CN107505276A - A kind of high throughput assay method of citron acid content in solution - Google Patents
A kind of high throughput assay method of citron acid content in solution Download PDFInfo
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- CN107505276A CN107505276A CN201710646028.6A CN201710646028A CN107505276A CN 107505276 A CN107505276 A CN 107505276A CN 201710646028 A CN201710646028 A CN 201710646028A CN 107505276 A CN107505276 A CN 107505276A
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- citric acid
- liquid
- standard items
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- 238000000034 method Methods 0.000 title claims abstract description 49
- 239000002253 acid Substances 0.000 title claims abstract description 26
- 240000004307 Citrus medica Species 0.000 title claims abstract description 20
- 238000012203 high throughput assay Methods 0.000 title claims abstract description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 198
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 33
- 239000007788 liquid Substances 0.000 claims abstract description 32
- 238000012360 testing method Methods 0.000 claims abstract description 30
- 239000012295 chemical reaction liquid Substances 0.000 claims abstract description 29
- 238000002835 absorbance Methods 0.000 claims abstract description 22
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims abstract description 14
- 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 abstract description 14
- 102000004317 Lyases Human genes 0.000 claims abstract description 11
- 108090000856 Lyases Proteins 0.000 claims abstract description 11
- 210000002700 urine Anatomy 0.000 claims description 29
- 239000000243 solution Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 238000003556 assay Methods 0.000 claims description 13
- 239000012153 distilled water Substances 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 239000007999 glycylglycine buffer Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 5
- 235000015203 fruit juice Nutrition 0.000 claims description 3
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- 230000035484 reaction time Effects 0.000 claims description 2
- 238000003780 insertion Methods 0.000 abstract description 3
- 230000037431 insertion Effects 0.000 abstract description 3
- 108020005199 Dehydrogenases Proteins 0.000 abstract 1
- 102000013460 Malate Dehydrogenase Human genes 0.000 abstract 1
- 108010026217 Malate Dehydrogenase Proteins 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 57
- 238000001514 detection method Methods 0.000 description 18
- 238000006243 chemical reaction Methods 0.000 description 11
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 8
- 235000013305 food Nutrition 0.000 description 7
- 235000002710 Ilex cornuta Nutrition 0.000 description 6
- 241001310146 Ilex cornuta Species 0.000 description 6
- 235000010326 Osmanthus heterophyllus Nutrition 0.000 description 6
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 230000000875 corresponding effect Effects 0.000 description 6
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 6
- 239000011575 calcium Substances 0.000 description 5
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
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- 239000013062 quality control Sample Substances 0.000 description 3
- 230000002485 urinary effect Effects 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 208000015924 Lithiasis Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- QXDMQSPYEZFLGF-UHFFFAOYSA-L calcium oxalate Chemical compound [Ca+2].[O-]C(=O)C([O-])=O QXDMQSPYEZFLGF-UHFFFAOYSA-L 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
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- 238000011835 investigation Methods 0.000 description 2
- 150000002500 ions Chemical group 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
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- 238000011282 treatment Methods 0.000 description 2
- WXHLLJAMBQLULT-UHFFFAOYSA-N 2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-yl]amino]-n-(2-methyl-6-sulfanylphenyl)-1,3-thiazole-5-carboxamide;hydrate Chemical compound O.C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1S WXHLLJAMBQLULT-UHFFFAOYSA-N 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010021703 Indifference Diseases 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- 101000745256 Rhodotorula graminis (S)-mandelate dehydrogenase, mitochondrial Proteins 0.000 description 1
- 101001004672 Schizosaccharomyces pombe (strain 972 / ATCC 24843) Probable L-lactate dehydrogenase Proteins 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- -1 acyl glycine Chemical compound 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 230000001458 anti-acid effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000010165 autogamy Effects 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 235000012970 cakes Nutrition 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 235000013877 carbamide Nutrition 0.000 description 1
