CN107084938A - The alkaline phosphatase assay method of oxidizing ferment is simulated based on chitosan platinum - Google Patents
The alkaline phosphatase assay method of oxidizing ferment is simulated based on chitosan platinum Download PDFInfo
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- CN107084938A CN107084938A CN201710298329.4A CN201710298329A CN107084938A CN 107084938 A CN107084938 A CN 107084938A CN 201710298329 A CN201710298329 A CN 201710298329A CN 107084938 A CN107084938 A CN 107084938A
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- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 title claims abstract description 68
- 229910052697 platinum Inorganic materials 0.000 title claims abstract description 64
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 48
- 230000001590 oxidative effect Effects 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000008940 Alkaline Phosphatase assay kit Methods 0.000 title claims abstract description 11
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims abstract description 66
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims abstract description 66
- 238000004088 simulation Methods 0.000 claims abstract description 47
- 238000006243 chemical reaction Methods 0.000 claims abstract description 32
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 31
- -1 ascorbic acid phosphoric acid esters Chemical class 0.000 claims abstract description 27
- 229960005070 ascorbic acid Drugs 0.000 claims abstract description 26
- 235000010323 ascorbic acid Nutrition 0.000 claims abstract description 21
- 239000011668 ascorbic acid Substances 0.000 claims abstract description 21
- 238000002835 absorbance Methods 0.000 claims abstract description 20
- 239000003112 inhibitor Substances 0.000 claims abstract description 14
- 230000008859 change Effects 0.000 claims abstract description 12
- 238000000862 absorption spectrum Methods 0.000 claims abstract description 8
- 238000001514 detection method Methods 0.000 claims abstract description 7
- CMZYGFLOKOQMKF-UHFFFAOYSA-N 1-(3,5-dimethylphenyl)-3,5-dimethylbenzene Chemical group CC1=CC(C)=CC(C=2C=C(C)C=C(C)C=2)=C1 CMZYGFLOKOQMKF-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 110
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 34
- 102000004190 Enzymes Human genes 0.000 claims description 30
- 108090000790 Enzymes Proteins 0.000 claims description 30
- 238000003756 stirring Methods 0.000 claims description 23
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- 230000003647 oxidation Effects 0.000 claims description 19
- 238000007254 oxidation reaction Methods 0.000 claims description 19
- 239000002253 acid Substances 0.000 claims description 18
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 14
- 239000012279 sodium borohydride Substances 0.000 claims description 14
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 14
- 239000007853 buffer solution Substances 0.000 claims description 12
- 210000002966 serum Anatomy 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 11
- 239000008351 acetate buffer Substances 0.000 claims description 10
- 150000007513 acids Chemical class 0.000 claims description 8
- 230000009467 reduction Effects 0.000 claims description 7
- 239000003381 stabilizer Substances 0.000 claims description 7
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- 239000002211 L-ascorbic acid Substances 0.000 claims description 5
- 235000000069 L-ascorbic acid Nutrition 0.000 claims description 5
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- 150000008547 L-phenylalanines Chemical class 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 238000003556 assay Methods 0.000 claims description 4
- 239000007795 chemical reaction product Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 3
- 235000008729 phenylalanine Nutrition 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 239000000052 vinegar Substances 0.000 claims description 3
- 235000021419 vinegar Nutrition 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 238000006911 enzymatic reaction Methods 0.000 claims description 2
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 claims description 2
- GDSOZVZXVXTJMI-SNAWJCMRSA-N (e)-1-methylbut-1-ene-1,2,4-tricarboxylic acid Chemical compound OC(=O)C(/C)=C(C(O)=O)\CCC(O)=O GDSOZVZXVXTJMI-SNAWJCMRSA-N 0.000 claims 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims 1
- 239000005864 Sulphur Substances 0.000 claims 1
- 150000002148 esters Chemical class 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 230000007062 hydrolysis Effects 0.000 abstract description 2
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract 1
- 230000003197 catalytic effect Effects 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 23
- 102000004316 Oxidoreductases Human genes 0.000 description 5
- 108090000854 Oxidoreductases Proteins 0.000 description 5
- 238000011161 development Methods 0.000 description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- RUAXWVDEYJEWRY-UHFFFAOYSA-N 4-(4-aminophenyl)aniline;dihydrochloride Chemical compound Cl.Cl.C1=CC(N)=CC=C1C1=CC=C(N)C=C1 RUAXWVDEYJEWRY-UHFFFAOYSA-N 0.000 description 2
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- ALLIZEAXNXSFGD-UHFFFAOYSA-N 1-methyl-2-phenylbenzene Chemical group CC1=CC=CC=C1C1=CC=CC=C1 ALLIZEAXNXSFGD-UHFFFAOYSA-N 0.000 description 1
