CN107238598A - The Assay of acid phosphatase content method of oxidizing ferment is simulated based on chitosan platinum - Google Patents
The Assay of acid phosphatase content method of oxidizing ferment is simulated based on chitosan platinum Download PDFInfo
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- CN107238598A CN107238598A CN201710298324.1A CN201710298324A CN107238598A CN 107238598 A CN107238598 A CN 107238598A CN 201710298324 A CN201710298324 A CN 201710298324A CN 107238598 A CN107238598 A CN 107238598A
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- 102000013563 Acid Phosphatase Human genes 0.000 title claims abstract description 76
- 108010051457 Acid Phosphatase Proteins 0.000 title claims abstract description 76
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 title claims abstract description 69
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 title claims abstract description 65
- 229910052697 platinum Inorganic materials 0.000 title claims abstract description 65
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 50
- 230000001590 oxidative effect Effects 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000003556 assay Methods 0.000 title claims abstract description 16
- 238000004088 simulation Methods 0.000 claims abstract description 50
- 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 34
- 238000006243 chemical reaction Methods 0.000 claims abstract description 29
- 229960005070 ascorbic acid Drugs 0.000 claims abstract description 28
- -1 ascorbic acid phosphoric acid esters Chemical class 0.000 claims abstract description 26
- 235000010323 ascorbic acid Nutrition 0.000 claims abstract description 22
- 239000011668 ascorbic acid Substances 0.000 claims abstract description 22
- 238000002835 absorbance Methods 0.000 claims abstract description 18
- 230000008859 change Effects 0.000 claims abstract description 15
- 239000003112 inhibitor Substances 0.000 claims abstract description 14
- 238000000862 absorption spectrum Methods 0.000 claims abstract description 11
- 238000001514 detection method Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 101
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 36
- 239000008351 acetate buffer Substances 0.000 claims description 25
- 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
- 102000004190 Enzymes Human genes 0.000 claims description 21
- 108090000790 Enzymes Proteins 0.000 claims description 21
- 239000002253 acid Substances 0.000 claims description 19
- 230000003647 oxidation Effects 0.000 claims description 17
- 238000007254 oxidation reaction Methods 0.000 claims description 17
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical class [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 claims description 14
- 239000012279 sodium borohydride Substances 0.000 claims description 13
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 13
- 210000002966 serum Anatomy 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 11
- 235000013024 sodium fluoride Nutrition 0.000 claims description 9
- 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
- 239000002211 L-ascorbic acid Substances 0.000 claims description 6
- 235000000069 L-ascorbic acid Nutrition 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- 239000011775 sodium fluoride Substances 0.000 claims description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 4
- MMCPOSDMTGQNKG-UHFFFAOYSA-N anilinium chloride Chemical compound Cl.NC1=CC=CC=C1 MMCPOSDMTGQNKG-UHFFFAOYSA-N 0.000 claims description 4
- 239000007795 chemical reaction product Substances 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 238000011084 recovery Methods 0.000 claims description 3
- 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 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 230000003111 delayed effect Effects 0.000 claims description 2
- 238000011010 flushing procedure Methods 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims 2
- 229910019142 PO4 Inorganic materials 0.000 claims 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims 1
- 150000002148 esters Chemical class 0.000 claims 1
- 150000004676 glycans Chemical class 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 1
- 239000010452 phosphate Substances 0.000 claims 1
- 230000007062 hydrolysis Effects 0.000 abstract description 5
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 5
- 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 abstract description 3
- 230000003197 catalytic effect Effects 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 15
- 102000004316 Oxidoreductases Human genes 0.000 description 6
- 108090000854 Oxidoreductases Proteins 0.000 description 6
- 238000011161 development Methods 0.000 description 5
- 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 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- ALLIZEAXNXSFGD-UHFFFAOYSA-N 1-methyl-2-phenylbenzene Chemical group CC1=CC=CC=C1C1=CC=CC=C1 ALLIZEAXNXSFGD-UHFFFAOYSA-N 0.000 description 2
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 229910052796 boron Inorganic materials 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 102000012440 Acetylcholinesterase Human genes 0.000 description 1
- 108010022752 Acetylcholinesterase Proteins 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 108010000659 Choline oxidase 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
- 241000196324 Embryophyta Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 201000002980 Hyperparathyroidism Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 1
- 241000124008 Mammalia Species 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
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 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
- 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
- 229940022698 acetylcholinesterase Drugs 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000003745 diagnosis Methods 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
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000000686 essence Substances 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- 230000003902 lesion Effects 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
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 150000003014 phosphoric acid esters Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000104 sodium hydride Inorganic materials 0.000 description 1
- 239000012312 sodium hydride Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 235000021419 vinegar Nutrition 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
Landscapes
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a kind of Assay of acid phosphatase content method that oxidizing ferment is simulated based on chitosan platinum, it is characterized in that generating ascorbic acid using acid 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 acid phosphatase and its inhibitor content.In the range of 0.25 ~ 2.5 U/L, absorbance change value △ A450Linear with acid phosphatase enzyme concentration, detection is limited to 0.0161 U/L.Easy to operate, sensitivity of the invention is high, and the measure of acid phosphatase and its inhibitor content in environment and life science system can be applied to as analysis method.
