CN102466684A - Ammonia (ammonia ion) determination method and ammonia (ammonia ion) diagnosis/determination kit - Google Patents
Ammonia (ammonia ion) determination method and ammonia (ammonia ion) diagnosis/determination kit Download PDFInfo
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- CN102466684A CN102466684A CN2010105373537A CN201010537353A CN102466684A CN 102466684 A CN102466684 A CN 102466684A CN 2010105373537 A CN2010105373537 A CN 2010105373537A CN 201010537353 A CN201010537353 A CN 201010537353A CN 102466684 A CN102466684 A CN 102466684A
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- ferredoxin
- damping fluid
- nitrite reductase
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 title claims abstract description 137
- 229910021529 ammonia Inorganic materials 0.000 title claims abstract description 66
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000003745 diagnosis Methods 0.000 title claims description 12
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 93
- 102000004190 Enzymes Human genes 0.000 claims abstract description 9
- 108090000790 Enzymes Proteins 0.000 claims abstract description 9
- 238000013016 damping Methods 0.000 claims description 54
- 239000012530 fluid Substances 0.000 claims description 54
- 239000003381 stabilizer Substances 0.000 claims description 44
- 239000000523 sample Substances 0.000 claims description 40
- 238000012360 testing method Methods 0.000 claims description 38
- MWUXSHHQAYIFBG-UHFFFAOYSA-N nitrogen oxide Inorganic materials O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 claims description 34
- 239000005515 coenzyme Substances 0.000 claims description 32
- 108010085487 Ferredoxin-Nitrite Reductase Proteins 0.000 claims description 27
- 238000002835 absorbance Methods 0.000 claims description 26
- 108010025915 Nitrite Reductases Proteins 0.000 claims description 25
- 108010028143 Dioxygenases Proteins 0.000 claims description 24
- 102000016680 Dioxygenases Human genes 0.000 claims description 24
- 108010074122 Ferredoxins Proteins 0.000 claims description 24
- 238000003556 assay Methods 0.000 claims description 19
- 239000000872 buffer Substances 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 19
- 230000005477 standard model Effects 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 16
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 14
- 239000012496 blank sample Substances 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 11
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 9
- 239000008363 phosphate buffer Substances 0.000 claims description 9
- 235000011187 glycerol Nutrition 0.000 claims description 8
- OPGYRRGJRBEUFK-UHFFFAOYSA-L disodium;diacetate Chemical compound [Na+].[Na+].CC([O-])=O.CC([O-])=O OPGYRRGJRBEUFK-UHFFFAOYSA-L 0.000 claims description 7
- NQMRYBIKMRVZLB-UHFFFAOYSA-N methylamine hydrochloride Chemical compound [Cl-].[NH3+]C NQMRYBIKMRVZLB-UHFFFAOYSA-N 0.000 claims description 7
- 239000001632 sodium acetate Substances 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 235000017454 sodium diacetate Nutrition 0.000 claims description 7
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- IOVCWXUNBOPUCH-UHFFFAOYSA-N Nitrous acid Chemical compound ON=O IOVCWXUNBOPUCH-UHFFFAOYSA-N 0.000 claims description 5
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 5
- 108010075031 Cytochromes c Proteins 0.000 claims description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 4
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 230000002421 anti-septic effect Effects 0.000 claims description 3
- 229960002319 barbital Drugs 0.000 claims description 3
- 239000012490 blank solution Substances 0.000 claims description 3
- GHXRKGHKMRZBJH-UHFFFAOYSA-N boric acid Chemical compound OB(O)O.OB(O)O GHXRKGHKMRZBJH-UHFFFAOYSA-N 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 claims description 3
- 239000007981 phosphate-citrate buffer Substances 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Substances [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 3
- FYKDNWHPKQOZOT-UHFFFAOYSA-M sodium;dihydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].OP(O)([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O FYKDNWHPKQOZOT-UHFFFAOYSA-M 0.000 claims description 3
- OTNVGWMVOULBFZ-UHFFFAOYSA-N sodium;hydrochloride Chemical compound [Na].Cl OTNVGWMVOULBFZ-UHFFFAOYSA-N 0.000 claims description 3
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 238000006555 catalytic reaction Methods 0.000 claims description 2
- -1 ferrocyanide C Proteins 0.000 claims description 2
- 229910017604 nitric acid Inorganic materials 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000005070 sampling Methods 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 229940064004 antiseptic throat preparations Drugs 0.000 claims 1
- 238000005259 measurement Methods 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 230000002255 enzymatic effect Effects 0.000 abstract description 3
- 238000004737 colorimetric analysis Methods 0.000 abstract description 2
- 238000010168 coupling process Methods 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000007689 inspection Methods 0.000 abstract description 2
- 210000002381 plasma Anatomy 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- FDONOVUTWDJOQT-UHFFFAOYSA-N Cl.Cl.Cl.Cl.CN Chemical compound Cl.Cl.Cl.Cl.CN FDONOVUTWDJOQT-UHFFFAOYSA-N 0.000 description 4
- 235000013772 propylene glycol Nutrition 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- 108010090768 Nitric oxide dioxygenase Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 235000002639 sodium chloride Nutrition 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000007824 enzymatic assay Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- ICIWUVCWSCSTAQ-UHFFFAOYSA-M iodate Chemical compound [O-]I(=O)=O ICIWUVCWSCSTAQ-UHFFFAOYSA-M 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000011430 maximum method Methods 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a determination method of ammonia (ammonium ion) content by utilizing an enzymatic colorimetric method and an enzyme coupling method technology, and composition and components of a reagent. The determination method of the invention has high sensitivity and small error, so the determination method and the kit of the invention can be widely applied to clinical medicine/food inspection.