- 102000012440 Acetylcholinesterase Human genes 0.000 description 1
- 108010022752 Acetylcholinesterase Proteins 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 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
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- NYNRGZULARUZCC-UHFFFAOYSA-N [4-(4-azaniumyl-3,5-dimethylphenyl)-2,6-dimethylphenyl]azanium;dichloride Chemical compound Cl.Cl.CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 NYNRGZULARUZCC-UHFFFAOYSA-N 0.000 description 1
- QYSYEILYXGRUOM-UHFFFAOYSA-N [Cl].[Pt] Chemical compound [Cl].[Pt] QYSYEILYXGRUOM-UHFFFAOYSA-N 0.000 description 1
- 159000000021 acetate salts Chemical class 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 229940022698 acetylcholinesterase Drugs 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- MMCPOSDMTGQNKG-UHFFFAOYSA-N anilinium chloride Chemical compound Cl.NC1=CC=CC=C1 MMCPOSDMTGQNKG-UHFFFAOYSA-N 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 229910052796 boron Inorganic materials 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- KDLRVYVGXIQJDK-AWPVFWJPSA-N clindamycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 description 1
- 229960002227 clindamycin Drugs 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 239000000686 essence Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- NICDRCVJGXLKSF-UHFFFAOYSA-N nitric acid;trihydrochloride Chemical compound Cl.Cl.Cl.O[N+]([O-])=O NICDRCVJGXLKSF-UHFFFAOYSA-N 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 150000003014 phosphoric acid esters Chemical class 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 229910000104 sodium hydride Inorganic materials 0.000 description 1
- 239000012312 sodium hydride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
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- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a kind of alkaline phosphatase assay method that oxidizing ferment is simulated based on chitosan platinum, it is characterized in that generating ascorbic acid using alkaline phosphatase enzyme hydrolysis ascorbic acid phosphoric acid esters, influence chitosan platinum simulation oxidizing ferment/3,3 ', 5,5 ' tetramethyl biphenyl amine hydrochlorate Catalytic color reaction systems, the change of binding soln color and ultra-violet absorption spectrum feature is directly used in the measure of alkaline phosphatase and its inhibitor content.In the range of 0.5 ~ 20 U/L, absorbance A450Linear with alkaline phosphatase concentration, detection is limited to 0.34 U/L.Easy to operate, sensitivity of the invention is high, and the measure of environment and life science system alkaline phosphatase and its inhibitor content can be applied to as analysis method.
Description
Technical field
The alkaline phosphatase of oxidase catalyzed color development system and its measure of inhibitor are simulated the present invention relates to chitosan-platinum
Method, belongs to analytical chemistry and field of nanometer technology.
Background technology
Alkaline phosphatase is to be distributed widely in the tissue such as human liver, bone, intestines, kidney and placenta to discharge to outside courage through liver
A kind of enzyme, it is the antidiastole of the important indicator for judging some diseases in the liver and gallbladder, especially jaundice.Alkaline phosphatase is used as one
Plant and the dephosphorylized enzyme of correspondence substrate can be removed the phosphate group on substrate molecule by hydrolyzing phosphate monoester, generation
Phosphate anion and free hydroxyl, this kind of substrate include nucleic acid, albumen and biomolecule etc..Because alkaline phosphatase removes phosphoric acid
Change is acted on so that it is widely applied in molecular biology and ELISA, is clinically hydrolyzed using it gram
Woods mycin phosphate, the clindamycin of generation has antibiosis.In immunoreagent product, alkaline phosphatase is by as most normal
One of marker enzyme, compared with horseradish peroxidase, alkaline phosphatase, which is used as marker enzyme, has high stability, high sensitivity
The advantages of.More and more extensive with the utilization of alkaline phosphatase, the detection for alkaline phosphatase also becomes further important.Mesh
Before, China is also relatively single to the species of alkaline phosphatase enzyme assay method, therefore sets up a kind of new and feasible measure alkalescence
The method of phosphatase has important practical significance in scientific research instantly.
The present invention simulates oxidase catalyzed oxygen using ascorbic acid phosphoric acid esters as alkaline phosphatase substrate with chitosan-platinum
It is sensed signal sources to aoxidize TMB hydrochloride color development system, establishes a kind of alkaline phosphorus of colorimetric estimation
The method of sour enzyme.This law is easy to operate, sensitivity is high, strong antijamming capability, favorable reproducibility, is expected to be used for actual clinical diagnosis.
The content of the invention
It is an object of the invention to provide one kind oxidase catalyzed dioxygen oxidation 3,3 ', 5,5 '-four is simulated using chitosan-platinum
Methyl biphenyl amine hydrochlorate color development system, using ascorbic acid phosphoric acid esters as alkaline phosphatase substrate, so as to determine alkaline phosphatase
And its method for inhibitor.
