CN107238598B - Based on chitosan-platinum simulation oxidizing ferment Assay of acid phosphatase content method - Google Patents
Based on chitosan-platinum simulation oxidizing ferment Assay of acid phosphatase content method Download PDFInfo
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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
- 229910052697 platinum Inorganic materials 0.000 title claims abstract description 66
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Substances [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 title claims abstract description 66
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 238000004088 simulation Methods 0.000 title claims abstract description 46
- 230000001590 oxidative effect Effects 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000003556 assay Methods 0.000 title claims abstract description 12
- 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 32
- 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
- 238000005259 measurement Methods 0.000 claims abstract description 20
- 238000002835 absorbance Methods 0.000 claims abstract description 17
- 239000003112 inhibitor Substances 0.000 claims abstract description 14
- 238000000862 absorption spectrum Methods 0.000 claims abstract description 11
- 230000008859 change Effects 0.000 claims abstract description 9
- 238000001514 detection method Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 101
- 229920001661 Chitosan Polymers 0.000 claims description 48
- 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
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 24
- 238000002156 mixing Methods 0.000 claims description 24
- 238000003756 stirring Methods 0.000 claims description 24
- 102000004190 Enzymes Human genes 0.000 claims description 22
- 108090000790 Enzymes Proteins 0.000 claims description 22
- 239000002253 acid Substances 0.000 claims description 18
- 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
- 235000013024 sodium fluoride Nutrition 0.000 claims description 9
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 claims description 8
- 150000007513 acids Chemical class 0.000 claims description 8
- 230000009467 reduction Effects 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
- 239000003381 stabilizer Substances 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- MMCPOSDMTGQNKG-UHFFFAOYSA-N anilinium chloride Chemical compound Cl.NC1=CC=CC=C1 MMCPOSDMTGQNKG-UHFFFAOYSA-N 0.000 claims description 5
- 239000011775 sodium fluoride Substances 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 4
- 239000007795 chemical reaction product Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 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 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims 1
- 238000011010 flushing procedure Methods 0.000 claims 1
- 150000003840 hydrochlorides Chemical class 0.000 claims 1
- 230000007062 hydrolysis Effects 0.000 abstract description 5
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 5
- 230000003197 catalytic effect Effects 0.000 abstract description 3
- 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 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 16
- OBWSOTREAMFOCQ-UHFFFAOYSA-N 4-(4-amino-3,5-dimethylphenyl)-2,6-dimethylaniline;hydrochloride Chemical compound Cl.CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 OBWSOTREAMFOCQ-UHFFFAOYSA-N 0.000 description 6
- 102000004316 Oxidoreductases Human genes 0.000 description 6
- 108090000854 Oxidoreductases Proteins 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- ALLIZEAXNXSFGD-UHFFFAOYSA-N 1-methyl-2-phenylbenzene Chemical group CC1=CC=CC=C1C1=CC=CC=C1 ALLIZEAXNXSFGD-UHFFFAOYSA-N 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 3
- 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
- WBODDOZXDKQEFS-UHFFFAOYSA-N 1,2,3,4-tetramethyl-5-phenylbenzene Chemical group CC1=C(C)C(C)=CC(C=2C=CC=CC=2)=C1C WBODDOZXDKQEFS-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
- 239000000872 buffer Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000523 sample 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
- 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
- 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
- 150000001412 amines Chemical class 0.000 description 1
- 239000003795 chemical substances by application Substances 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
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000012467 final product 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
- 238000007654 immersion Methods 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
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000012488 sample solution 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
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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- 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
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- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a kind of based on chitosan-platinum simulation oxidizing ferment Assay of acid phosphatase content method, it is characterized in that generating ascorbic acid using acid phosphatase enzyme hydrolysis ascorbic acid phosphoric acid esters, it influences chitosan-platinum and simulates oxidizing ferment/3,3 ', 5,5 '-tetramethyl biphenyl amine hydrochlorate Catalytic color reaction systems, the variation of binding soln color and ultra-violet absorption spectrum feature are directly used in the measurement of acid phosphatase and its inhibitor content.Within the scope of 0.25 ~ 2.5 U/L, absorbance change value △ A450In a linear relationship with acid phosphatase enzyme concentration, detection is limited to 0.0161 U/L.Operation of the present invention simplicity, high sensitivity can be applied to the measurement of acid phosphatase and its inhibitor content in environment and life science system as analysis method.
