrode.Above-mentioned electrode all has certain life-span, because electrode uses a period of time will automatic aging, the term of validity is different in size.

Atom spectrophotometric method atom spectrophotometric method can be used for detecting serum K+, Na+, trivial operations, and error is bigger, and is easy not as good as flame photometry.

The decisive method that potassium is measured is isotope dilution mass spectrometry and neutron activation method.The method that WHO recommends is a flare photometer.Ion selective electrode method application at present is comparatively general, but electrode cost costliness.

Enzyme assay and flare photometer or ion selective electrode method all have consistance preferably, and strong interference immunity, and good stability has remedied the deficiency that Biochemical Analyzer can not measured potassium sodium simultaneously.Analytical performance is preferably arranged, be easy to robotization, must replace flare photometer in the clinical chemistry analysis becomes the conventional analysis project.

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 potassium (ion) concentration, simultaneously, the present invention also will provide potassium (ion) diagnosis/determination kit in order to realize this method, adopt this reagent not only can be ultraviolet analyser or half, carry out potassium (ion) concentration determination on the automatic clinical chemistry analyzer, and finding speed is fast, the accuracy height, thereby can obtain practical applying.

Potassium of the present invention (ion) method for measurement of concentration principle is as follows:

Adenosine diphosphate+phosphoenolpyruvate Pyruvic acid pyruvate kinase/K ⁺ Adenosine triphosphate+

Pyruvic acid

Pyruvic acid+ferricytochrome b1+ water Pyruvic dehydrogenaseAcetate+carbon dioxide

+ ferricytochrome b1

Carbon dioxide+ketoglutaric acid+reduced coenzyme Isocitric dehydrogenaseIsocitric acid

+ coenzyme

Pyruvate kinase (the pyruv Aunar kinase that this method application need potassium ion activates; EC2.7.1.40) enzyme (idol) connection pyruvic dehydrogenase (Pyruvate dehydrogenase; EC 1.2.2.2), isocitric dehydrogenase (isocitrate dehydrogenase; EC 1.1.1.41; EC 1.1.1.42) enzymatic reaction continuous monitoring/speed ratio color method.Under the activation of potassium (ion), the reaction of pyruvate kinase enzymolysis phosphoenolpyruvic acid produces pyruvic acid, the effect of uniting pyruvic dehydrogenase, isocitric 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 speed that reduced coenzyme descends in 340nm place absorbance, by measuring the speed that 340nm place absorbance descends, can calculate the concentration of potassium (ion).

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 potassium of the present invention (ion) diagnosis/determination kit of following composition relation is comparatively desirable:

Damping fluid 100mmol/L

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Pyruvate kinase 6000U/L

Pyruvic dehydrogenase 8000U/L

Isocitric dehydrogenase 10000U/L

Adenosine diphosphate 3mmol/L

Phosphoenolpyruvic acid 3mmol/L

Ferricytochrome b1 4mmol/L

Ketoglutaric acid 10mmol/L

Potassium of the present invention (ion) diagnosis/determination kit can be single agent, comprising:

Damping fluid, stabilizing agent, reduced coenzyme, pyruvate kinase, pyruvic dehydrogenase, isocitric dehydrogenase, adenosine diphosphate, phosphoenolpyruvic acid, ferricytochrome b1, ketoglutaric 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, adenosine diphosphate, phosphoenolpyruvic acid, ferricytochrome b1, ketoglutaric acid.

Reagent 2

Damping fluid, stabilizing agent, pyruvate kinase, pyruvic dehydrogenase, isocitric dehydrogenase.

Reduced coenzyme, pyruvate kinase, pyruvic dehydrogenase, isocitric dehydrogenase, adenosine diphosphate, phosphoenolpyruvic acid, ferricytochrome b1, the position of ketoglutaric 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, adenosine diphosphate, phosphoenolpyruvic acid, ferricytochrome b1, ketoglutaric acid.

Reagent 2

Damping fluid, stabilizing agent, pyruvic dehydrogenase, isocitric dehydrogenase.

Reagent 3

Damping fluid, stabilizing agent, pyruvate kinase.

Reduced coenzyme, pyruvate kinase, pyruvic dehydrogenase, isocitric dehydrogenase, adenosine diphosphate, phosphoenolpyruvic acid, ferricytochrome b1, the position of ketoglutaric 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 potassium (ion) 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 potassium of present embodiment (ion) diagnosing/determining reagent is single reagent, comprising:

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 500mmol/L

Reduced coenzyme 0.25mmol/L

Pyruvate kinase 6000U/L

Pyruvic dehydrogenase 8000U/L

Isocitric dehydrogenase 10000U/L

Adenosine diphosphate 3mmol/L

Phosphoenolpyruvic acid 3mmol/L

Ferricytochrome b1 4mmol/L

Ketoglutaric acid 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 potassium (ion) sample and reagent is 1/25, the Direction of Reaction is negative reaction (reaction descends), about about 2 minutes 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 descends, thereby calculates the concentration of potassium (ion).