Description
Technical field
The acid 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
Acid phosphatase is a kind of a kind of hydrolase of the monoesters of catalytic phosphatase in acid condition hydrolysis generation inorganic phosphate,
In the tissue for being widely distributed in some plants or mammal.Agriculture aspect, acid phosphatase is to judging crop growth
Optimal pH plays an important role.In terms of medical science, its can as the diseases such as hepatitis, hyperparathyroidism, red blood cell lesion weight
Want diagnosis index.Therefore, the measure of acid phosphatase content has far reaching significance.But the measure of current acid phosphatase is also present
Many limitations, for example, existing method is mainly from 4-NPP as substrate, but the nitro that hydrolysis is produced
Benzene needs just have stronger signal in the basic conditions, this most suitable catalytic condition with acid phosphatase（pH = 5.0）Xiang Mao
Shield.Therefore a kind of more effective, accurate colorimetric method of development has very important practical significance to determine acid phosphatase.
Acid phosphatase is 37 DEG C in temperature, and pH is that can hydrolyze ascorbic acid phosphoric acid esters under conditions of 5.0, is generated anti-bad
Hematic acid.And the ascorbic acid with strong reduced form can suppress oxidizing ferment and TMB hydrochloride is urged
Oxidation so that the colour developing of TMB hydrochloride is not obvious.The content of acid phosphatase is higher, body
The absorption angle value of system is smaller.Based on this, it can be achieved to quantitative determine acid phosphatase.
The present invention simulates oxidase catalyzed oxidation using ascorbic acid phosphoric acid esters as acid phosphatase zymolyte with chitosan-platinum
TMB hydrochloride color development system is sensed signal sources, establishes a kind of colorimetric estimation acid phosphatase
Method.Acid phosphatase enzyme catalyzed hydrolysis and the oxidase catalyzed oxidation 3,3 ', 5 of chitosan-platinum simulation, 5 '-tetramethyl connection
Anilinechloride chromogenic reaction can be carried out under same pH.This method is easy, quick, and any complicated, valuable instrument is not related to,
Testing cost is low.
The content of the invention
The oxidase catalyzed oxidation 3 of chitosan-platinum simulation, 3 ', 5,5 '-tetramethyl are utilized it is an object of the invention to provide one kind
Benzidine dihydrochloride color development system, using ascorbic acid phosphoric acid esters as substrate, so as to determine the side of acid phosphatase and its inhibitor
Method.
To achieve these goals, the present invention uses following technical scheme：
The Assay of acid phosphatase content 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 acid phosphatase 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 acid phosphatase content；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, chitosan-platinum simulation oxidizing ferment is obtained, products therefrom lucifuge is stored refrigerated.
The described Assay of acid phosphatase content method that oxidizing ferment is simulated based on chitosan-platinum, it is characterized in that by 50 μ L concentration
It is 5 that the acid 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
In acetate buffer for 50 mmol/L, reacted 30 minutes at 37 DEG C after being well mixed, then sequentially add 632.5 μ L
PH is 5, and concentration is that 50 mmol/L acetate buffer, 50 μ L concentration are the 3 of 3 mmol/L, 3 ', 5,5 '-tetramethyl connection
Anilinechloride solution and 17.5 μ L chitosans-platinum simulation oxidation enzyme solutions, react 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, the absorbance A of solution is determined450。
The described Assay of acid phosphatase content method that oxidizing ferment is simulated based on chitosan-platinum, it is characterized in that 0.25 ~
In the range of 2.5 U/L, absorbance change value △ A450Linear with acid phosphatase enzyme concentration, detection is limited to 0.0161 U/L.