To achieve these goals, the present invention uses following technical scheme：
The alkaline phosphatase assay method of the present invention that oxidizing ferment is simulated based on chitosan-platinum, it is characterized in that in Vitamin C
TMB HCI solution and chitosan-platinum are added in acid phosphoric acid ester and alkaline phosphatase enzyme reaction product
Simulation oxidation enzyme solutions, add sulfuric acid solution terminating reaction, according to the change of solution colour and ultra-violet absorption spectrum feature after reaction
Change, the measure for content of alkaline phosphatase；Used chitosan-platinum simulation oxidizing ferment be with chitosan as stabilizer,
Sodium borohydride reduction chloroplatinic acid is obtained, and specific synthesis step is as follows：Weigh 0.1 g chitosans and be dissolved in 50 mL concentration for 1 v/v
In % acetic acid, stirring makes chitosan be completely dissolved the chitosan solution for obtaining that concentration is 0.2 m/v % for 15 minutes, by 2 mL
Concentration is that 10 mmol/L chloroplatinic acids are added to during 47 mL concentration are 0.2 m/v % chitosan solutions, stirs 30 minutes, then by
1 mL concentration is added dropwise to for the 0.2 mol/L sodium borohydride solutions newly prepared and was added within 5 minutes, dark place stirring 90 is placed in
Minute, obtain chitosan-platinum simulation oxidizing ferment.
The described alkaline phosphatase assay method that oxidizing ferment is simulated based on chitosan-platinum, it is characterized in that by 50 μ L concentration
It is 9.10 that the alkaline phosphatase of ascorbic acid phosphoric acid esters and 50 μ L various concentrations for 4 mmol/L, which is added to 200 μ L pH,
Concentration for 10 mmol/L Tris-HCl buffer solutions in, be well mixed after at 37 DEG C react 30 minutes, then sequentially add
632.5 μ L pH are 5, concentration is 50 mmol/L acetate buffer, 50 μ L concentration are 3 mmol/L 3,3 ', 5,5 '-
Tetramethyl biphenyl amide hydrochloride and 17.5 μ L chitosans-platinum simulation oxidation enzyme solutions, it is anti-at 37 DEG C again after being well mixed
Answer 5 minutes, add the sulfuric acid solution terminating reaction that 200 μ L concentration are 2 mol/L, determine the absorbance A of solution450。
The described alkaline phosphatase assay method that oxidizing ferment is simulated based on chitosan-platinum, it is characterized in that 0.5 ~ 20
In the range of U/L, absorbance A450Linear with alkaline phosphatase concentration, detection is limited to 0.34 U/L.
The method of measure human serum alkaline phosphatase of the present invention based on chitosan-platinum simulation oxidizing ferment, it is special
Levy is to comprise the following steps：50 μ L concentration are added to 200 for 4 mmol/L ascorbic acid phosphoric acid esters and 50 μ L human serums
μ L pH be 9.10, concentration for 10 mmol/L Tris-HCl buffer solutions in, be well mixed after at 37 DEG C react 30 minutes,
632.5 μ L pH are sequentially added for 5, concentration is that 50 mmol/L acetate buffer, 50 μ L concentration are the 3 of 3 mmol/L,
3 ', 5,5 '-tetramethyl biphenyl amide hydrochloride and 17.5 μ L chitosans-platinum simulation aoxidize enzyme solutions, exist again after being well mixed
Reacted 5 minutes at 37 DEG C, add the sulfuric acid solution terminating reaction that 200 μ L concentration are 2 mol/L, determine the absorbance of solution
A450, the content of alkaline phosphatase in human serum is calculated according to alkaline phosphatase standard curve；Used chitosan-platinum simulation
Oxidizing ferment is that sodium borohydride reduction chloroplatinic acid is obtained, and specific synthesis step is as follows as stabilizer with chitosan：Weigh 0.1 g
Chitosan is dissolved in the acetic acid that 50 mL concentration are 1 v/v %, and stirring is completely dissolved 15 minutes chitosan to obtain concentration and be
2 mL concentration are that to be added to 47 mL concentration be 0.2 m/v % shells to 10 mmol/L chloroplatinic acids by 0.2 m/v % chitosan solution
In glycan solution, stir 30 minutes, be then added dropwise sodium borohydride solution that 1 mL concentration newly prepared for 0.2 mol/L and
Added within 5 minutes, be placed in dark place and stir 90 minutes, obtain chitosan-platinum simulation oxidizing ferment.