- 238000005266 casting Methods 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 235000020965 cold beverage Nutrition 0.000 description 1
- 239000008139 complexing agent Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
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- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
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- 238000002845 discoloration Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000003349 gelling agent Substances 0.000 description 1
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical class [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 235000015094 jam Nutrition 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 229940116298 l- malic acid Drugs 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
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- 230000009965 odorless effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000007833 oxidative deamination reaction Methods 0.000 description 1
- 239000003002 pH adjusting agent Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002133 sample digestion Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
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- 239000004575 stone Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 235000012976 tarts Nutrition 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
Abstract
The present invention relates to a kind of high throughput assay method of citron acid content in solution, including：Prepare citric acid standard items；Prepare the enzyme digestion reaction liquid A liquid containing L malic dehydrogenases, L lactic dehydrogenases and NADH；Prepare the enzyme digestion reaction liquid B liquid containing citric acid lyases；Enzyme digestion reaction liquid A liquid is added in each hole of porous ultraviolet microwell plate；Citric acid standard items and testing sample are separately added into each hole of porous ultraviolet microwell plate, is placed on ELIASA and reads the absorbance A of each citric acid standard items microporeSTD1And the absorbance A of testing sample microporeSP1；Enzyme digestion reaction liquid B liquid is added in each hole of porous ultraviolet microwell plate, is placed on ELIASA and reads the absorbance A of each citric acid standard items microporeSTD2And the absorbance A of testing sample microporeSP2；By the A of each citric acid standard items microporeSTD1And ASTD2The concentration returns of difference and each citric acid standard items be fitted to build standard curve；By ASP1And ASP2Difference insertion standard curve, obtain corresponding concentration.
Description
Technical field
The invention belongs to biochemical analysis and field of medical examination, and in particular to the high flux of citron acid content in a kind of solution
Assay method.
Background technology
Chinese holly edge acid is also known as citric acid (H3cit), is a kind of important organic acid, clear crystal, often contains a molecular crystalline water,
It is odorless, there is very strong tart flavour, it is soluble in water.The purposes of Chinese holly edge acid is very extensive, for food industry account for the 75% of output with
On, can as the acid of food, antioxidant, pH adjusting agent, in the food such as cold drink, jam, fruit and cake.
10% or so is accounted for for medical industry, is mainly used as anticoagulant, antiacid, flavouring, cosmetics etc..For chemical industry etc.
15% or so is accounted for, as buffer, complexing agent, metal cleaner, mordant, gelling agent, toner etc..In electronics, weaving, stone
There is very broad application value in the industrial circles such as oil, leather, building, photography, plastics, casting and ceramics.In view of Chinese holly edge is sour
Value so is widely applied, then is detected as citric acid required for multiple industries.
In medical domain, citric acid is again healthy closely bound up with the mankind.If citric acid and its salt are in calculi in urinary system
Formation and prevention in play a very important role.Citric acid, it is with 3 carboxyls and 1 the distance between hydroxyl, carboxyl
3 C-C key lengths, in the solution, H3cit can be with Ca2+Ion forms the chelate containing 1 five-membered ring and 1 hexatomic ring, its
Stability constant is 4.79 × 104.H3cit and its salt and Ca in urine2+Ion forms the highly soluble citric acid for being difficult to dissociate
After calcium, it can be excreted with urine, so as to reduce the concentration of urinary calcium and urinate the saturation degree of calcium oxalate crystal growth in healthy and calcium phosphate.Calculus is suffered from
In the urine of person, H3cit concentration reduces, and this causes it to urinate the rise of intermediate ion calcium fraction, CaOXaThe degree of supersaturation of (calcium oxalate) increases
Big formed contains calcium calculus.Research shows that some urinary systems calcium salt calculus patient often has hypocitruria disease.Therefore detection urine
Middle citron acid content is assessed for the effect of medicine of the diagnosis of lithiasis and some treatment lithiasises all has important value.