Description
Technical field
The present invention relates to the measurements that chitosan-platinum simulates the acid phosphatase and its inhibitor of oxidase catalyzed color development system
Method belongs to analytical chemistry and field of nanometer technology.
Background technique
Acid phosphatase is a kind of a kind of hydrolase of the hydrolysis of catalytic phosphatase monoesters in acid condition generation inorganic phosphate,
It is widely distributed in the tissue of some plants or mammal.Agriculture aspect, acid phosphatase is to judging crop growth
Optimal pH plays an important role.In terms of medicine, the weight of the diseases such as hepatitis, hyperparathyroidism, red blood cell lesion can be used as
Want diagnosis index.Therefore, the measurement of acid phosphatase content has far reaching significance.But the measurement of current acid phosphatase there is also
Many limitations, for example, existing method mainly selects 4-NPP as substrate, however the nitro that hydrolysis generates
Benzene needs just have stronger signal under alkaline condition, most suitable catalytic condition (pH=5.0) Xiang Mao of this and acid phosphatase
Shield.Therefore develop more effective, the accurate colorimetric method of one kind to measure acid phosphatase with very important practical significance.
Acid phosphatase is 37 DEG C in temperature, and pH can hydrolyze ascorbic acid phosphoric acid esters under conditions of being 5.0, is generated anti-bad
Hematic acid.And the ascorbic acid with strong reduced form can inhibit oxidizing ferment to urge 3,3',5,5'-tetramethylbenzidine hydrochloride
Oxidation, so that the colour developing of 3,3',5,5'-tetramethylbenzidine hydrochloride is unobvious.The content of acid phosphatase is higher, body
The absorption angle value of system is smaller.Based on this, it can be achieved that being quantitative determined to acid phosphatase.
The present invention simulates oxidase catalyzed oxidation using ascorbic acid phosphoric acid esters as acid phosphatase zymolyte, with chitosan-platinum
3,3',5,5'-tetramethylbenzidine hydrochloride color development system is sensed signal sources, establishes a kind of colorimetric estimation acid phosphatase
Method.Acid phosphatase enzyme catalyzed hydrolysis and chitosan-platinum simulation 3,3 ', 5,5 '-tetramethyls of oxidase catalyzed oxidation connection
Anilinechloride chromogenic reaction can be carried out at same pH.This method is easy, quick, is not related to any complicated, valuable instrument,
Testing cost is low.
Summary of the invention
3,3 ', 5,5 '-tetramethyls of oxidase catalyzed oxidation are simulated using chitosan-platinum the object of the present invention is to provide a kind of
Benzidine dihydrochloride color development system, using ascorbic acid phosphoric acid esters as substrate, to measure the side of acid phosphatase and its inhibitor
Method.
To achieve the goals above, the invention adopts the 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 anti-
3,3',5,5'-tetramethylbenzidine HCI solution is added in bad hematic acid phosphate and acid phosphatase reaction product and shell is poly-
Sugar-platinum simulation oxidation enzyme solutions, is added sulfuric acid solution and terminates reaction, according to solution colour and ultra-violet absorption spectrum feature after reaction
Variation, the measurement for acid phosphatase content；Used chitosan-platinum simulation oxidizing ferment is to use chitosan as stabilization
Agent, sodium borohydride reduction chloroplatinic acid obtain, and specific synthesis step is as follows: weighing 0.1 g chitosan and being dissolved in 50 mL concentration is 1%
In the acetic acid of v/v, stirring makes chitosan be completely dissolved the chitosan solution for obtaining 0.002 g/mL of concentration in 15 minutes, by 2
ML concentration is that 10 mmol/L chloroplatinic acids are added to 47 mL concentration and are and stir 30 minutes in 0.002 g/mL chitosan solution, so
1 mL concentration is added dropwise afterwards as the 0.2 mol/L sodium borohydride solution newly prepared and is added within 5 minutes, is placed in dark place and stirs
It mixes 90 minutes, obtains chitosan-platinum simulation oxidizing ferment, products therefrom is protected from light stored refrigerated.