The method of the described measure human serum alkaline phosphatase based on chitosan-platinum simulation oxidizing ferment, it is characterized in that
The rate of recovery that human serum sample determines is 97.1% ~ 102.5%, and relative standard deviation is 1.6 ~ 6.8%.
The assay method of Inhibitors of Alkaline Phosphatase of the present invention based on chitosan-platinum simulation oxidizing ferment, it is special
Levy is that 3,3 ', 5,5 '-four are added in the reaction product of ascorbic acid phosphoric acid esters, alkaline phosphatase and Inhibitors of Alkaline Phosphatase
Methyl biphenyl amide hydrochloride and chitosan-platinum simulation oxidation enzyme solutions, add sulfuric acid solution terminating reaction after reaction, according to
The change of solution colour and ultra-violet absorption spectrum feature, the measure for the inhibiting rate of Inhibitors of Alkaline Phosphatase；It is used
Chitosan-platinum simulation oxidizing ferment is that sodium borohydride reduction chloroplatinic acid is obtained, and specific synthesis step is such as stabilizer with chitosan
Under：Weigh 0.1 g chitosans to be dissolved in the acetic acid that 50 mL concentration are 1 v/v %, stirring is completely dissolved chitosan in 15 minutes
The chitosan solution that concentration is 0.2 m/v % is obtained, is that 10 mmol/L chloroplatinic acids are added to 47 mL concentration by 2 mL concentration
In 0.2 m/v % chitosan solutions, to stir 30 minutes, it is the boron that 0.2 mol/L is newly prepared that 1 mL concentration, which is then added dropwise,
Sodium hydride solution was simultaneously added within 5 minutes, is placed in dark place and is stirred 90 minutes, obtained chitosan-platinum simulation oxidation enzyme solutions.
The assay method of the described Inhibitors of Alkaline Phosphatase based on chitosan-platinum simulation oxidizing ferment, it is characterized in that will
The alkaline phosphatase enzyme solutions that the ascorbic acid phosphoric acid esters and 50 μ L concentration that 50 μ L concentration are 4 mmol/L are 0.4 U/mL are added
To 200 μ L phenylalanines containing various concentrations pH be 9.10, concentration for 10 mmol/L Tris-HCl buffer solutions in, mixing
Reacted 30 minutes at 37 DEG C after uniform, it is 5 to sequentially add 632.5 μ L pH, concentration is 50 mmol/L acetate salt buffer
Liquid, 50 μ L concentration are 3 mmol/L TMB HCI solution and 17.5 μ L chitosans-platinum mould
Intend oxidation enzyme solutions, reacted 5 minutes at 37 DEG C again after being well mixed, add the sulfuric acid solution that 200 μ L concentration are 2 mol/L
Terminating reaction, determines the absorbance A of solution450, inhibiting rate is calculated, the IC for obtaining phenylalanine is fitted by software50For 0.77
mmoL/L。
Specifically, the present invention uses following technical scheme：
（One）Chitosan-platinum simulates the preparation of oxidizing ferment：It is 1% to weigh 0.1 g chitosans and be dissolved in 50 mL concentration（v/v）Vinegar
In acid, stirring is completely dissolved 15 minutes chitosan to obtain concentration for 0.2%（m/v）Chitosan solution.It is by 2 mL concentration
10 mmol/L chloroplatinic acids are added to 47 mL concentration in 0.2% chitosan solution, to stir 30 minutes, 1 mL is then added dropwise
Concentration is the sodium borohydride solution that 0.2 mol/L is newly prepared（Added within 5 minutes）, it is placed in dark place and stirs 90 minutes, obtains shell
Glycan-platinum simulation oxidizing ferment, products therefrom lucifuge is stored refrigerated.
（Two）The measure of alkaline phosphatase：First, by ascorbic acid phosphoric acid esters and 50 μs of the 50 μ L concentration for 4 mmol/L
The alkaline phosphatase of L various concentrations is added to 200 μ L Tris-HCl buffer solutions（The mmol/L of pH 9.10,10）In, mixing is equal
Reacted 30 minutes at 37 DEG C after even；Then, successively by 632.5 μ L acetate buffers（The mmol/L of pH 5,50）、50 μl
TMB HCI solution and 17.5 μ l steps that concentration is 3 mmol/L（One）The shell of preparation gathers
Sugar-platinum simulation oxidation enzyme solutions are added in above-mentioned reaction solution, are reacted 5 minutes at 37 DEG C again after being well mixed；Add 200 μ
L concentration is 2 mol/L sulfuric acid solution terminating reaction, determines absorbance of the solution at 450 nm wavelength（A450）, draw mark
Directrix curve carries out content of alkaline phosphatase measure.