Embodiment two

The potassium of present embodiment (ion) diagnosing/determining reagent is double reagent, comprising:

Reagent 1

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 50mmol/L

Reduced coenzyme 0.25mmol/L

Adenosine diphosphate 3mmol/L

Phosphoenolpyruvic acid 3mmol/L

Ferricytochrome b1 4mmol/L

Ketoglutaric acid 10mmol/L

Reagent 2

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 500mmol/L

Pyruvate kinase 6000U/L

Pyruvic dehydrogenase 8000U/L

Isocitric 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 potassium (ion) sample and reagent 1, reagent 2 is 2/20/5, the Direction of Reaction is negative reaction (reaction descends), about about 2 minutes 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 descends, thereby calculates the concentration of potassium (ion).

Embodiment three

The potassium of present embodiment (ion) diagnosing/determining reagent is three reagent, comprising:

Reagent 1

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 50mmol/L

Reduced coenzyme 0.25mmol/L

Adenosine diphosphate 3mmol/L

Phosphoenolpyruvic acid 3mmol/L

Ferricytochrome b1 4mmol/L

Ketoglutaric acid 10mmol/L

Reagent 2

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 500mmol/L

Pyruvic dehydrogenase 8000U/L

Isocitric dehydrogenase 10000U/L

Reagent 3

Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L

Stabilizing agent 500mmol/L

Pyruvate kinase 6000U/L

Reagent divides the bottle of packing into after all dissolving and preparing, and makes liquid three reagent, can directly use.

When measuring potassium (ion) 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 potassium (ion) 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 2 minutes 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 descends, thereby calculates the concentration of potassium (ion).

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)≤0.016; Absorbance time response curve should be decline curve until terminal point; Reagent can be surveyed effectively, and (R 〉=0.99) linear range can reach 10mmol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 5%; The coefficient of variation (CV)≤3% of the precision of reagent test (repeatability); The sensitivity of reagent can reach that 0.016 ± 0.008 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. potassium (ion) method for measurement of concentration that utilizes enzymic colorimetric and enzyme-linked method technology, its method principle is as follows:

Adenosine diphosphate+phosphoenolpyruvic acid Pyruvate kinase/K ⁺ Adenosine triphosphate+

Pyruvic acid

Pyruvic acid+ferricytochrome b1+ water Pyruvic dehydrogenaseAcetate+carbon dioxide

+ ferricytochrome b1

Carbon dioxide+ketoglutaric acid+reduced coenzyme Isocitric dehydrogenaseIsocitric acid

+ 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 descends, calculate the concentration measurement result of potassium (ion).

2. a potassium (ion) diagnosis/determination kit, principal ingredient comprises:

Damping fluid 20---500mmol/L

Stabilizing agent 1---4000mmol/L

Reduced coenzyme 0.1---0.35mmol/L

Pyruvate kinase 1000---80000U/L

Pyruvic dehydrogenase 1000---80000U/L

Isocitric dehydrogenase 1000---80000U/L

Adenosine diphosphate 1---50mmol/L

Phosphoenolpyruvic acid 1---50mmol/L

Ferricytochrome b1 1---50mmol/L

Ketoglutaric acid 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 potassium of claim 2 (ion) diagnosis/determination kit, it is characterized in that:

Form single agent reagent by damping fluid, stabilizing agent, reduced coenzyme, pyruvate kinase, pyruvic dehydrogenase, isocitric dehydrogenase, adenosine diphosphate, phosphoenolpyruvic acid, ferricytochrome b1, ketoglutaric acid.

4. according to the described potassium of claim 2 (ion) diagnosis/determination kit, it is characterized in that:

Form two agent reagent by damping fluid, stabilizing agent, reduced coenzyme, pyruvate kinase, pyruvic dehydrogenase, isocitric dehydrogenase, adenosine diphosphate, phosphoenolpyruvic acid, ferricytochrome b1, ketoglutaric acid; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, adenosine diphosphate, phosphoenolpyruvic acid, ferricytochrome b1, ketoglutaric acid; Reagent 2 is made up of damping fluid, stabilizing agent, pyruvate kinase, pyruvic dehydrogenase, isocitric dehydrogenase.Reduced coenzyme, pyruvate kinase, pyruvic dehydrogenase, isocitric dehydrogenase, adenosine diphosphate, phosphoenolpyruvic acid, ferricytochrome b1, the position of ketoglutaric acid in reagent 1 or reagent 2 can not limit.

5. according to the described potassium of claim 2 (ion) diagnosis/determination kit, it is characterized in that:

Form multi-agent reagent by damping fluid, stabilizing agent, reduced coenzyme, pyruvate kinase, pyruvic dehydrogenase, isocitric dehydrogenase, adenosine diphosphate, phosphoenolpyruvic acid, ferricytochrome b1, ketoglutaric acid; Reagent 1 is made up of damping fluid, stabilizing agent, reduced coenzyme, adenosine diphosphate, phosphoenolpyruvic acid, ferricytochrome b1, ketoglutaric acid; Reagent 2 is made up of damping fluid, stabilizing agent, pyruvic dehydrogenase, isocitric dehydrogenase; Reagent 3 is made up of damping fluid, stabilizing agent, pyruvate kinase.Reduced coenzyme, pyruvate kinase, pyruvic dehydrogenase, isocitric dehydrogenase, adenosine diphosphate, phosphoenolpyruvic acid, ferricytochrome b1, the position of ketoglutaric acid in reagent 1, reagent 2 or reagent 3 can not limit.

6. according to the described potassium of claim 2 (ion) diagnosis/determination kit, 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.