The 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
Being added to 200 μ L pH for the ascorbic acid phosphoric acid esters of 4 mmol/L and the acid phosphatase of 50 μ L various concentrations is 5, concentration
In acetate buffer for 50 mmol/L, is reacted 30 minutes at 37 DEG C after mixing, then sequentially add 632.5 μ L
PH is 5,3,3 ', 5, the 5 '-tetramethyls connection that concentration is the acetate buffer of 50 mmol/L, 50 μ L concentration are 3 mmol/L
Anilinechloride solution and 17.5 μ L chitosans-platinum simulation oxidation enzyme solutions, react 5 minutes at 37 DEG C again after mixing,
The sulfuric acid solution that 200 μ L concentration are 2 mol/L is added and terminates reaction, measures the absorbance value A of solution450。
It is described based on chitosan-platinum simulation oxidizing ferment Assay of acid phosphatase content method, it is characterized in that 0.25 ~
Within the scope of 2.5 U/L, absorbance change value △ A450In a linear relationship with acid phosphatase enzyme concentration, detection is limited to 0.0161 U/L.
Method of the present invention based on chitosan-platinum simulation oxidizing ferment measurement human serum acid phosphatase, it is special
Sign is to include the following steps: 50 μ L concentration being added to 200 for the ascorbic acid phosphoric acid esters of 4 mmol/L and 50 μ L human serums
μ L pH is 5, and concentration is to react at 37 DEG C 30 minutes, successively will after mixing in the acetate buffer of 50 mmol/L
632.5 μ L pH are 5, and concentration is the acetate buffer of 50 mmol/L, 50 μ L concentration are 3,3 ', 5, the 5 '-of 3 mmol/L
Tetramethyl biphenyl amide hydrochloride and 17.5 μ L chitosans-platinum simulation oxidation enzyme solutions are added in above-mentioned reaction solution, are mixed
It is reacted 5 minutes at 37 DEG C again after uniformly, the sulfuric acid solution that 200 μ L concentration are 2 mol/L is added and terminates reaction, measures solution
Absorbance value A450, the content of alkaline phosphatase in human serum is calculated according to acid phosphatase standard curve；Used shell is poly-
Sugar-platinum simulation oxidizing ferment is to use chitosan as stabilizer, and sodium borohydride reduction chloroplatinic acid obtains, and specific synthesis step is as follows:
It weighs 0.1 g chitosan to be dissolved in the acetic acid that 50 mL concentration are 1 %v/v, stirring is completely dissolved chitosan in 15 minutes to obtain the final product
2 mL concentration are that 10 mmol/L chloroplatinic acids are added to 47 mL concentration and are by the chitosan solution for being 0.002 g/mL to concentration
It in 0.002 g/mL chitosan solution, stirs 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, obtains chitosan-platinum simulation oxidizing ferment.
The method based on chitosan-platinum simulation oxidizing ferment measurement human serum acid phosphatase, it is characterized in that
The rate of recovery of human serum sample's measurement is 97.4% ~ 107.8%, and relative standard deviation is 2.4 ~ 6.4%.
Measuring method of the present invention based on chitosan-platinum simulation oxidizing ferment acid phosphatase inhibitors, it is special
Sign 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, are added sulfuric acid solution and terminate reaction after reaction, according to
The variation of solution colour and ultra-violet absorption spectrum feature, the measurement for Inhibitors of Alkaline Phosphatase inhibiting rate；Used shell
Glycan-platinum simulation oxidizing ferment is to use chitosan as stabilizer, and sodium borohydride reduction chloroplatinic acid obtains, and specific synthesis step is such as
Under: it weighs 0.1 g chitosan and is 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 0.002 g/mL is obtained, is that 10 mmol/L chloroplatinic acids are added to 47 mL concentration by 2 mL concentration
To stir 30 minutes in 0.002 g/mL chitosan solution, it is the boron that 0.2 mol/L is newly prepared that 1 mL concentration, which is then added dropwise,
Sodium hydride solution simultaneously adds within 5 minutes, is placed in dark place and stirs 90 minutes, obtains chitosan-platinum simulation oxidizing ferment.