24h is urinated in biochemical indicator at present, and the detection of inorganic ions is relatively simple, and it is then organic acid to urinate citric acid, content
It is low, check that difficulty is larger, existing common method is colorimetric method and titration both at home and abroad at present, and the two methods sensitivity is low, difficult
To carry out accurate quantitative analysis to oxalic acid micro in urine sample, citric acid, and conventional discoloration acid colorimetric method need to be with dense
Sulfuric acid, chemical colorimetry need to use bromine, have an impact to staff and environment, and both measuring method division operations are complicated outer, also
A sample, the flux finite for being detected, disposably being detected single successively of sample.Before HPLC method determination samples,
Urine need to be pre-processed and be intended to prevent vitamin C etc. from converting under neutral or alkaline environment, it is necessary to add concentrated hydrochloric acid acidifying
Measurement result is influenceed for oxalic acid, also to add sulfosalicylic acid can remove albumen a small amount of in urine sample, reduce the interference of miscellaneous peak,
The preprocessing process complexity of sample is cumbersome, and the addition of concentrated hydrochloric acid needs to operate in the specific facilities such as ventilating kitchen, otherwise easily
Volatilization stimulates body effect's environment, and (HPLC methods determine the evaluation of methodology of oxalate crystal growth in healthy urine and citron acid content simultaneously and clinic should
With).Though in the market has kit to be based on enzyme digestion reaction, measure reacts ultraviolet light absorption shading value one by one again, but the kit needs
By means of cuvette, the consumption that cuvette detects not only sample is all very big, uneconomical, somewhat expensive, but also is to utilize
The method that formula calculates sample result one by one detects to citric acid, operates time-consuming inconvenience, and efficiency is also low.In a word, it is above-mentioned this
There is certain deficiency, application lacks in terms of medical science and biochemical investigation facilitates a little existing citric acid detection methods
Property, practical operation difficulty is big.
The content of the invention
In view of the above-mentioned problems, it is an object of the invention to provide a kind of more convenient, high efficiency, the economic solution for saving sample
The detection method of middle citron acid content.
The present invention provides a kind of high throughput assay method of citron acid content in solution, comprises the following steps：
(1) the citric acid standard items of various concentrations gradient are prepared；
(2) the enzyme digestion reaction liquid A containing EC 1.1.1.39, LDH and NADH is prepared
Liquid；
(3) the enzyme digestion reaction liquid B liquid containing citric acid lyases is prepared；
(4) the enzyme digestion reaction liquid A liquid is added in each hole of porous ultraviolet microwell plate；
(5) citric acid standard items and more than one testing sample are separately added into each hole of the porous ultraviolet microwell plate, instead
After a period of time, it should be placed on ELIASA and the absorbance of each citric acid standard items micropore is read under specific UV wavelength
ASTD1And the absorbance A of testing sample microporeSP1；
(6) enzyme digestion reaction liquid B liquid is added in each hole of the porous ultraviolet microwell plate, after reacting a period of time, is placed in enzyme
The absorbance A of each citric acid standard items micropore is read on mark instrument under the specific UV wavelengthSTD2And testing sample is micro-
The absorbance A in holeSP2；
(7) by the A of each citric acid standard items microporeSTD1And ASTD2The concentration of difference DELTA A and each citric acid standard items returned
Return fitting to build standard curve；
(8) by ASP1And ASP2Difference insert the standard curve, obtain corresponding concentration, you can obtain the citron of testing sample
Acid content.
The present invention is based on citritase cracking principle, innovatively real using porous ultraviolet microwell plate combination with standard curve method
Existing multiple samples detect simultaneously, synchronous to produce multiple sample results.Based on microwell plate, then amount of samples is few, the enzyme separately reacted
Demand volume is also accordingly reduced, and greatlys save financial cost, economy.It is moreover, high the invention enables the detection of citric acid
Flux, the measure of more than one sample citron acid content can be carried out simultaneously, inspection is greatlyd save than cuvette method, titration etc.
The time is surveyed, and is different from equation, the present invention can disposably produce multiple sample results based on standard curve, than equation section
About detection time and calculate time.Sample pre-treatments are also more simple and convenient than HPLC detection method, and this method to sample without carrying out
Specially treated, greatly reduce cumbersome sample handling procedure.