Description
[technical field]
The present invention relates to medical science/Food Inspection determination techniques field, more specifically, the present invention relates to ammonia (ammonium ion) diagnosing/determining method and kit thereof.
[background technology]
The ammonia method for measuring has microdiffusion, ion exchange process, enzyme process and ammonia electrode method etc.Use at present maximum methods and be enzyme process and based on the determination of blood ammonia appearance analytic approach of ion-selective electrode.
Diffusion method discharges NH after being the sample alkalization
3, the ammonia that discharges with acidometric titration, or form the two mercury amine of pale brown look iodate with the Nessler reaction and carry out colorimetric.These methods need alkalization, and endogenic ammonia forms and impacts, and its accuracy and precision are affected, and seldom use at present; Ion exchange process is more accurate than diffusion method, and CV is 8%～13%; Ion selective electrode method is to utilize NH
3Be diffused into electrode surface, the pH that causes electrode changes and measures, and the CV of this method is 3.5%～4.8%, and the recovery is high.In conjunction with concrete actual, should be practical with the enzymatic assays.
The retrieval Chinese patent is only found 87105593.7 patented claims and is disclosed a kind of rapid freezing cup for blood ammonia determination, does not but find more satisfactory determination of blood ammonia method.
[summary of the invention]
[technical matters that will solve]
The assay method that the purpose of this invention is to provide a kind of ammonia (ammonium ion).
Another object of the present invention provides a kind of ammonia (ammonium ion) diagnosis/determination kit.
[technical scheme]
The present invention realizes through following technical proposals.
Method of the present invention is the coupling technique of a kind of employing enzymic colorimetric (Enzymatic Colorimetric Method) and enzyme (even) united method (Couple Reaction), utilizes reduced form nicotinamide coenzyme (reduced coenzyme) changes mensuration ammonia (ammonium ion) in the absorbance of 340nm wavelength the method for measuring.
Ammonia of the present invention (ammonium ion) assay method know-why be to accomplish according to the serial catalytic reaction of following ferredoxin-nitrite reductase, nitrite reductase, nitrogen monoxide dioxygenase:
Ammonia+2 water+6 oxidized form ferredoxin
Ferredoxin-nitrite reductase
Nitrous acid+6 reduced form ferredoxins
Nitrous acid+ferricytochrome c
Nitrite reductaseNitrogen monoxide+water+
Ferricytochrome c
2 nitrogen monoxides+2 oxygen+reduced coenzyme
The nitrogen monoxide dioxygenase2 nitric acid+
Coenzyme
Method of the present invention is utilized ferredoxin-nitrite reductase (ferredoxin-nitrite reductase; EC1.7.7.1) enzyme (idol) joins nitrite reductase (nitrite reductase; EC 1.7.2.1), nitrogen monoxide dioxygenase (nitric oxide dioxygenase; EC 1.14.12.17) enzymatic inverse ratio look end-point method.Ferredoxin-nitrite reductase enzymolysis ammonia react produces nitrous acid; The effect of uniting nitrite reductase, nitrogen monoxide dioxygenase again through (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 be able to measure the reduced coenzyme degree that absorbance descends at the 340nm place, can calculate the concentration of ammonia (ammonium ion) through measuring the degree that 340nm place absorbance descends like this.
The present invention realizes through following technical proposals.