The measuring method based on chitosan-platinum simulation oxidizing ferment acid phosphatase inhibitors, 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
PH to 200 μ L sodium fluorides containing various concentration is 5, and concentration is to exist after mixing in the acetate buffer of 50 mmol/L
It is reacted 30 minutes at 37 DEG C, sequentially adding 632.5 μ L pH is 5, and concentration is acetate buffer, the 50 μ L of 50 mmol/L
The 3,3',5,5'-tetramethylbenzidine HCI solution and 17.5 μ L chitosans-platinum that concentration is 3 mmol/L simulate oxidizing ferment
Solution reacts 5 minutes at 37 DEG C again after mixing, and the sulfuric acid solution that 200 μ L concentration are 2 mol/L is added and terminates reaction,
Measure the absorbance value A of solution450, inhibiting rate is calculated, is fitted to obtain the IC of sodium fluoride by software50For 0.896 mmoL/L.
Specifically, the present invention adopts the following technical scheme:
(1) chitosan-platinum simulation oxidizing ferment preparation: weighing 0.1 g chitosan and being dissolved in 50 mL concentration is 1%v/v's
In acetic acid, stirring makes chitosan be completely dissolved the chitosan solution for obtaining 0.002 g/mL of concentration in 15 minutes.2 mL are dense
Degree is that 10 mmol/L chloroplatinic acids are added to 47 mL concentration and are and stir 30 minutes in 0.002 g/mL chitosan solution, then by
Being added dropwise to 1 mL concentration is the sodium borohydride solution (adding within 5 minutes) that 0.2 mol/L is newly prepared, and is placed in dark place and stirs 90 points
Clock obtains chitosan-platinum simulation oxidizing ferment, and products therefrom is protected from light stored refrigerated.
(2) measurement of acid phosphatase: 50 μ L concentration are different for the ascorbic acid phosphoric acid esters of 4 mmol/L and 50 μ L
The acid phosphatase of concentration is added in 200 μ L acetate buffers (5,50 mmol/L of pH), after mixing at 37 DEG C
Reaction 30 minutes；Sequentially add 632.5 μ L acetate buffers (5,50 mmol/L of pH), 50 μ l concentration are 3 mmol/L's
3,3',5,5'-tetramethylbenzidine HCI solution and the chitosan of 17.5 μ l step (1) preparation-platinum simulation oxidizing ferment are molten
Liquid reacts 5 minutes at 37 DEG C again after mixing, and the sulfuric acid solution that 200 μ L concentration are 2 mol/L is added and terminates reaction, surveys
Determine absorbance value (A of the solution at 450 nm wavelength450), it draws standard curve and carries out acid phosphatase assay.
(3) measurement of acid phosphatase inhibitors: the acid phosphatase enzyme solutions and 50 for being 0.1 U/mL by 50 μ L concentration
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 concentration
In liquid (5,50 mmol/L of pH), reacted 30 minutes at 37 DEG C after shaking up.Sequentially add 632.5 μ L acetate buffer (pH
5,50 mmol/L), 50 μ L concentration be 3 mmol/L 3,3',5,5'-tetramethylbenzidine HCI solution and 17.5 μ L step
Suddenly the chitosan of (one) preparation-platinum simulation oxidation enzyme solutions, react 5 minutes at 37 DEG C again after mixing, 200 μ L are added
The sulfuric acid solution that concentration is 2 mol/L terminates reaction, measures solution absorbance value A450.It is fitted by software, obtains sodium fluoride
503nhibiting concentration IC50。
Advantages of the present invention
(1) present invention generates ascorbic acid using acid phosphatase enzyme hydrolysis ascorbic acid phosphoric acid esters, to inhibit chitosan-
Platinum simulates oxidase catalyzed coloring reaction system, and the variation of binding soln color and ultra-violet absorption spectrum feature is directly used in acid
The measurement of acid phosphatase and its inhibitor content.
(2) chitosan prepared by the present invention-platinum simulation oxidizing ferment is by chitosan as stabilizer, sodium borohydride reduction chlorine platinum
Acid obtains, and preparation process is simple and quick.
(3) detecting step of the present invention is simple, is not related to any complicated, valuable instrument, and 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.