It is preferred that in step (1), compound concentration scope is 50000~1000ng/mL citric acid standard items more than 5,
Preferably, compound concentration be respectively 40300,32240,24180,16120,8060,4030,2015,1007.5ng/mL citron
Sour standard items quantitatively use standard curve to build.It is preferred that solution to be measured is urine, fruit juice, wine, the homogeneous solution of solid food
In any one, it is preferable that the solution to be measured is urine, and the testing sample is that urine to be measured is diluted with water and obtains,
Preferably, 10 times are at least diluted；
The citron acid content of solution to be measured is multiplied by extension rate by the concentration of gained in step (8) and obtained.The present invention need not treat
Survey solution (such as urine) sample and carry out specially treated, need to be only diluted with water, greatly reduce cumbersome sample treatment step
Suddenly.The preprocessing process of sample can be optimized, make the detection of citric acid more convenient and simplify.
It is preferred that in step (2), in enzyme digestion reaction liquid A liquid, solvent is glycylglycine buffers, and L MALIC ACID takes off
The concentration of hydrogen enzyme is 10~15U/mL, and the concentration of LDH is 20~30U/mL, NADH
Concentration is 0.3~0.6g/L.
It is preferred that in step (3), in enzyme digestion reaction liquid B liquid, solvent is water, preferably distilled water, citric acid lyases
Concentration is 30~50U/mL.
It is preferred that in step (4), the dosage of enzyme digestion reaction liquid A liquid is below 100 microlitres/hole；In step (5), citric acid
The dosage of standard items and/or testing sample is below 200 microlitres/hole；In step (6), the dosage of enzyme digestion reaction liquid B liquid is micro- for 2
Below liter/hole.In the present invention, based on microwell plate, then amount of samples is few, and a microwell plate at most needs 200 microlitres of sample size,
The demand volume of the enzyme separately reacted is also accordingly reduced, and the amount for often surveying reagent needed for a sample is only the 1/ of spectrophotometer method
10, greatly save financial cost, economy.
It is preferred that in step (5), the quantity of testing sample is more than 30.The invention enables the detection high pass of citric acid
Amount, the measure of the sample citron acid content of more than 30 can be carried out simultaneously.
It is preferred that the specific UV wavelength is 320~360nm, preferably 340nm.Citric acid is in citric acid lyases
(CL) oxaloacetic acid and acetate are produced under catalysis；Oxidative deamination generation pyruvic acid easily occurs for oxaloacetic acid；Oxaloacetic acid, third
Ketone acid and NADH react under EC 1.1.1.39 and LDH catalysis, the NADH reactions being oxidized in reaction
Caused signal can be detected under the conditions of 320~360nm (preferably 340nm) by instrument.
It is preferred that the porous ultraviolet microwell plate is the ultraviolet microwell plate in 96 holes.
It is preferred that in step (5) and/or step (6), the reaction time is more than 5 minutes.
It is preferred that step (7) and step (8) are carried out using microwell plate data acquisition and analysis software, the software is preferred
For SoftMax softwares.Using microwell plate data acquisition and analysis software can rapidly one-time calculation produces multiple (examples automatically
Such as more than 30) testing result, save detection time and calculate the time.
The present invention has advantages below：
1) consumption of sample dosage and reaction reagent is saved,
2) optimize the preprocessing process of sample, make the detection of citric acid more convenient and simplify,
3) higher sensitivity is lifted, possesses lower lower limit of quantitation (reaching ng levels),
4) detection efficiency is improved, high pass quantifies, and disposable multiple samples detect simultaneously, while produce the result of multiple samples, carry
The detection efficiency of high practical application.
Brief description of the drawings
Fig. 1 is the canonical plotting of an example of the present invention.
Embodiment
The present invention is further illustrated below in conjunction with accompanying drawing and following embodiments, it should be appreciated that accompanying drawing and following embodiments
The present invention is merely to illustrate, is not intended to limit the present invention.
The invention provides a kind of high throughput assay method of citron acid content in solution.The principle that the present invention is utilized
For：Citric acid produces oxaloacetic acid and acetate under citric acid lyases (CL) catalysis；It is de- that oxidation easily occurs for oxaloacetic acid
Carboxylic generates pyruvic acid；Oxaloacetic acid, pyruvic acid and NADH (NADH) are in EC 1.1.1.39 and L-
Reacted under lactate dehydrogenase catalyzed, signal caused by the NADH reactions being oxidized in reaction can be in specific UV wavelength
Detected under (such as 320~360nm, preferably 340nm) by instrument.In the present invention, as long as solution to be measured contains the molten of citric acid
Liquid, including but not limited to urine, fruit juice, wine, the homogeneous solution etc. of solid food.