（Three）The measure of Inhibitors of Alkaline Phosphatase：By the alkaline phosphatase enzyme solutions and 50 that 50 μ L concentration are 0.4 U/mL
μ L concentration is delayed for the Tris-HCl that 4 mmol/L ascorbic acid phosphoric acid esters solution are added to 200 μ L phenylalanines containing various concentrations
Rush in solution（The mmol/L of pH 9.10,10）, reacted 30 minutes at 37 DEG C after shaking up.Then, successively by 632.5 μ L acetic acid
Salt buffer（The mmol/L of pH 5,50）, 50 μ L concentration for 3 mmol/L TMB hydrochloride it is molten
Liquid and 17.5 μ L steps（One）The chitosan of preparation-platinum simulation oxidation enzyme solutions are added in above-mentioned reaction solution, after being well mixed
Reacted 5 minutes at 37 DEG C again.The sulfuric acid solution terminating reaction that 200 μ L concentration are 2 mol/L is added, solution absorbance is determined
Value A450.It is fitted by software, obtains the 503nhibiting concentration IC of phenylalanine50。
Advantages of the present invention
（1）The present invention is using alkaline phosphatase enzyme hydrolysis ascorbic acid phosphoric acid esters generation ascorbic acid, so as to suppress chitosan-platinum mould
Intend oxidase catalyzed coloring reaction system, the change of binding soln color and ultra-violet absorption spectrum feature is directly used in alkaline phosphorus
The measure of sour enzyme and its inhibitor content.
（2）Chitosan prepared by the present invention-platinum simulation oxidizing ferment is used as stabilizer, sodium borohydride reduction chlorine platinum by chitosan
Acid is obtained, and its preparation process is simple and quick.
（3）Detecting step of the present invention is simple, any complicated, valuable instrument is not related to, testing cost is low.
（4）Detection sensitivity of the present invention is high, and the detection of alkaline phosphatase is limited to 0.34 U/L.
Brief description of the drawings
Fig. 1 is outside drawing.A control groups in figure：Chitosan-platinum simulation oxidizing ferment+3,3 ', 5,5 '-tetramethyl biphenyl
Amine hydrochlorate；B control groups in figure:Chitosan-platinum simulation oxidizing ferment+TMB hydrochloride+
Ascorbic acid phosphoric acid esters；C control groups in figure:Chitosan-platinum simulation oxidizing ferment+TMB salt
Hydrochlorate+alkaline phosphatase；D experimental groups in figure：Chitosan-platinum simulation oxidizing ferment+TMB
Hydrochloride+ascorbic acid phosphoric acid esters+alkaline phosphatase.
Fig. 2 is absorbance A450Figure.A control groups in figure：Chitosan-platinum simulation oxidizing ferment+3,3 ', 5,5 '-tetramethyl
Base benzidine dihydrochloride；B control groups in figure:Chitosan-platinum simulation oxidizing ferment+TMB salt
Hydrochlorate+ascorbic acid phosphoric acid esters；C control groups in figure:Chitosan-platinum simulation oxidizing ferment+3,3 ', 5,5 '-tetramethyl connection
Anilinechloride+alkaline phosphatase；D experimental groups in figure：Chitosan-platinum simulation oxidizing ferment+3,3 ', 5,5 '-tetramethyl
Benzidine dihydrochloride+ascorbic acid phosphoric acid esters+alkaline phosphatase.
Fig. 3 is that alkaline phosphatase concentration simulates oxidase catalyzed color development system absorbance A with chitosan-platinum450It is linear
Graph of a relation.
Fig. 4 is alkaline phosphatase assay interference experiment.Numeral 0 ~ 9 represents blank group, alkaline phosphatase, burnt phosphorus respectively successively
Sour hydrolase, protease k, catalase, HRPO, lysozyme, urase, acetylcholinesterase, grape are glycoxidative
Enzyme.
Fig. 5 is phenylalanine inhibiting rate curve map.
Embodiment
Embodiment 1：
It is 1% to weigh 0.1 g chitosans and be dissolved in 50 mL concentration（v/v）Acetic acid in, stirring make chitosan completely molten within 15 minutes
It is 0.2% that solution, which obtains concentration,（m/v）Chitosan solution.It is that to be added to 47 mL dense for 10 mmol/L chloroplatinic acids by 2 mL concentration
Spend for 0.2%（m/v）In chitosan solution, stir 30 minutes, be then added dropwise what 1 mL concentration was newly prepared for 0.2 mol/L
Sodium borohydride solution（Added within 5 minutes）, it is placed in dark place and stirs 90 minutes, obtains auburn chitosan-platinum simulation oxidation
Enzyme solutions.Product lucifuge is stored refrigerated.All glasswares used in above procedure soak by chloroazotic acid, and use distilled water
Thoroughly cleaning, dries.