The present invention relates to the assay method of a kind of ammonia (ammonium ion).The step of this ammonia (ammonium ion) assay method is following:
A, sample are prepared:
A.1 the preparation of standard model
A certain amount of ammonia salt in the water-soluble or damping fluid, is adjusted to 100 micromoles per liter with ammonia concentration again, and the solution that obtains is as standard model;
A.2 the preparation of testing sample
The testing liquid sample is directly tested, need not pre-service; With a certain amount of solid sample to be measured as the preparation standard sample in the water-soluble or damping fluid;
A.3 blank sample
Described water or damping fluid are as blank sample, and its ammonia (ammonium ion) concentration is 0 micromoles per liter;
The preparation of B, reagent solution:
Pipette or take by weighing damping fluid, stabilizing agent, reduced coenzyme, ferredoxin-nitrite reductase, nitrite reductase, nitrogen monoxide dioxygenase, oxidized form ferredoxin and ferrocyanide C respectively; Then they are mixed; The water dissolving obtains described reagent solution, and their concentration is respectively 20-500mmol/L, 0.001-7mol/L, 0.1-0.35mmol/L, 1000-80000U/L, 1000-80000U/L, 1000-80000U/L, 1-100mmol/L and 1-100mmol/L;
C, testing sample with at step B) reagent solution that obtains mixes according to volume ratio 1/10 to 1/500; Reacted 5-60 minute down at temperature 15-45 ℃; Under predominant wavelength 340nm and commplementary wave length 405nm (can not establish commplementary wave length), measure, measure its absorbance over time if receive the instrument restriction;
D, with step C) determination step A under the same condition) and standard model absorbance over time;
E, with step C) be determined at steps A under the same condition) absorbance of blank sample over time;
F, data processing
By step C-E) absorbance of the predominant wavelength 340nm of said mensuration over time, obtain the content of ammonia according to computes:
In the formula:
The absorbance of the testing sample that Δ A (sample) expression step C) obtains changes;
Δ A (blank) representes step e) absorbance of the blank sample that obtains changes;
Δ A (standard) expression step D) absorbance of standard model changes.
According to the present invention, in described ammonia (ammonium ion) assay method, described damping fluid should be appreciated that it is to make it measure the solution of the pH kept stable (generally being 6.0-9.0) of medium.If this pH be higher than 9.0 or pH be lower than 6.0, then the activity of the enzyme that uses of this assay method does not reach the active effect of expection, therefore needs to add more medium and enzyme, just might reach the active effect of expection.
In the present invention, described damping fluid is one or more damping fluids that are selected from three (ethyloic) aminomethane-hydrochloride buffer, phosphate buffer, imidazoles-hydrochloride buffer, sodium hydrogen phosphate-citrate buffer solution, borax-hydrochloride buffer, glycocoll-sodium hydrate buffer solution, barbital sodium-hydrochloride buffer, boric acid-borate buffer solution, diethanolamine buffer or " PBS " damping fluid.
Preferably, described damping fluid for example is selected from three (ethyloic) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, phosphate buffer or " PBS " damping fluid.
More preferably, described damping fluid for example is selected from three (ethyloic) aminomethane-hydrochloric acid (Tris-HCl) damping fluid or phosphate buffer.
According to the present invention; In described ammonia (ammonium ion) assay method, described stabilizing agent should be appreciated that it is a kind of medium (substrate) and the enzyme that can protect in the reagent, makes it can not change its character as time passes; And then deactivated material; It can make reagent possess very long active lifetime, reaches the several months usually, even one, two year.If there is not described stabilizing agent, then the activity of reagent can only be kept tens of hours in solution, a couple of days at most, will lose activity gradually and no longer possessed the detection performance.In the present invention, described stabilizing agent use amount is 0.01-7mol/L.If described stabilizing agent use amount is not enough, then the life-span of agent of activity will shorten; If described stabilizing agent use amount is too high, then can increase cost.
In the present invention, described stabilizing agent is one or more stabilizing agents that are selected from sodium chloride, monoethylene glycol, propylene glycol, glycerine or sodium Diacetate or Sodium azide antiseptic.
Preferably, described stabilizing agent for example is one or more stabilizing agents that are selected from sodium chloride, propylene glycol, glycerine or sodium Diacetate or Sodium azide.
More preferably, described stabilizing agent for example is one or more stabilizing agents that are selected from glycerine or sodium Diacetate.
In the present invention, described reduced coenzyme is one or more reduced coenzymes that are selected from NADPH, NADH or thio-NADH.
According to a kind of preferred implementation of the present invention, when the present invention used automatic clinical chemistry analyzer to measure ammonia (ammonium ion), testing sample, said standard model and said blank sample were by (parameter) sampling automatically under imposing a condition of this analyser; Under following condition, measure then: assay method is 2 end-point method/end-point methods, 37 ℃ of temperature, 10 minutes reaction time; Test predominant wavelength 340nm; Test commplementary wave length 405nm, the volume ratio of tested ammonia (ammonium ion) sample and reagent is 1/10-1/500, the Direction of Reaction is negative reaction; 1/0 minute time delay, 4/5 minute detection time.