In an embodiment of the present invention, the ELIASA that can be detected based on microwell plate progress under the conditions of ultraviolet is employed, is matched somebody with somebody
The citric acid standard items of various concentrations gradient processed, using detected signal value of the citric acid standard items under specific UV wavelength with it is right
The theoretical concentration answered generates Log-log mark under microwell plate data acquisition and analysis software (such as SoftMax softwares)
Directrix curve, detection signal insertion standard curve can be by microwell plate Data acquisition and issuance under specific UV wavelength for testing sample
The concentration results of the automatic one-time calculation all samples of software (such as SoftMax softwares).So flux is big, and detection is fast, enzyme with
Amount of samples is few.
Multiple samples are realized present invention employs porous ultraviolet microwell plate combination with standard curve method while are detected, it is synchronous to produce
Multiple sample results.Based on microwell plate, then amount of samples is few, and a microwell plate at most needs 200 microlitres of sample size, another reaction
The demand volume of enzyme also accordingly reduce, the amount for often surveying reagent needed for a sample is only the 1/10 of spectrophotometer method, significantly
Financial cost economy is saved.
Here, " porous ultraviolet microwell plate " refer to containing it is multiple it is ultraviolet can micropore thoroughly microwell plate, hole count is, for example, 96,
384 etc..In one example, the UV-96 orifice plates of CORNING companies production can be used.Also ultraviolet 96 can be surveyed using other
Hole microwell plate.
In the present invention, ELIASA can be all-wave length ELIASA or other can at least survey the ultraviolet ELIASAs of 340nm.
The preparation of enzyme digestion reaction liquid
In the present invention, Chinese holly is obtained using the absorbance difference before and after citric acid enzyme digestion reaction and the corresponding relation of citron acid concentration
Rafter acid concentration.In the present invention, use the enzyme digestion reaction liquid A liquid containing EC 1.1.1.39, LDH and NADH and
Enzyme digestion reaction liquid B liquid containing citric acid lyases.Specifically, bioassay standard product, testing sample and enzyme digestion reaction liquid A first
Reacted first absorbance of liquid, then at wherein addition enzyme digestion reaction liquid B liquid, and determine reacted second absorbance.
Using the difference of the first absorbance and the second absorbance as above-mentioned absorbance difference.
In enzyme digestion reaction liquid A liquid, solvent can be glycylglycine buffers, and the concentration of EC 1.1.1.39 can be 10
~15U/mL, the concentration of LDH can be 20~30U/mL, and NADH concentration can be 0.3~0.6g/L.Wherein, sweet ammonia
The pH of acyl glycine buffer can be 7.8.Glycylglycine buffers can autogamy also can purchase, such as purchased from good intelligent biology.
In one example, glycylglycine buffers (PH7.8) are prepared by the following method：7.13g glycylglycines are dissolved in
In about 70mL distilled waters, pH to 7.8 is modulated with NaOH, adds about 8mg ZnCl2, finally plus distilled water is settled to 100mL.Its
It can be stored 1 month at 2-8 DEG C.EC 1.1.1.39, LDH, NADH are purchased from commercial (such as Sigma companies).
In one example, enzyme digestion reaction liquid A liquid is prepared by the following method：By about 140U EC 1.1.1.39 (L-MDH), 280U
LDH (L-LDH) and 5mg NADH be dissolved in glycylglycine buffers, final volume 12mL.Its
2-8 DEG C can store two weeks.
In enzyme digestion reaction liquid B liquid, solvent can be water, preferably distilled water, the concentration of citric acid lyases can be 30~
50U/mL.Citric acid lyases is purchased from commercial (such as Sigma companies).In one example, enzyme digestion reaction liquid B liquid passes through as follows
Method is prepared：12U lyophilized citric acid lyases is dissolved in 0.3mL distilled water.It can be stored 14 days at 2-8 DEG C.