The method of measure human serum acid 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 are 5, and concentration successively will in 50 mmol/L acetate buffer, to be reacted after being well mixed at 37 DEG C 30 minutes
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 are added in above-mentioned reaction solution, are mixed
Reacted 5 minutes at 37 DEG C again after uniform, add the sulfuric acid solution terminating reaction that 200 μ L concentration are 2 mol/L, determine solution
Absorbance A450, the content of alkaline phosphatase in human serum is calculated according to acid phosphatase standard curve；Used shell gathers
Sugar-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 chitosans to be dissolved in the acetic acid that 50 mL concentration are 1 v/v %, stirring makes chitosan be completely dissolved i.e. 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 and are by 2 mL concentration
In 0.2 m/v % chitosan solutions, stir 30 minutes, it is the boron hydrogen that 0.2 mol/L is newly prepared that 1 mL concentration, which is then added dropwise,
Change sodium solution 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 acid phosphatase based on chitosan-platinum simulation oxidizing ferment, it is characterized in that
The rate of recovery that human serum sample determines is 97.4% ~ 107.8%, and relative standard deviation is 2.4 ~ 6.4%.
The assay method of acid phosphatase inhibitors 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, acid phosphatase and acid phosphatase inhibitors
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 Inhibitors of Alkaline Phosphatase inhibiting rate；Used shell
Glycan-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 oxidizing ferment.
The assay method of the described acid phosphatase inhibitors based on chitosan-platinum simulation oxidizing ferment, it is characterized in that will
The acid 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 0.1 U/mL are added
To 200 μ L sodium fluorides containing various concentrations pH be 5, concentration for 50 mmol/L acetate buffer in, be well mixed after
Reacted 30 minutes at 37 DEG C, it is 5 to sequentially add 632.5 μ L pH, concentration is 50 mmol/L acetate buffer, 50 μ L
Concentration is 3 mmol/L TMB HCI solution and 17.5 μ L chitosans-platinum simulation oxidizing ferment
Solution, reacts 5 minutes at 37 DEG C again after being well mixed, and adds the sulfuric acid solution terminating reaction that 200 μ L concentration are 2 mol/L,
Determine the absorbance A of solution450, inhibiting rate is calculated, the IC for obtaining sodium fluoride is fitted by software50For 0.896 mmoL/L.
Specifically, the present invention is adopted the following technical scheme that：
（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 acid phosphatase：50 μ L concentration are different for 4 mmol/L ascorbic acid phosphoric acid esters and 50 μ L
The acid phosphatase of concentration is added to 200 μ L acetate buffers（The mmol/L of pH 5,50）In, after being well mixed at 37 DEG C
Reaction 30 minutes；Sequentially add 632.5 μ L acetate buffers（The mmol/L of pH 5,50）, 50 μ l concentration is 3 mmol/L
TMB HCI solution and 17.5 μ l steps（One）The chitosan of preparation-platinum simulation oxidizing ferment is molten
Liquid, reacts 5 minutes at 37 DEG C again after being well mixed, and adds the sulfuric acid solution terminating reaction that 200 μ L concentration are 2 mol/L, surveys
Determine absorbance of the solution at 450 nm wavelength（A450）, draw standard curve and carry out acid phosphatase assay.
（Three）The measure of acid phosphatase inhibitors：By the acid phosphatase enzyme solutions and 50 that 50 μ L concentration are 0.1 U/mL
μ L concentration is molten for the acetate salt buffer that 4 mmol/L ascorbic acid phosphoric acid esters solution are added to 200 μ L sodium fluorides containing various concentrations
In liquid（The mmol/L of pH 5,50）, reacted 30 minutes at 37 DEG C after shaking up.Sequentially add 632.5 μ L acetate buffers（pH
5,50 mmol/L）, 50 μ L concentration for 3 mmol/L TMB HCI solution and 17.5 μ L step
Suddenly（One）The chitosan of preparation-platinum simulation oxidation enzyme solutions, reacts 5 minutes at 37 DEG C, adds 200 μ L again after being well mixed
Concentration is 2 mol/L sulfuric acid solution terminating reaction, determines solution absorbance value A450.It is fitted by software, obtains sodium fluoride
503nhibiting concentration IC50。
Advantages of the present invention
（1）The present invention is using acid 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 acid 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 acid phosphatase is limited to 0.0161 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+acid phosphatase；D experimental groups in figure：Chitosan-platinum simulation oxidizing ferment+TMB
Hydrochloride+ascorbic acid phosphoric acid esters+acid phosphatase.
Fig. 2 is uv absorption spectra.A control groups in figure：Chitosan-platinum simulation oxidizing ferment+3,3 ', 5,5 '-four
Methyl 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+3,3 ', 5,5 '-tetramethyl
Benzidine dihydrochloride+acid phosphatase；D experimental groups in figure：Chitosan-platinum simulation oxidizing ferment+3,3 ', 5,5 '-tetramethyl
Base benzidine dihydrochloride+ascorbic acid phosphoric acid esters+acid phosphatase.