Embodiment 2：
The alkaline phosphatase that the ascorbic acid phosphoric acid esters for being 4 mmol/L by 50 μ L concentration and 50 μ L concentration are 0.4 U/mL adds
Enter to 200 μ L Tris-HCl buffer solutions（The mmol/L of pH 9.10,10）In, reacted 30 minutes at 37 DEG C after being well mixed.
Then successively by 632.5 μ L acetate buffers（The mmol/L of pH 5,50）, 50 μ L concentration be 3 mmol/L 3,3 ', 5,
Chitosan made from 5 '-tetramethyl biphenyl amide hydrochloride and 17.5 μ L embodiments 1-platinum simulation oxidation enzyme solutions are added to
In above-mentioned reaction solution, reacted 5 minutes at 37 DEG C again after being well mixed.Add the sulfuric acid solution that 200 μ L concentration are 2 mol/L
The uv-visible absorption spectra of terminating reaction, the visually change of observation color or measure solution.Visually during observation color change,
Control group solution colour is buff, and experimental group solution colour is colourless（See Fig. 1）.When determining uv-visible absorption spectra,
The absorbance A of control group450Change is little, and the absorbance A of experimental group450It is obviously reduced（See Fig. 2）.
Embodiment 3：
It is that 4 mmol/L ascorbic acid phosphoric acid esters and the alkaline phosphatase of 50 μ L various concentrations are added to 200 by 50 μ L concentration
μ L Tris-HCl buffer solutions（The mmol/L of pH 9.10,10）In, reacted 30 minutes at 37 DEG C after being well mixed.Then successively
By 632.5 μ L acetate buffers（The mmol/L of pH 5,50）, 50 μ L concentration be 3 mmol/L 3,3 ', 5,5 '-tetramethyl
Chitosan made from benzidine dihydrochloride solution and 17.5 μ L embodiments 1-platinum simulation oxidation enzyme solutions are added to above-mentioned reaction solution
In, reacted 5 minutes at 37 DEG C again after being well mixed.The sulfuric acid solution terminating reaction that 200 μ L concentration are 2 mol/L is added, is surveyed
Determine the absorbance A of solution450.From the figure 3, it may be seen that with the increase of alkaline phosphatase concentration, absorbance A450It is gradually reduced.
In the range of 0.5 ~ 20 U/L, A450Linear with alkaline phosphatase concentration, detection is limited to 0.34 U/L.
Embodiment 4：
The alkaline phosphatase that the ascorbic acid phosphoric acid esters for being 4 mmol/L by 50 μ L concentration and 50 μ L concentration are 0.2 U/mL adds
Enter to 200 μ L Tris-HCl buffer solutions（The mmol/L of pH 9.10,10）In, reacted 30 minutes at 37 DEG C after being well mixed.
Then successively by 632.5 μ L acetate buffers（The mmol/L of pH 5,50）, 50 μ L concentration be 3 mmol/L 3,3 ', 5,
Chitosan made from 5 '-tetramethyl biphenyl amide hydrochloride and 17.5 μ L embodiments 1-platinum simulation oxidation enzyme solutions are added to
In above-mentioned reaction solution, reacted 5 minutes at 37 DEG C again after being well mixed.Add the sulfuric acid solution that 200 μ L concentration are 2 mol/L
Terminating reaction, determines the absorbance A of solution450.The above-mentioned experimental procedure of repetition 10 times, obtains relative standard deviation（RSD）For
7.0%, show that this method reappearance is good.
Embodiment 5：
Normal human serum is taken, the alkaline phosphatase with various concentrations is according to 1:4 volume ratio is sufficiently mixed, and obtains sample solution.
50 μ L concentration are added to 200 μ L Tris-HCl for 4 mmol/L ascorbic acid phosphoric acid esters and the 50 above-mentioned sample solutions of μ L
Buffer solution（The mmol/L of pH 9.10,10）In, reacted 30 minutes at 37 DEG C after being well mixed.Then successively by 632.5 μ L vinegar
Phthalate buffer（The mmol/L of pH 5,50）, 50 μ L concentration be 3 mmol/L TMB hydrochloride
Chitosan made from solution and 17.5 μ L embodiments 1-platinum simulation oxidation enzyme solutions are added in above-mentioned reaction solution, are well mixed
Reacted 5 minutes at 37 DEG C again afterwards.The sulfuric acid solution terminating reaction that 200 μ L concentration are 2 mol/L is added, the suction of solution is determined
Shading value A450.In conjunction with the embodiments 3 calculate normal human serum alkaline phosphatases content, determine the rate of recovery be 97.1% ~
102.5%, relative standard deviation is 1.6 ~ 6.8%.