The preparation of standard items
Multiple citric acid standard items of various concentrations gradient are prepared, for building standard curve.For example, can be with compound concentration model
Enclose the citric acid standard items more than 5 for 50000~1000ng/mL.In one example, lyophilized citric acid standard items are used double
After steaming water dissolving, 40300 are diluted to using distilled water, 32240,24180,16120,8060,4030,2015,1007.5ng/mL
Standard curve is quantitatively used with structure.Citric acid standard items are purchased from National Institute for Food and Drugs Control.
Urine sample pre-treatment
In the present invention, without carrying out specially treated to urine sample, it need to be only diluted with water.In one example, urine
Sample is diluted with distilled water or distilled water before testing.For example, sample is at least diluted with water 10 times, if Chinese holly in sample
The estimation of rafter acid concentration is higher, can carry out the dilution more than 10 times.Here, with urine as an example, however, it is understood that as above institute
State, the present invention can be not only used for surveying urine sample, can also survey other samples containing citric acid, such as contain Chinese holly in field of food
Solution of rafter acid etc..That is, the present invention can apply not only in clinical field in field of food etc..When solution to be measured
For other samples containing citric acid when, solution to be measured also can according to circumstances be diluted with water.
Hereinafter, the specific detecting step in an embodiment of the present invention is illustrated.
Add substrate and sample
The ultraviolet microwell plate in 96 holes is taken out, 100 μ L enzyme reaction solution A liquid are added in the hole needed to use being pre-designed, are then pressed
Standard items, quality-control sample and testing sample, each 200 μ L/ holes, piping and druming are added on the position of the corresponding aperture preplaned according to microwell plate
Mix, be placed in and react at least 5min at room temperature, carry out enzymatic lysis reaction abundant.Here, quality-control sample is to be used to verify this hair
The measurement stability and accuracy of bright method, not measure institute is required.In addition, injection volume can be according to the hole of ultraviolet microwell plate
Count and difference (i.e. different and different according to pore volume), that is to say, that at least within the open ended amount in hole.For example, if with
384 orifice plates, corresponding injection volume can be respectively the 1/4 of 96 orifice plates.
Upper machine-readable plate
The orifice plates of complete UV -96 will be reacted and be placed on ELIASA the OD that each micropore containing standard items is read under the conditions of 340nm
(absorbance) value, and calculate average OD (absorbance) the values A between multiple holes1.In addition, read the OD values of the micropore containing testing sample
ASP1。
Add enzyme
Take out the ultraviolet microwell plate in 96 holes then according to corresponding to microwell plate sample arrangement table on position add enzyme reaction solution B liquid, each 2
μ L/ holes, piping and druming mix, are placed in and react at least 5min at room temperature.
Upper machine-readable plate
Complete 96 hole microwell plate plate will be reacted it is placed on ELIASA under the conditions of 340nm and reads the OD of each micropore containing standard items
(absorbance) value, and calculate average OD (absorbance) the values A between multiple holes2.In addition, read the OD values of the micropore containing testing sample
ASP2。
Data analysis
The component of standard curve
Handled using SoftMax softwares, with Log-log parameters to each concentration point instrumental response value enzyme digestion reaction of standard items before
Difference DELTA A (i.e. A afterwards1- A2) carry out regression fit with concentration relationship and determine standard curve.Fig. 1 shows an example of the present invention
Canonical plotting.As can be seen that the concentration of Δ A and mark song point is into extraordinary linear relation.
Obtain the concentration of testing sample
The concentration value of testing sample can be by difference (A before and after its instrumental response value enzyme digestion reactionSP1- ASP2) insertion standard curve, it is soft
Part is calculated automatically, is finally multiplied by corresponding extension rate to obtain final sample concentration value.
It is (including multiple for the detection of the standard items that build standard curve and the detection of testing sample in present embodiment
The detection of testing sample) it can be carried out simultaneously on a porous ultraviolet microwell plate, can more it save the time.