Fig. 3 is that acid phosphatase enzyme concentration simulates oxidase catalyzed color development system absorbance A with chitosan-platinum450It is linear
Graph of a relation.
Fig. 4 is Assay of acid phosphatase content interference experiment.Numeral 0 ~ 11 represents blank, acid phosphatase, alkalescence respectively successively
Phosphatase, horseradish peroxidase, pyrophosphohydrolase, acetylcholinesterase, alpha-glucosidase, choline oxidase, albumen
Enzyme k, catalase, lysozyme and urase.
Fig. 5 is sodium fluoride 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 acid phosphatase that the ascorbic acid phosphoric acid esters for being 4 mmol/L by 50 μ L concentration and 50 μ L concentration are 0.1 U/mL adds
Enter to 200 μ L acetate buffers（The mmol/L of pH 5,50）In, reacted 30 minutes at 37 DEG C after being well mixed.Successively plus
Enter 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, exists 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, visually the change of observation color
Or determine the uv-visible absorption spectra of solution.Visually during observation color change, control group solution colour is buff, real
A group solution colour is tested to become colorless（See Fig. 1）.When determining uv-visible absorption spectra, the absorption spectrum of control group is hardly sent out
Changing, and absorption of the absorption spectrum of experimental group in 350 ~ 550 nm regions is obviously reduced（See Fig. 2）.
Embodiment 3：
It is that 4 mmol/L ascorbic acid phosphoric acid esters and the acid phosphatase of 50 μ L various concentrations are added to 200 by 50 μ L concentration
μ L acetate buffers（The mmol/L of pH 5,50）In, reacted 30 minutes at 37 DEG C after being well mixed.Sequentially add 632.5 μ
L acetate buffers（The mmol/L of pH 5,50）, 50 μ L concentration be 3 mmol/L TMB hydrochloric acid
Chitosan made from salting liquid and 17.5 μ L embodiments 1-platinum simulation oxidation enzyme solutions, 5 are reacted after being well mixed at 37 DEG C again
Minute, the sulfuric acid solution terminating reaction that 200 μ L concentration are 2 mol/L is added, the absorbance A of solution is determined450.Can by Fig. 3
Know, with the increase of acid phosphatase enzyme concentration, absorbance A450Changing value（△A450）Gradually increase.0.25 ~ 2.5
In the range of U/L, △ A450Linear with acid phosphatase enzyme concentration, lowest detection is limited to 0.0161 U/L.
Embodiment 4：
The acid phosphatase that the ascorbic acid phosphoric acid esters for being 4 mmol/L by 50 μ L concentration and 50 μ L concentration are 0.0125 U/mL
It is added to 200 μ L acetate buffers（The mmol/L of pH 5,50）In, reacted 30 minutes at 37 DEG C after being well mixed.Successively
Add 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 base benzidine dihydrochloride solution and 17.5 μ L embodiments 1-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.The above-mentioned experimental procedure of repetition 9 times, obtains relative standard deviation（RSD）For 3.9%, show that this method reappearance is good.
Embodiment 5：
Normal human serum is taken, the acid 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 acetate and delayed for 4 mmol/L ascorbic acid phosphoric acid esters and the 50 above-mentioned sample solutions of μ L
Fliud flushing（The mmol/L of pH 5,50）In, reacted 30 minutes at 37 DEG C after being well mixed.Sequentially added in above-mentioned reaction solution
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 connection
Chitosan made from anilinechloride solution and 17.5 μ L embodiments 1-platinum simulation oxidation enzyme solutions, again 37 after being well mixed
Reacted 5 minutes at DEG C, add the sulfuric acid solution terminating reaction that 200 μ L concentration are 2 mol/L, determine the absorbance of solution
A450.In conjunction with the embodiments 3 calculate normal human serums in acid phosphatase content, determine the rate of recovery be 97.4% ~ 107.8%, relatively
Standard deviation is 2.4 ~ 6.4%.
Embodiment 6：
Assay of acid phosphatase content 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 1 U/mL are added to 200 μ L acetate buffers（The mmol/L of pH 5,50）In, after being well mixed at 37 DEG C
Reaction 30 minutes.Sequentially add 632.5 μ L acetate buffers（The mmol/L of pH 5,50）, 50 μ L concentration is 3 mmol/L
Chitosan made from TMB HCI solution and 17.5 μ L embodiments 1-platinum simulation oxidizing ferment is molten
Liquid, is reacted 5 minutes at 37 DEG C again after being well mixed, and the sulfuric acid solution that 200 μ L concentration are 2 mol/L is added immediately and is terminated instead
Should, determine the absorbance A of solution450.As shown in Figure 4, even if the concentration ratio acid phosphatase of other enzymes is high 10 times, still can not
Obvious interference is produced, shows that this method has good selectivity to acid phosphatase.