According to another kind preferred implementation of the present invention, the reagent solution that the present invention uses is mixed with following two agent reagent:
The reagent of forming by described damping fluid, stabilizing agent, reduced coenzyme, oxidized form ferredoxin and ferrocyanide C 1;
The reagent of forming by described damping fluid, stabilizing agent, ferredoxin-nitrite reductase, nitrite reductase and nitrogen monoxide dioxygenase 2;
Wherein reduced coenzyme, ferredoxin-nitrite reductase, nitrite reductase, nitrogen monoxide dioxygenase, oxidized form ferredoxin, the position of ferrocyanide C in reagent 1 or reagent 2 do not limit.
According to another kind preferred implementation of the present invention, the reagent that the present invention uses is mixed with following three doses of reagent:
The reagent of forming by described damping fluid, stabilizing agent, reduced coenzyme, oxidized form ferredoxin and ferrocyanide C 1;
The reagent of forming by described damping fluid, stabilizing agent, nitrite reductase and nitrogen monoxide dioxygenase 2;
The reagent of forming by described damping fluid, stabilizing agent and ferredoxin-nitrite reductase 3;
Wherein reduced coenzyme, ferredoxin-nitrite reductase, nitrite reductase, nitrogen monoxide dioxygenase, oxidized form ferredoxin, the position of ferrocyanide C in reagent 1, reagent 2 or reagent 3 do not limit.
The determining instrument that in ammonia of the present invention (ammonium ion) assay method, uses can be the ultraviolet analyser, for example 723 visible spectrophotometers of the ultraviolet-visible pectrophotometer of Shanghai precision instrumentation company limited sale, the sale of Tianjin Ka Nasi optic analytical instrument company limited; Semi-automatic biochemical analyzer, for example the BTS-330 semi-automatic biochemical analyzer of medical equipment company limited of Shanghai Victory-idea; Automatic clinical chemistry analyzer, for example: the automatic clinical chemistry analyzer of selling with trade name CX20 with trade name CB8000 or Beckman company with trade name 7600, Abbott with trade name 120, Hitachi, Ltd with trade name AU400, Toshiba with trade name BS-300, Olympus company by Mai Rui company.
The invention still further relates to ammonia (ammonium ion) diagnosis/determination kit.This ammonia (ammonium ion) diagnosis/determination kit is made up of following powdered reagent, and water dissolves the liquid reagent that can directly use that obtains having following concentration range with their in use:
Damping fluid 20-500mmol/L
Stabilizing agent 0.001-7mol/L
Reduced coenzyme 0.1-0.35mmol/L
Ferredoxin-nitrite reductase 1000-80000U/L
Nitrite reductase 1000-80000U/L
Nitrogen monoxide dioxygenase 1000-80000U/L
Oxidized form ferredoxin 1-100mmol/L
Ferrocyanide C 1-100mmol/L.
According to a kind of preferred implementation of the present invention, ammonia of the present invention (ammonium ion) diagnosis/determination kit has:
Reagent 1:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Oxidized form ferredoxin 5mmol/L
Ferrocyanide C 5mmol/L;
Form following reagent 2:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Ferredoxin-nitrite reductase 16000U/L
Nitrite reductase 18000U/L
Nitrogen monoxide dioxygenase 12000U/L.
According to another kind preferred implementation of the present invention, ammonia of the present invention (ammonium ion) diagnosis/determination kit has:
Reagent 1:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Reduced coenzyme 0.25mmol/L
Oxidized form ferredoxin 5mmol/L
Ferrocyanide C 5mmol/L;
Form following reagent 2:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Nitrite reductase 18000U/L
Nitrogen monoxide dioxygenase 12000U/L;
Form following reagent 3:
Damping fluid 100mmol/L
Stabilizing agent 500mmol/L
Ferredoxin-nitrite reductase 16000U/L.
According to another kind preferred implementation of the present invention; In ammonia of the present invention (ammonium ion) diagnosis/determination kit, described damping fluid is one or more damping fluids that are selected from three (ethyloic) aminomethane-hydrochloride buffer, phosphate buffer, imidazoles-hydrochloride buffer, sodium hydrogen phosphate-citrate buffer solution, borax-hydrochloride buffer, glycocoll-sodium hydrate buffer solution, barbital sodium-hydrochloride buffer, boric acid-borate buffer solution, diethanolamine buffer or " PBS " damping fluid.
Preferably, described damping fluid for example is selected from three (ethyloic) aminomethane-hydrochloric acid (Tris-HCl) damping fluid, phosphate buffer or " PBS " damping fluid.
More preferably, described damping fluid for example is selected from three (ethyloic) aminomethane-hydrochloric acid (Tris-HCl) damping fluid or phosphate buffer.