Detailed description of the invention
Fig. 1 is outside drawing.A control group in figure: chitosan-platinum simulates+3,3 ', 5,5 '-tetramethyl biphenyl of oxidizing ferment
Amine hydrochlorate；B control group in figure: chitosan-platinum simulation oxidizing ferment+3,3',5,5'-tetramethylbenzidine hydrochloride+
Ascorbic acid phosphoric acid esters；C control group in figure: chitosan-platinum simulates oxidizing ferment+3,3',5,5'-tetramethylbenzidine salt
Hydrochlorate+acid phosphatase；D experimental group in figure: chitosan-platinum simulates oxidizing ferment+3,3',5,5'-tetramethylbenzidine
Hydrochloride+ascorbic acid phosphoric acid esters+acid phosphatase.
Fig. 2 is uv absorption spectra.A control group in figure: chitosan-platinum simulates oxidizing ferment+3,3 ', 5,5 '-four
Methyl biphenyl amine hydrochlorate；B control group in figure: chitosan-platinum simulates oxidizing ferment+3,3',5,5'-tetramethylbenzidine
Hydrochloride+ascorbic acid phosphoric acid esters；C control group in figure: chitosan-platinum simulates+3,3 ', 5,5 '-tetramethyl of oxidizing ferment
Benzidine dihydrochloride+acid phosphatase；D experimental group in figure: chitosan-platinum simulates+3,3 ', 5,5 '-tetramethyl of oxidizing ferment
Base benzidine dihydrochloride+ascorbic acid phosphoric acid esters+acid phosphatase.
Fig. 3 is that acid phosphatase enzyme concentration and chitosan-platinum simulate oxidase catalyzed color development system absorbance value A450It is linear
Relational graph.
Fig. 4 is Assay of acid phosphatase content interference experiment.Number 0 ~ 11 successively respectively represents blank, acid phosphatase, alkalinity
Phosphatase, horseradish peroxidase, pyrophosphohydrolase, acetylcholinesterase, alpha-glucosidase, choline oxidase, albumen
Enzyme k, catalase, lysozyme and urase.
Fig. 5 is sodium fluoride inhibiting rate curve graph.
Specific embodiment
Embodiment 1:
It weighs 0.1 g chitosan to be dissolved in acetic acid of the 50 mL concentration for 1%(v/v), stirring keeps chitosan complete in 15 minutes
Fully dissolved is to obtain the chitosan solution that concentration is 0.002 g/mL.It is that 10 mmol/L chloroplatinic acids are added to 47 by 2 mL concentration
ML concentration is to stir 30 minutes in 0.002 g/mL chitosan solution, and it is that 0.2 mol/L newly matches that 1 mL concentration, which is then added dropwise,
The sodium borohydride solution (adding within 5 minutes) of system is placed in dark place and stirs 90 minutes, obtains auburn chitosan-platinum simulation
Aoxidize enzyme solutions.Product is protected from light stored refrigerated.All glasswares used in above procedure pass through chloroazotic acid immersion, and with pair
It steams water thoroughly to clean, dry.
Embodiment 2:
By 50 μ L concentration be 4 mmol/L ascorbic acid phosphoric acid esters and 50 μ L concentration be 0.1 U/mL acid phosphatase
Enzyme is added in 200 μ L acetate buffers (5,50 mmol/L of pH), is reacted 30 minutes at 37 DEG C after mixing.According to
Secondary addition 632.5 μ L acetate buffers (5,50 mmol/L of pH), 3,3 ', 5,5 '-four that 50 μ L concentration are 3 mmol/L
Chitosan made from methyl biphenyl amide hydrochloride and 17.5 μ L embodiments 1-platinum simulation oxidation enzyme solutions, after mixing
It is reacted 5 minutes at 37 DEG C again, the sulfuric acid solution that 200 μ L concentration are 2 mol/L is added and terminates reaction, visually observes color
The uv-visible absorption spectra of variation or measurement solution.Visually when observation color change, control group solution colour is deep yellow
Color, experimental group solution colour become colorless (see figure 1).When measuring uv-visible absorption spectra, the absorption spectrum of control group is almost
It does not change, and (see figure 2) is obviously reduced in absorption of the absorption spectrum of experimental group in 350 ~ 550 regions nm.