Embodiment 6：
Alkaline phosphatase assay interference experiment：The ascorbic acid phosphoric acid esters for being 4 mmol/L by 50 μ L concentration and 50 μ L concentration are
Other enzyme solutions of 4 U/mL are added to 200 μ L Tris-HCl buffer solutions（The mmol/L of pH 9.10,10）In, be well mixed after
Reacted 30 minutes at 37 DEG C.Then successively by 632.5 μ L acetate buffers（The mmol/L of pH 5,50）, 50 μ L concentration be 3
Chitosan made from mmol/L TMB HCI solution and 17.5 μ L embodiments 1-platinum simulation oxygen
Change enzyme solutions to be added in above-mentioned reaction solution, reacted 5 minutes at 37 DEG C again after being well mixed.It is 2 to add 200 μ L concentration
Mol/L sulfuric acid solution terminating reaction, determines the absorbance A of solution450.As shown in Figure 4, even if the concentration ratio alkali of other enzymes
Acid phosphatase is high 10 times, can not still produce obvious interference, shows that this method has good selectivity to alkaline phosphatase.
Embodiment 7：
The alkaline phosphatase that the ascorbic acid phosphoric acid esters for being 4 mmol/L by 50 μ L concentration and 50 μ L concentration are 0.4 U/mL is molten
Liquid is added to the Tris-HCl buffer solutions of 200 μ L phenylalanines containing various concentrations（The mmol/L of pH 9.10,10）In, mixing is equal
Reacted 30 minutes at 37 DEG C after even.Then successively by 632.5 μ L acetate buffers（The mmol/L of pH 5,50）, 50 μ L it is dense
Spend chitosan-platinum made from 3 mmol/L TMB HCI solution and 17.5 μ L embodiments 1
Simulation oxidation enzyme solutions are added in above-mentioned reaction solution, are reacted 5 minutes at 37 DEG C again after being well mixed.Add 200 μ L concentration
For 2 mol/L sulfuric acid solution terminating reaction, the absorbance A of solution is determined450, calculate inhibiting rate.As a result as shown in figure 5, logical
Cross the IC that software fitting obtains phenylalanine50For 0.77 mmoL/L.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
God and any modification made within principle, equivalent substitution and improvement etc., should be included in the scope of the protection.
Claims (7)
1. a kind of alkaline phosphatase assay method that oxidizing ferment is simulated based on chitosan-platinum, it is characterized in that in ascorbic acid phosphoric acid
TMB HCI solution and chitosan-platinum simulation oxygen are added in ester and alkaline phosphatase enzyme reaction product
Change enzyme solutions, sulfuric acid solution terminating reaction is added after reaction, according to the change of solution colour and ultra-violet absorption spectrum feature, is used for
The measure of content of alkaline phosphatase；Used chitosan-platinum simulation oxidizing ferment is as stabilizer, sodium borohydride with chitosan
Reduction chloroplatinic acid is obtained, and specific synthesis step is as follows：Weigh 0.1 g chitosans and be dissolved in the acetic acid that 50 mL concentration are 1 v/v %
In, stirring makes chitosan be completely dissolved the chitosan solution for obtaining that concentration is 0.2 m/v % for 15 minutes, is 10 by 2 mL concentration
Mmol/L chloroplatinic acids are added to 47 mL concentration in 0.2 m/v % chitosan solutions, to stir 30 minutes, be then added dropwise 1
ML concentration is the sodium borohydride solution that 0.2 mol/L is newly prepared and added within 5 minutes, is placed in dark place and stirs 90 minutes, obtains
To chitosan-platinum simulation oxidizing ferment.