In the present invention, described stabilizing agent is one or more stabilizing agents that are selected from sodium chloride, monoethylene glycol, propylene glycol, glycerine or sodium Diacetate or Sodium azide antiseptic.
Preferably, described stabilizing agent for example is one or more stabilizing agents that are selected from sodium chloride, propylene glycol, glycerine, sodium Diacetate or Sodium azide.
More preferably, described stabilizing agent for example is one or more stabilizing agents that are selected from glycerine or sodium Diacetate.
In the present invention, no matter be single agent reagent, two agent reagent or three doses of reagent, to measure in the method for ammonia (ammonium ion) in the present invention, described reduced coenzyme can be one or more reduced coenzymes that are selected from NADPH, NADH or thio-NADH.
When using ammonia of the present invention (ammonium ion) diagnosis/determination kit; Can use the ultraviolet analyser, for example 723 visible spectrophotometers of the ultraviolet-visible pectrophotometer of Shanghai precision instrumentation company limited sale, the sale of Tianjin Ka Nasi optic analytical instrument company limited; Semi-automatic biochemical analyzer, for example the BTS-330 semi-automatic biochemical analyzer of medical equipment company limited of Shanghai Victory-idea; Automatic clinical chemistry analyzer, for example: the automatic clinical chemistry analyzer of selling with trade name CX20 with trade name CB8000 or Beckman company with trade name 7600, Abbott with trade name 120, Hitachi, Ltd with trade name AU400, Toshiba with trade name BS-300, Olympus company by Mai Rui company.
When adopting the inventive method to measure ammonia (ammonium ion), carry out test of many times according to requirement of experiment, these test findings that will obtain then go out precision (CV) according to computes:
In the formula:
Each time of Xi-test findings;
N-test number (TN) .N >=10
These test findings that obtain are gone out relative extreme difference according to computes:
the-the 3rd batch of each time test findings mean value
-be X; Y, the minimum value among the Z
Every Lot sample number is got n >=3
Confirming through a large amount of tests, is the sample of 0-1000 μ mol/L for ammonia (ammonium ion) content, its analytical error can reach≤and 5%.
The sensitivity of the inventive method can reach 1 μ mol/L.
Confirm that through a large amount of tests it is 0-1000 μ mol/L that the inventive method is measured ammonia (ammonium ion) content range, the inventive method is suitable for clinical medicine/food diagnosing.
[embodiment]
Following embodiment explains the present invention and does not limit protection scope of the present invention.
Embodiment 1: ammonia in the blood plasma (ammonium ion) Determination on content
1) preparation of standard model
A certain amount of ammonia salt in the water-soluble or damping fluid, is adjusted to 100 micromoles per liter with ammonia concentration again;
2) testing sample pre-service
Plasma sample is as testing sample, need not pre-service;
3) blank sample
Described water or damping fluid are as blank sample, and its ammonia concentration is 0 micromoles per liter;
The preparation of B, reagent solution:
The ammonia diagnosing/determining reagent of present embodiment is single agent reagent, and it contains:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Glycerine 1mol/L
NADPH 0.25mmol/L
Ferredoxin-nitrite reductase 16000U/L
Nitrite reductase 18000U/L
Nitrogen monoxide dioxygenase 12000U/L
Oxidized form ferredoxin 5mmol/L
Ferrocyanide C 4mmol/L.
According to above-mentioned concentration these reagent being added the whole dissolvings of deionized water prepares for use.
C, plasma sample to be measured; With at step B) reagent solution that obtains mixes according to volume ratio 1/20; In the reaction 5 minutes down of 37 ℃ of temperature, under predominant wavelength 340nm and commplementary wave length 405nm, measure, measure its absorbance over time Δ A (sample) be 0.0277;
D, with step C) determination step A under the same condition) and standard model absorbance over time Δ A (standard) be 0.0523;
E, with step C) be determined at steps A under the same condition) and the water that uses as the absorbance of blank solution over time Δ A (blank) be 0.0106;
F, data processing
By step C-E) absorbance of the predominant wavelength 340nm of said mensuration over time, obtain the content of ammonia (ammonium ion) according to computes:
In the formula:
The absorbance of the testing sample that Δ A (sample) expression step C) obtains changes;
Δ A (blank) representes step e) absorbance of the blank sample that obtains changes;
Δ A (standard) expression step D) absorbance of standard model changes.
Ammonia (ammonium ion) content that calculates this plasma sample through following formula is 82 μ mol/L, and its error is ± 2 μ mol/L.