Embodiment 3:
The acid phosphatase of ascorbic acid phosphoric acid esters and 50 μ L various concentrations that 50 μ L concentration are 4 mmol/L is added
To in 200 μ L acetate buffers (5,50 mmol/L of pH), reacted 30 minutes at 37 DEG C after mixing.It sequentially adds
632.5 μ L acetate buffers (5,50 mmol/L of pH), 3,3 ', 5, the 5 '-tetramethyls that 50 μ L concentration are 3 mmol/L join
Chitosan made from anilinechloride solution and 17.5 μ L embodiments 1-platinum simulation oxidation enzyme solutions, after mixing again 37
It is reacted 5 minutes at DEG C, the sulfuric acid solution that 200 μ L concentration are 2 mol/L is added and terminates reaction, measures the absorbance value of solution
A450.From the figure 3, it may be seen that with the increase of acid phosphatase enzyme concentration, absorbance value A450Changing value (△ A450) be gradually increased.?
Within the scope of 0.25 ~ 2.5 U/L, △ A450In a linear relationship with acid phosphatase enzyme concentration, lowest detection is limited to 0.0161 U/L.
Embodiment 4:
By 50 μ L concentration be 4 mmol/L ascorbic acid phosphoric acid esters and 50 μ L concentration be 0.0125 U/mL acid phosphorus
Sour enzyme is added in 200 μ L acetate buffers (5,50 mmol/L of pH), is reacted 30 minutes at 37 DEG C after mixing.
Sequentially add 632.5 μ L acetate buffers (5,50 mmol/L of pH), 3,3 ', 5,5 '-that 50 μ L concentration are 3 mmol/L
Chitosan made from tetramethyl biphenyl amide hydrochloride and 17.5 μ L embodiments 1-platinum simulation oxidation enzyme solutions, is uniformly mixed
It is reacted 5 minutes at 37 DEG C again afterwards, the sulfuric acid solution that 200 μ L concentration are 2 mol/L is added and terminates reaction, measures the suction of solution
Shading value A450.It repeats above-mentioned experimental procedure 9 times, obtaining relative standard deviation (RSD) is 3.9%, shows that this method reproducibility is good.
Embodiment 5:
Normal human serum is taken, is sufficiently mixed with the acid phosphatase of various concentration according to the volume ratio of 1:4, it is molten to obtain sample
Liquid.50 μ L concentration are added to 200 μ L acetate for the ascorbic acid phosphoric acid esters of 4 mmol/L and the 50 above-mentioned sample solutions of μ L
In buffer (5,50 mmol/L of pH), reacted 30 minutes at 37 DEG C after mixing.It is sequentially added in above-mentioned reaction solution
632.5 μ L acetate buffers (5,50 mmol/L of pH), 3,3 ', 5, the 5 '-tetramethyls that 50 μ L concentration are 3 mmol/L join
Chitosan made from anilinechloride solution and 17.5 μ L embodiments 1-platinum simulation oxidation enzyme solutions, after mixing again 37
It is reacted 5 minutes at DEG C, the sulfuric acid solution that 200 μ L concentration are 2 mol/L is added and terminates reaction, measures the absorbance value of solution
A450.In conjunction with the embodiments 3 calculate normal human serums in acid phosphatase content, measurement 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: 50 μ L concentration are dense for the ascorbic acid phosphoric acid esters of 4 mmol/L and 50 μ L
Degree is that other enzyme solutions of 1 U/mL are added in 200 μ L acetate buffers (5,50 mmol/L of pH), after mixing 37
It is reacted 30 minutes at DEG C.Sequentially add 632.5 μ L acetate buffers (5,50 mmol/L of pH), 50 μ L concentration are 3
Chitosan made from the 3,3',5,5'-tetramethylbenzidine HCI solution of mmol/L and 17.5 μ L embodiments 1-platinum simulates oxygen
Change enzyme solutions, reacted 5 minutes at 37 DEG C again after mixing, the sulfuric acid solution that 200 μ L concentration are 2 mol/L is added immediately
Reaction is terminated, the absorbance value A of solution is measured450.As shown in Figure 4, even if the concentration of other enzymes is 10 times higher than acid phosphatase,
Apparent interference cannot be still generated, shows that this method has good selectivity acid phosphatase.