2. the alkaline phosphatase assay method according to claim 1 that oxidizing ferment is simulated based on chitosan-platinum, it is characterized in that
It is that 4 mmol/L ascorbic acid phosphoric acid esters and the alkaline phosphatase of 50 μ L various concentrations are added to 200 μ L by 50 μ L concentration
PH is 9.10, concentration for 10 mmol/L Tris-HCl buffer solutions in, be well mixed after at 37 DEG C react 30 minutes, so
After sequentially add 632.5 μ L pH for 5, concentration is that 50 mmol/L acetate buffer, 50 μ L concentration are 3 mmol/L
TMB HCI solution and 17.5 μ L chitosans-platinum simulation oxidation enzyme solutions, after being well mixed again
Reacted 5 minutes at 37 DEG C, add the sulfuric acid solution terminating reaction that 200 μ L concentration are 2 mol/L, determine the absorbance of solution
Value A450。
3. the alkaline phosphatase assay method according to claim 1 or 2 that oxidizing ferment is simulated based on chitosan-platinum, it is special
It is the absorbance A in the range of 0.5 ~ 20 U/L to levy450Linear with alkaline phosphatase concentration, detection is limited to 0.34 U/
L。
4. a kind of method of the measure human serum alkaline phosphatase based on chitosan-platinum simulation oxidizing ferment, it is characterized in that including such as
Lower step：The ascorbic acid phosphoric acid esters for being 4 mmol/L by 50 μ L concentration and 50 μ L human serums are added to 200 μ L pH
9.10, concentration for 10 mmol/L Tris-HCl buffer solutions in, be well mixed after at 37 DEG C react 30 minutes, sequentially add
632.5 μ L pH are 5, concentration is 50 mmol/L acetate buffer, 50 μ L concentration are 3 mmol/L 3,3 ', 5,5 '-
Tetramethyl biphenyl amide hydrochloride and 17.5 μ L chitosans-platinum simulation oxidation enzyme solutions, it is anti-at 37 DEG C again after being well mixed
Answer 5 minutes, add the sulfuric acid solution terminating reaction that 200 μ L concentration are 2 mol/L, determine the absorbance A of solution450, according to
Alkaline phosphatase standard curve calculates the content of alkaline phosphatase in human serum；Used chitosan-platinum simulates oxidizing ferment
With chitosan as stabilizer, sodium borohydride reduction chloroplatinic acid is obtained, and specific synthesis step is as follows：Weigh 0.1 g chitosans molten
Solution is in 50 mL concentration in 1 v/v % acetic acid, and stirring is completely dissolved 15 minutes chitosan to obtain concentration for 0.2 m/v
2 mL concentration are that to be added to 47 mL concentration be 0.2 m/v % chitosan solutions to 10 mmol/L chloroplatinic acids by % chitosan solution
In, stir 30 minutes, be then added dropwise sodium borohydride solution that 1 mL concentration newly prepared for 0.2 mol/L and 5 minutes it
Inside add, be placed in dark place and stir 90 minutes, obtain chitosan-platinum simulation oxidizing ferment.
5. the method for the measure human serum alkaline phosphatase according to claim 4 based on chitosan-platinum simulation oxidizing ferment,
It is characterized in that the rate of recovery for obtaining human serum sample's measure is 97.1% ~ 102.5%, relative standard deviation is 1.6 ~ 6.8%.
6. a kind of assay method of the Inhibitors of Alkaline Phosphatase based on chitosan-platinum simulation oxidizing ferment, it is characterized in that anti-bad
TMB is added in the reaction product of hematic acid phosphate, alkaline phosphatase and Inhibitors of Alkaline Phosphatase
HCI solution and chitosan-platinum simulation oxidation enzyme solutions, add sulfuric acid solution terminating reaction after reaction, according to solution colour and
The change of ultra-violet absorption spectrum feature, the measure for the inhibiting rate of Inhibitors of Alkaline Phosphatase；Used chitosan-platinum mould
It is that sodium borohydride reduction chloroplatinic acid is obtained, and specific synthesis step is as follows as stabilizer with chitosan to intend oxidizing ferment：Weigh 0.1
G chitosans are dissolved in the acetic acid that 50 mL concentration are 1 v/v %, and stirring is completely dissolved chitosan in 15 minutes to obtain concentration
It is that to be added to 47 mL concentration be 0.2 m/v % to 10 mmol/L chloroplatinic acids by 2 mL concentration for 0.2 m/v % chitosan solution
In chitosan solution, stir 30 minutes, sodium borohydride solution that 1 mL concentration newly prepared for 0.2 mol/L is then added dropwise simultaneously
Added within 5 minutes, be placed in dark place and stir 90 minutes, obtain chitosan-platinum simulation oxidation enzyme solutions.
7. the assay method of the Inhibitors of Alkaline Phosphatase according to claim 6 based on chitosan-platinum simulation oxidizing ferment,
It is characterized in that the alkaline phosphatase that the ascorbic acid phosphoric acid esters and 50 μ L concentration that are 4 mmol/L by 50 μ L concentration are 0.4 U/mL
The pH that enzyme solutions are added to 200 μ L phenylalanines containing various concentrations is 9.10, and concentration buffers for 10 mmol/L Tris-HCl
In liquid, reacted 30 minutes at 37 DEG C after being well mixed, it is 5 to sequentially add 632.5 μ L pH, concentration is 50 mmol/L vinegar
Phthalate buffer, 50 μ L concentration are poly- for 3 mmol/L TMB HCI solution and 17.5 μ L shells
Sugar-platinum simulation oxidation enzyme solutions, reacts 5 minutes at 37 DEG C, adds the sulphur that 200 μ L concentration are 2 mol/L again after being well mixed
Acid solution terminating reaction, determines the absorbance A of solution450, inhibiting rate is calculated, the IC for obtaining phenylalanine is fitted by software50
For 0.77 mmoL/L.
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