Embodiment 2: ammonia in the blood plasma (ammonium ion) Determination on content
1) preparation of standard model
A certain amount of ammonia salt is soluble in water, again ammonia (ammonium ion) concentration is adjusted to 100 micromoles per liter, as standard model;
2) preparation of testing sample
Plasma sample is as testing sample, need not pre-service;
3) blank sample
Described water is as blank sample, and its ammonia (ammonium ion) concentration is 0 micromoles per liter;
The preparation of B, reagent solution:
Ammonia (ammonium ion) diagnosing/determining reagent is two agent reagent, and it contains:
Form following reagent 1:
Phosphate buffer 1 00mmol/L
NADH 0.25mmol/L
Oxidized form ferredoxin 5mmol/L
Ferrocyanide C 5mmol/L;
Form following reagent 2:
Phosphate buffer 1 00mmol/L
Monoethylene glycol 5mol/L
Ferredoxin-nitrite reductase 16000U/L
Nitrite reductase 32000U/L
Nitrogen monoxide dioxygenase 40000U/L.
According to above-mentioned concentration these reagent being added the whole dissolvings of deionized water prepares for use.
C, sample use Hitachi's 7080 automatic clinical chemistry analyzers to measure
This automatic clinical chemistry analyzer main operating parameters is following:
Metering method: 2 end-point methods
Test and measuring time point: 21,31
Predominant wavelength: 340
Commplementary wave length: 405
Temperature of reaction: 37
Sample volume: 20
Reagent 1 (R1) volume: 200
Reagent 2 (R3) volume: 50
The Direction of Reaction: negative
Standard model 1: 0
Standard model 2: 100
The calibration result shows that the K value is-5011, and being converted into sensitivity is 0.00020 Δ A/ μ mol/L.
Test result shows that containing ammonia (ammonium ion) in this blood plasma is 21 μ mol/L.Its error is ± 1 μ mol/L.
Embodiment 3: ammonia in the plasma sample (ammonium ion) Determination on content
The mode of operation of this embodiment is identical with embodiment 2, just present embodiment:
The preparation of B, reagent solution:
Ammonia (ammonium ion) diagnosing/determining reagent is three doses of reagent, and it contains:
Form following reagent 1:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
NADH 0.25mmol/L
Oxidized form ferredoxin 6mmol/L
Ferrocyanide C 4mmol/L;
Form following reagent 2:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Sodium chloride 2mol/L
Nitrite reductase 28000U/L
Nitrogen monoxide dioxygenase 50000U/L;
Form following reagent 3:
Three (ethyloic) aminomethane-hydrochloride buffer 100mmol/L
Sodium chloride 2mol/L
Ferredoxin-nitrite reductase 26000U/L.
According to above-mentioned concentration these reagent being added the whole dissolvings of deionized water prepares for use.
C, sample use Hitachi's 7080 automatic clinical chemistry analyzers to measure
This automatic clinical chemistry analyzer main operating parameters is following:
Metering method: 2 end-point methods
Test and measuring time point: 21,31
Predominant wavelength: 340
Commplementary wave length: 405
Temperature of reaction: 37
Sample volume: 20
Reagent 1 (R1) volume: 20
Reagent 2 (R2) volume: 180
Reagent 3 (R3) volume: 50
The Direction of Reaction: just
Standard model 1: 0
Standard model 2: 100
The calibration result shows that the K value is-4705, and being converted into sensitivity is 0.00021 Δ A/ μ mol/L.
Test result shows that containing ammonia (ammonium ion) in this blood plasma is 78 μ mol/L.Its error is ± 2 μ mol/L.
Embodiment 4: stability test
The mode of operation of this embodiment is identical with embodiment 1, and just after embodiment 1 implemented, the reagent that embodiment 1 uses had been deposited under 2-8 ℃ half a year and 1 year in airtight reagent bottle.
Use the same standard sample, use with embodiment 2 same novel agent and the above-mentioned reagent of depositing half a year and 1 year and measure respectively, other condition is all identical with embodiment 2, and it is following that it measures the result:
Table 1
| ? | Ammonia (ammonium ion) content | Analytical error |
| Novel agent | 201μmol/L | ±4μmol/L |
| Deposit the reagent of half a year | 202μmol/L | ±4μmol/L |
[0230]
| Deposit the reagent in 1 year | 205 μ mol/L | ± 4 μ mol/L |
Table 1 is the result show, uses kit of the present invention, adopts assay method of the present invention can guarantee to obtain stable result, and its stability is more than at least one year.
Embodiment 5: linear test
The mode of operation of this embodiment is identical with embodiment 2, just prepares new standard model concentration and reaches 1200 μ mol/L, and the ammonia (ammonium ion) of 800 μ mol/L according to doubling dilution, is tested then, and it is following that it measures the result:
Table 2
| Ammonia (ammonium ion) desired value | Ammonia (ammonium ion) test value |
| 0 | 0 |
| 50 | 49 |
| 100 | 99 |
| 200 | 98 |
| 400 | 399 |
| 600 | 601 |
| 800 | 798 |
| 1000 | 1003 |
| 1200 | 1150 |
Table 2 is the result show, uses kit of the present invention, adopts assay method of the present invention can guarantee that linearity can reach 1000 μ mol/L.