Embodiment 7:
By 50 μ L concentration be 4 mmol/L ascorbic acid phosphoric acid esters and 50 μ L concentration be 0.1 U/mL acid phosphatase
Enzyme solutions are added in the acetate buffer (5,50 mmol/L of pH) of 200 μ L sodium fluorides containing various concentration, after mixing
It is reacted 30 minutes at 37 DEG C.Sequentially add 632.5 μ L acetate buffers (5,50 mmol/L of pH), 50 μ L concentration are 3
Chitosan made from the 3,3',5,5'-tetramethylbenzidine HCI solution of mmol/L and 17.5 μ L embodiments 1-platinum simulates oxygen
Change enzyme solutions, reacted 5 minutes at 37 DEG C again after mixing, the sulfuric acid solution that 200 μ L concentration are 2 mol/L is added and terminates
Reaction, measures the absorbance value A of solution450, calculate inhibiting rate.As a result as shown in figure 5, being fitted to obtain sodium fluoride by software
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 in essence of the invention
Made any modification within mind and principle, equivalent replacement and improvement etc., should all be included in the protection scope of the present invention.
Claims (7)
1. it is a kind of based on chitosan-platinum simulation oxidizing ferment Assay of acid phosphatase content method, it is characterized in that in ascorbic acid phosphoric acid
3,3',5,5'-tetramethylbenzidine HCI solution is added in ester and acid phosphatase reaction product and chitosan-platinum simulates oxygen
Change enzyme solutions, sulfuric acid solution is added after reaction and terminates reaction, according to the variation of solution colour and ultra-violet absorption spectrum feature, is used for
The measurement of acid phosphatase content；Used chitosan-platinum simulation oxidizing ferment is to use chitosan as stabilizer, sodium borohydride
Reduction chloroplatinic acid obtains, and specific synthesis step is as follows: weighing 0.1 g chitosan and is 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 0.002 g/mL of concentration in 15 minutes, is by 2 mL concentration
It is to stir 30 minutes, then add dropwise in 0.002 g/mL chitosan solution that 10 mmol/L chloroplatinic acids, which are added to 47 mL concentration,
Enter 1 mL concentration for the 0.2 mol/L sodium borohydride solution newly prepared and added within 5 minutes, is placed in dark place and stirs 90 points
Clock obtains chitosan-platinum simulation oxidizing ferment, and products therefrom is protected from light stored refrigerated.
2. it is according to claim 1 based on chitosan-platinum simulation oxidizing ferment Assay of acid phosphatase content method, it is characterized in that
The acid phosphatase of ascorbic acid phosphoric acid esters and 50 μ L various concentrations that 50 μ L concentration are 4 mmol/L is added to 200 μ L
PH is 5, and concentration is to react 30 minutes at 37 DEG C after mixing, then successively in the acetate buffer of 50 mmol/L
It is 5 that 632.5 μ L pH, which are added, and concentration is the acetate buffer of 50 mmol/L, 50 μ L concentration are the 3,3 ' of 3 mmol/L,
5,5 '-tetramethyl biphenyl amide hydrochlorides and 17.5 μ L chitosans-platinum simulation oxidation enzyme solutions, after mixing again 37
It is reacted 5 minutes at DEG C, the sulfuric acid solution that 200 μ L concentration are 2 mol/L is added and terminates reaction, measures the absorbance value of solution
A450。
3. it is according to claim 1 or 2 based on chitosan-platinum simulation oxidizing ferment Assay of acid phosphatase content method, it is special
Sign is the absorbance change value △ A within the scope of 0.25 ~ 2.5 U/L450It is in a linear relationship with acid phosphatase enzyme concentration, detection limit
For 0.0161 U/L.