The checking of method performance
Detect the robustness of sample concentration standard curve fit
The analysis of 6 different batches has been carried out continuously using above method flow, the investigations of analyses batch different to 6 compare (see
Table 1), between 95.9%-104.0%, description standard curve matching is sane for the degree of accuracy between its batch, and model of fit selection is proper,
The measurement that citron acid concentration is carried out using mark song method is suitable；
The comparison of the different batches standard curve of table 1
The degree of accuracy of method is evaluated with accuracy
Using citric acid standard items, the sample of 5 concentration knowns, i.e. SA1 (40300.0ng/mL), SA2 have been prepared
(30225.0ng/mL), SA3 (12090.0ng/mL), SA4 (3022.5ng/mL), SA5 (1511.25ng/mL) carry out repeating survey
It is fixed with determine the durability of method, the degree of accuracy and the checking of precision by not on the same day it is interior by different analysis personnel different
The measure of at least six difference analysis batch is carried out on instrument, 3 sets of concentration known samples so independently prepared are tested in each analysis batch
Product, in last statistical estimation batch with criticize between the degree of accuracy and accuracy (being shown in Table 2 and table 3)；
2 batches of interior degrees of accuracy of table and precision
3 batches of degrees of accuracy of table and precision
6 analysis batch in each concentration level concentration known sample batch in the degree of accuracy be respectively：SA1 is 84.9%~
105.8%th, SA2 is that 83.2%~92.8%, SA3 is that 87.6%~91.6%, SA4 is that 92.1%~103.1%, SA5 is
97.2%~102.8%；Withinrun precision is respectively：SA1 is that 0.7%~13.2%, SA2 is that 0.4%~5.8%, SA3 is
1.0%~4.4%, SA4 is that 1.7%~19.1%, SA5 is 1.0%~14%, and details are shown in Table 2.It is all dense in 6 analyses batch
The horizontal quality-control sample of degree batch between the degree of accuracy be：In the range of 86.7%~97.8%；Betweenrun precision is：3.3%~16%
In the range of, as a result illustrate the concentration known sample of each concentration level in 6 analyses batch batch between the degree of accuracy and precision all accord with
Splice grafting receives standard, details result table 3.As a result prove that the measurement between being criticized in this method batch to sample is all extremely stable accurate.
The selectivity evaluation of citron acidity test in human urine
To investigate influence of other main components to this citric acid content assaying method in urine, to determine the single-minded of the method
Property.Choose other similar with citric acid structure in urine and on the influential main component of citric acid assay, manually match somebody with somebody
Make a urine.Oxalic acid, lactic acid, urea, uric acid, ammonium chloride respectively about 0.1g are weighed respectively, is placed in the small graduated cylinders of same 10ml, are used
Water dissolves and is diluted to scale, shaking, survey pH be 1.7, with sodium hydroxide solution adjust pH to 6.5, filtering, as prepare urine,
Then the citric acid standard items that 30000ng/mL is added in urine are simulated at this, is measured by above-mentioned detecting step, investigates this
Method is to citric acid selectivity in human urine.Data are shown in Table 4：
The selectivity data of the citric acid assay of table 4
Measured concentration is that the rate of recovery is 95.9% to theoretical concentration, and as shown by data, other compositions do not have to the assay of citric acid
There is interference, this method selectivity is preferable, not other composition influences in by urine.
The contrast of the method and existing method of the present invention
It is respectively 40ug/L, 20ug/L, 10ug/L, 5ug/L, 2.5ug/L, 1.540ug/ that six theoretical concentrations are prepared in urine
L sample merges equation (abbreviation method 1, referring to prior art literature with ultraviolet specrophotometer respectively：Citric acid it is ultraviolet
Spectrophotometry, Jiangsu food with fermentation, the 1996, the 1st phase) merge with 96 microwell plates mark song method (i.e. the present invention method,
Abbreviation method 2) detected, as a result as shown in table 5：
The contrast of the result of 5 method of table 1 and the result of method 2
The results relevance of two methods is compared using SPSS softwares, the results are shown in Table 6：
The results relevance of the method 1 of table 6 and method 2
* are significantly correlated on .01 horizontal (bilateral).
As can be seen that two methods are significantly correlated to the testing result of sample, indifference.