The applicant adopts other assay methods of putting down in writing in the above summary of the invention all can reach the object of the 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 proof: adopt assay method of the present invention can draw required mensuration result through general biochemical analyzer fully---the blank reagent absorbance changes (Δ A)≤0.01; Absorbance time response curve should be decline curve; Reagent can be surveyed effectively, and (R>=0.99) linear range can reach 1000 μ mol/L; The inaccuracy of reagent test, its relative deviation be no more than ± and 5%; The coefficient of variation (CV)≤5% of the precision of reagent test (repeatability); The sensitivity of reagent can reach 0.0002 ± 0.0001A/ μ mol/L; Reagent is preserved down at 2-8 ℃, and activity can be stablized 1 year;---the present invention is highly sensitive, degree of accuracy good, and linear range is broad, and stationary phase is long, is enough to easy to utilize.
Claims (6)
1. one kind is utilized enzymic colorimetric and technological ammonia (ammonium ion) method for measurement of concentration of enzyme-linked method, and the know-why of its mensuration is to accomplish according to the serial catalytic reaction of following ferredoxin-nitrite reductase, nitrite reductase, nitrogen monoxide dioxygenase:
Ammonia+2 water+6 oxidized form ferredoxin
Ferredoxin-nitrite reductase
Nitrous acid+6 reduced form ferredoxins
Nitrous acid+ferricytochrome c
Nitrite reductaseNitrogen monoxide+water+
Ferricytochrome c
2 nitrogen monoxides+2 oxygen+reduced coenzyme
The nitrogen monoxide dioxygenase2 nitric acid+
Coenzyme
2. the assay method of an ammonia (ammonium ion) is characterized in that the step of this method is following:
2.1 sample is prepared:
2.1.1 the preparation of standard model
A certain amount of ammonia salt in the water-soluble or damping fluid, is adjusted to 100 micromoles per liter with its concentration again;
2.1.2 the preparation of testing sample
The testing liquid sample is directly tested, need not pre-service; With a certain amount of solid sample to be measured as the preparation standard sample in the water-soluble or damping fluid;
2.1.3 blank sample
Described water or damping fluid are as blank sample, and its ammonia (ammonium ion) concentration is 0 micromoles per liter;
2.2 the preparation of reagent solution:
Pipette or take by weighing damping fluid, stabilizing agent, reduced coenzyme, ferredoxin-nitrite reductase, nitrite reductase, nitrogen monoxide dioxygenase, oxidized form ferredoxin and ferrocyanide C respectively; Then they are mixed; The water dissolving obtains described reagent solution, and their concentration is respectively 20-500mmol/L, 0.001-7mol/L, 0.1-0.35mmol/L, 1000-80000U/L, 1000-80000U/L, 1000-80000U/L, 1-100mmol/L and 1-100mmol/L;
2.3 testing sample with in step 2.2) reagent solution that obtains mixes according to volume ratio 1/10 to 1/500; Reacted 5-60 minute down at temperature 15-45 ℃; At predominant wavelength 340nm and commplementary wave length 405nm (if limited by instrument; Can not establish commplementary wave length) under measure, measure its absorbance over time;
2.4 with step 2.3) determination step 2.1.1 under the same condition) and standard model absorbance over time;
2.5 with step 2.3) be determined at step 2.1.3 under the same condition) water that uses or damping fluid are as the absorbance of blank solution over time;
2.6 data processing
By step 2.3-2.5) absorbance of the predominant wavelength 340nm of said mensuration over time, obtain the content of ammonia according to computes:
In the formula:
Δ A (sample) representes step 2.3) absorbance of the testing sample that obtains changes;
Δ A (blank) representes step 2.5) absorbance of the blank solution that obtains changes;
Δ A (standard) representes step 2.4) absorbance of standard model changes.
3. according to claim 1,2 described assay methods, when it is characterized in that using automatic clinical chemistry analyzer to measure, testing sample, said standard model and said blank sample are by the sampling automatically under imposing a condition of this analyser; Under following condition, measure then: assay method is an end-point method, 37 ℃ of temperature, 10 minutes reaction time; Test predominant wavelength 340nm; Test commplementary wave length 405nm, the volume ratio of tested ammonia sample and reagent is 1/10-1/500, the Direction of Reaction is negative reaction; 1/0 minute time delay, 4/5 minute detection time.