4. a kind of method based on chitosan-platinum simulation oxidizing ferment measurement human serum acid phosphatase, it is characterized in that including such as
Lower step: by 50 μ L concentration be 4 mmol/L ascorbic acid phosphoric acid esters and 50 μ L human serums to be added to 200 μ L pH be 5,
Concentration is to react at 37 DEG C after mixing 30 minutes, in the acetate buffer of 50 mmol/L successively by 632.5 μ L
PH is 5,3,3 ', 5, the 5 '-tetramethyls connection that concentration is the acetate buffer of 50 mmol/L, 50 μ L concentration are 3 mmol/L
Anilinechloride solution and 17.5 μ L chitosans-platinum simulation oxidation enzyme solutions are added in above-mentioned reaction solution, after mixing again
It is reacted 5 minutes at 37 DEG C, the sulfuric acid solution that 200 μ L concentration are 2 mol/L is added and terminates reaction, measures 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
Quasi- oxidizing ferment is to use chitosan as stabilizer, and sodium borohydride reduction chloroplatinic acid obtains, and specific synthesis step is as follows: weighing 0.1
G chitosan is dissolved in the acetic acid that 50 mL concentration are 1 %v/v, and stirring, which is completely dissolved chitosan in 15 minutes, obtains concentration
It is that be added to 47 mL concentration be 0.002 g/ to 10 mmol/L chloroplatinic acids by 2 mL concentration for the chitosan solution of 0.002 g/mL
It in mL chitosan solution, stirs 30 minutes, it is the sodium borohydride solution that 0.2 mol/L is newly prepared that 1 mL concentration, which is then added dropwise,
And added within 5 minutes, it is placed in dark place and stirs 90 minutes, obtain chitosan-platinum simulation oxidizing ferment.
5. the method according to claim 4 based on chitosan-platinum simulation oxidizing ferment measurement human serum acid phosphatase,
It is characterized in that the rate of recovery of human serum sample's measurement is 97.4% ~ 107.8%, relative standard deviation is 2.4 ~ 6.4%.
6. a kind of measuring method based on chitosan-platinum simulation oxidizing ferment acid phosphatase inhibitors, it is characterized in that anti-bad
3,3',5,5'-tetramethylbenzidine 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, are added sulfuric acid solution and terminate reaction after reaction, according to solution colour and
The variation of ultra-violet absorption spectrum feature, the measurement for Inhibitors of Alkaline Phosphatase inhibiting rate；Used chitosan-platinum simulation
Oxidizing ferment is to use chitosan as stabilizer, and sodium borohydride reduction chloroplatinic acid obtains, and specific synthesis step is as follows: weighing 0.1 g
Chitosan is dissolved in the acetic acid that 50 mL concentration are 1 %v/v, and stirring, which is completely dissolved chitosan in 15 minutes, obtains concentration
2 mL concentration are that be added to 47 mL concentration be 0.002 g/mL to 10 mmol/L chloroplatinic acids by the chitosan solution of 0.002 g/mL
In chitosan solution, stir 30 minutes, be then added dropwise 1 mL concentration be the sodium borohydride solution newly prepared of 0.2 mol/L simultaneously
It is added within 5 minutes, is placed in dark place and stirs 90 minutes, obtain chitosan-platinum simulation oxidizing ferment.
7. the measuring method according to claim 6 based on chitosan-platinum simulation oxidizing ferment acid phosphatase inhibitors,
It is characterized in that by 50 μ L concentration be 4 mmol/L ascorbic acid phosphoric acid esters and 50 μ L concentration be 0.1 U/mL acid phosphatase
The pH that enzyme solutions are added to 200 μ L sodium fluorides containing various concentration is 5, and concentration is to mix in the acetate buffer of 50 mmol/L
It is reacted 30 minutes at 37 DEG C after closing uniformly, sequentially adding 632.5 μ L pH is 5, and the acetate that concentration is 50 mmol/L is slow
Fliud flushing, the 3,3',5,5'-tetramethylbenzidine HCI solution and 17.5 μ L chitosans-platinum that 50 μ L concentration are 3 mmol/L
Simulation oxidation enzyme solutions, react 5 minutes at 37 DEG C again after mixing, and it is molten that the sulfuric acid that 200 μ L concentration are 2 mol/L is added
Liquid terminates reaction, measures the absorbance value A of solution450, inhibiting rate is calculated, is fitted to obtain the IC of sodium fluoride by software50For
0.896 mmoL/L。
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