Embodiment 7：
The acid phosphatase that the ascorbic acid phosphoric acid esters for being 4 mmol/L by 50 μ L concentration and 50 μ L concentration are 0.1 U/mL is molten
Liquid is added to the acetate buffer of 200 μ L sodium fluorides containing various concentrations（The mmol/L of pH 5,50）In, 37 after being well mixed
Reacted 30 minutes at DEG C.Sequentially add 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, reacted 5 minutes at 37 DEG C again after being well mixed, add 200 μ L concentration and terminated for 2 mol/L sulfuric acid solution
Reaction, determines the absorbance A of solution450, calculate inhibiting rate.As a result as shown in figure 5, obtaining sodium fluoride by software fitting
IC50For 0.896 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 Assay of acid phosphatase content 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 acid phosphatase 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 acid phosphatase content；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
Oxidizing ferment is simulated to chitosan-platinum, products therefrom lucifuge is stored refrigerated.
2. the Assay of acid phosphatase content 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 acid phosphatase of 50 μ L various concentrations are added to 200 μ L by 50 μ L concentration
PH is 5, concentration for 50 mmol/L acetate buffer in, be well mixed after at 37 DEG C react 30 minutes, then successively
632.5 μ L pH are added for 5, concentration is that 50 mmol/L acetate buffer, 50 μ L concentration are the 3,3 ' of 3 mmol/L,
5,5 '-tetramethyl biphenyl amide hydrochloride and 17.5 μ L chitosans-platinum simulation aoxidize enzyme solutions, again 37 after being well mixed
Reacted 5 minutes at DEG C, add the sulfuric acid solution terminating reaction that 200 μ L concentration are 2 mol/L, determine the absorbance of solution
A450。
3. the Assay of acid phosphatase content method according to claim 1 or 2 that oxidizing ferment is simulated based on chitosan-platinum, it is special
It is the absorbance change value △ A in the range of 0.25 ~ 2.5 U/L to levy450It is linear with acid phosphatase enzyme concentration, detection
It is limited to 0.0161 U/L.
4. a kind of method of the measure human serum acid phosphatase based on chitosan-platinum simulation oxidizing ferment, it is characterized in that including such as
Lower step：It is 5 that the ascorbic acid phosphoric acid esters for being 4 mmol/L by 50 μ L concentration and 50 μ L human serums, which are added to 200 μ L pH,
Concentration for 50 mmol/L acetate buffer in, be well mixed after at 37 DEG C react 30 minutes, successively by 632.5 μ L
PH is 5, and concentration is that 50 mmol/L acetate buffer, 50 μ L concentration are the 3 of 3 mmol/L, 3 ', 5,5 '-tetramethyl connection
Anilinechloride solution and 17.5 μ L chitosans-platinum simulation oxidation enzyme solutions are added in above-mentioned reaction solution, 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, the content of alkaline phosphatase in human serum is calculated according to acid phosphatase standard curve；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 oxidizing ferment.
5. the method for the measure human serum acid 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 97.4% ~ 107.8%, relative standard deviation is 2.4 ~ 6.4%.
6. a kind of assay method of the acid phosphatase inhibitors 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, acid phosphatase and acid phosphatase inhibitors
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 Inhibitors of Alkaline Phosphatase inhibiting rate；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.
7. the assay method of the acid phosphatase inhibitors according to claim 6 based on chitosan-platinum simulation oxidizing ferment,
It is characterized in that the acid phosphatase that the ascorbic acid phosphoric acid esters and 50 μ L concentration that are 4 mmol/L by 50 μ L concentration are 0.1 U/mL
Enzyme solutions be added to 200 μ L sodium fluorides containing various concentrations pH be 5, concentration for 50 mmol/L acetate buffer in, mix
Reacted 30 minutes at 37 DEG C after closing uniformly, it is 5 to sequentially add 632.5 μ L pH, concentration is delayed for 50 mmol/L acetate
Fliud flushing, 50 μ L concentration are 3 mmol/L TMB HCI solution and 17.5 μ L chitosans-platinum
Simulation oxidation enzyme solutions, react 5 minutes at 37 DEG C, add 200 μ L concentration molten for 2 mol/L sulfuric acid again after being well mixed
Liquid terminating reaction, determines the absorbance A of solution450, inhibiting rate is calculated, the IC for obtaining sodium fluoride is fitted by software50For
0.896 mmoL/L。
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