Claims (10)
1. a kind of high throughput assay method of citron acid content in solution, it is characterised in that comprise the following steps：
（1）Prepare the citric acid standard items of various concentrations gradient；
（2）Prepare the enzyme digestion reaction liquid A containing EC 1.1.1.39, LDH and NADH
Liquid；
（3）Prepare the enzyme digestion reaction liquid B liquid containing citric acid lyases；
（4）The enzyme digestion reaction liquid A liquid is added in each hole of porous ultraviolet microwell plate；
（5）Citric acid standard items and more than one testing sample are separately added into each hole of the porous ultraviolet microwell plate, instead
After a period of time, it should be placed on ELIASA and the absorbance of each citric acid standard items micropore is read under specific UV wavelength
ASTD1And the absorbance A of testing sample microporeSP1；
（6）Enzyme digestion reaction liquid B liquid is added in each hole of the porous ultraviolet microwell plate, after reacting a period of time, is placed in enzyme
The absorbance A of each citric acid standard items micropore is read on mark instrument under the specific UV wavelengthSTD2And testing sample is micro-
The absorbance A in holeSP2；
（7）By the A of each citric acid standard items microporeSTD1And ASTD2Difference DELTA A and each citric acid standard items concentration returned
It is fitted to build standard curve；
（8）By ASP1And ASP2Difference insert the standard curve, obtain corresponding concentration, you can obtain the citron of testing sample
Acid content.
2. assay method according to claim 1, it is characterised in that step（1）In, compound concentration scope be 50000~
1000ng/mL citric acid standard items more than 5, it is preferable that compound concentration is respectively 40300,32240,24180,16120,
8060th, 4030,2015,1007.5ng/mL citric acid standard items quantitatively use standard curve to build.
3. assay method according to claim 1 or 2, it is characterised in that
Solution to be measured be urine, fruit juice, wine, solid food solution in any one, it is preferable that the solution to be measured be urine
Liquid, the testing sample are that urine to be measured is diluted with water and obtains, it is preferable that at least dilute 10 times；
The citron acid content of solution to be measured is by step（8）The concentration of middle gained is multiplied by extension rate and obtained.
4. assay method according to any one of claim 1 to 3, it is characterised in that step（2）In, enzyme digestion reaction liquid A
In liquid, solvent is glycylglycine buffers, and the concentration of EC 1.1.1.39 is 10~15U/mL, LDH
Concentration is 20~30 U/mL, and the concentration of NADH is 0.3~0.6g/L.
5. assay method according to any one of claim 1 to 4, it is characterised in that step（3）In, enzyme digestion reaction liquid B
In liquid, solvent is water, preferably distilled water, and the concentration of citric acid lyases is 30~50U/mL.
6. assay method according to any one of claim 1 to 5, it is characterised in that
The porous ultraviolet microwell plate is the ultraviolet microwell plate in 96 holes,
Step（4）In, the dosage of enzyme digestion reaction liquid A liquid is below 100 microlitres/hole；
Step（5）In, the dosage of citric acid standard items and/or testing sample is below 200 microlitres/hole；
Step（6）In, the dosage of enzyme digestion reaction liquid B liquid is below 2 microlitres/hole.
7. assay method according to any one of claim 1 to 6, it is characterised in that step（5）In, testing sample
Quantity is more than 30.
8. assay method according to any one of claim 1 to 7, it is characterised in that the specific UV wavelength is 320
~360nm, preferably 340nm.
9. assay method according to any one of claim 1 to 8, it is characterised in that step（5）And/or step（6）
In, the reaction time is more than 5 minutes.
10. assay method according to any one of claim 1 to 9, it is characterised in that step（7）And step（8）Using
Microwell plate data acquisition and analysis software is carried out, and the software is preferably SoftMax softwares.
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| CN101324505A (en) * | 2007-06-13 | 2008-12-17 | 苏州艾杰生物科技有限公司 | Method for determining citric acid concentration and citric acid determining reagent kit |
| CN106480164A (en) * | 2016-10-25 | 2017-03-08 | 深圳市澳华农牧有限公司 | A kind of method of high flux screening lactein |
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Application publication date: 20171222 |