4. according to claim 1,2 or 3 described assay methods, it is characterized in that described stabilizing agent is one or more stabilizing agents that are selected from sodium chloride, monoethylene glycol, propylene glycol, glycerine or antiseptics such as sodium Diacetate, Sodium azide; Described damping fluid is one or more damping fluids that are selected from three (ethyloic) aminomethane-hydrochloride buffer, phosphate buffer, imidazoles-hydrochloride buffer, sodium hydrogen phosphate-citrate buffer solution, borax-hydrochloride buffer, glycocoll-sodium hydrate buffer solution, barbital sodium-hydrochloride buffer, boric acid-borate buffer solution, diethanolamine buffer or " PBS " damping fluid; Described reduced coenzyme is one or more reduced coenzymes that are selected from NADPH, NADH or thio-NADH.
5. according to claim 1,2 or 3 described assay methods, it is characterized in that:
5.1 described reagent solution is mixed with like the agent reagent that places an order:
It is made up of described damping fluid, stabilizing agent, reduced coenzyme, oxidized form ferredoxin, ferrocyanide C, ferredoxin-nitrite reductase, nitrite reductase and nitrogen monoxide dioxygenase;
5.2 described reagent solution is mixed with following two agent reagent:
Reagent 1, it is made up of described damping fluid, stabilizing agent, reduced coenzyme, oxidized form ferredoxin and ferrocyanide C;
Reagent 2, it is made up of described damping fluid, stabilizing agent, ferredoxin-nitrite reductase, nitrite reductase and nitrogen monoxide dioxygenase;
Wherein reduced coenzyme, ferredoxin-nitrite reductase, nitrite reductase, nitrogen monoxide dioxygenase, oxidized form ferredoxin, the position of ferrocyanide C in reagent 1 or reagent 2 do not limit.
5.3 described reagent is mixed with following three doses of reagent:
Reagent 1, it is made up of described damping fluid, stabilizing agent, reduced coenzyme, oxidized form ferredoxin and ferrocyanide C;
Reagent 2, it is made up of described damping fluid, stabilizing agent, nitrite reductase and nitrogen monoxide dioxygenase;
Reagent 3, it is made up of described damping fluid, stabilizing agent and ferredoxin-nitrite reductase;
Wherein reduced coenzyme, ferredoxin-nitrite reductase, nitrite reductase, nitrogen monoxide dioxygenase, oxidized form ferredoxin, the position of ferrocyanide C in reagent 1, reagent 2 or reagent 3 do not limit.
6. an ammonia (ammonium ion) diagnosis/determination kit is characterized in that:
6.1 it is made up of following powdered reagent, water dissolves the liquid reagent that can directly use that obtains having following concentration range with their in use:
Damping fluid 20-500mmol/L
Stabilizing agent 0.001-7mol/L
Reduced coenzyme 0.1-0.35mmol/L
Ferredoxin-nitrite reductase 1000-80000U/L
Nitrite reductase 1000-80000/L
Nitrogen monoxide dioxygenase 1000-80000U/L
Oxidized form ferredoxin 1-100mmol/L
Ferrocyanide C 1-100mmol/L.
6.2, it is characterized in that it has according to the described ammonia of claim 6.1 (ammonium ion) diagnosis/determination kit:
Form following reagent 1:
Damping fluid 20-500mmol/L
Stabilizing agent 0.001-7mol/L
Reduced coenzyme 0.1-0.35mmol/L
Oxidized form ferredoxin 1-100mmol/L
Ferrocyanide C 1-100mmol/L;
Form following reagent 2:
Damping fluid 20-500mmol/L
Stabilizing agent 0.001-7mol/L
Ferredoxin-nitrite reductase 1000-80000U/L
Nitrite reductase 1000-80000U/L
Nitrogen monoxide dioxygenase 1000-80000U/L.
6.3, it is characterized in that it has according to the described ammonia of claim 6.1 (ammonium ion) diagnosis/determination kit:
Form following reagent 1:
Damping fluid 20-500mmol/L
Stabilizing agent 0.001-7mol/L
Reduced coenzyme 0.1-0.35mmol/L
Oxidized form ferredoxin 1-100mmol/L
Ferrocyanide C 1-100mmol/L;
Form following reagent 2:
Damping fluid 20-500mmol/L
Stabilizing agent 0.001-7mol/L
Nitrite reductase 1000-80000U/L
Nitrogen monoxide dioxygenase 1000-80000U/L;
Form following reagent 3:
Damping fluid 20-500mmol/L
Stabilizing agent 0.001-7mol/L
Ferredoxin-nitrite reductase 1000-80000U/L.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105373537A CN102466684A (en) | 2010-11-10 | 2010-11-10 | Ammonia (ammonia ion) determination method and ammonia (ammonia ion) diagnosis/determination kit |
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Application publication date: 20120523 |