CN103910828A - Polydiacetylene color-changing vesicle and application thereof in activity analysis of beta-glucuronidase - Google Patents
Polydiacetylene color-changing vesicle and application thereof in activity analysis of beta-glucuronidase Download PDFInfo
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Abstract
The invention discloses a polydiacetylene color-changing vesicle and an application thereof in activity analysis of beta-glucuronidase. The polydiacetylene color-changing vesicle is polymerized by two polydiacetylene monomers and can be applied to activity analysis of beta-glucuronidase and construction of a high throughput screening enzyme inhibitor. When the vesicle is applied to activity analysis of beta-glucuronidase, the enzyme can specifically hydrolyze the glucoside group on the surface of the polydiacetylene micro-capsule to initiate self-decomposition reaction of a 3-nitryl-4-hydroxyl benzyl alcohol connecting chain so as to initiate color change of the polydiacetylene micro-capsule and generate fluorescence. The activity of beta-glucuronidase can be detected in real time by analyzing the change degree of color and the degree of fluorescence enhancement. The result can be directly observed by naked eyes, and the application has the characteristics of simplicity, rapidness, high sensitivity and the like. Meanwhile, the vesicle disclosed by the invention can be compatible with a high throughput microplate mode so as to realize high throughput screen of the beta-glucuronidase inhibitor.
Description
Technical field
The invention belongs to biochemical analysis field, be specifically related to a kind of poly-diacetylene variable color vesica and the application for GRD beta-glucuronidase activation analysis thereof.
Background technology
Poly-diacetylene (PDA) is to be formed containing the compound polymerization of diacetylene structure by monomer, as the important conjugated polymers of a class, PDA has following advantage: 1) monomer of PDA can be by 254nm ultraviolet lamp or γ-ray-initiated polymerization, undertaken 1 by diacetylene, 4 add and are shaped as alkene-alkynes conjugated backbone, do not need catalyzer or initiator, the very simple and environmental protection of the preparation that makes PDA.2) PDA monomer is colourless before polymerization not, when becoming blueness after polymerization, occur at 640nm place absorbing the most by force, be subject to after the stimulation of external environment, polymer color can become redness from indigo plant, occur at 550nm place absorbing the most by force, the transformation of this color can be passed through naked eyes perception, makes PDA have unique advantage in colorimetric sensing.3) blue color states of PDAs does not have fluorescence, and the aobvious red fluorescence of its red status, therefore, can be for building taking fluorescence as basic sensor-based system.People utilize above feature to develop a large amount of vesicas based on PDA and film-sensing system for detection of molecule important in biological and chemical.At present, people utilize the variable color of PDA vesica and fluorescent characteristic to realize the detection to multiple analytes such as virus, protein, bacteriums.
Self-decomposition is eliminated reaction and is referred to, in the compound being formed by complex construction, in the time that certain group is destroyed or remove, spontaneous transfer transport in whole compound structure generation molecule, thereby cause the elimination reaction of total, make compound be decomposed into several simple structures by complex construction.
GRD beta-glucuronidase is a kind of acid hydrolase, is extensively present in organism, can contain β-D-Glucose aldehydic acid glycoside compound by catalytic hydrolysis.At present, this enzyme has been used as the marker of tumour, the saccharase of tumour prodrug treatment, and the tracking enzyme of gene transport agent and obtained research widely.Meanwhile, this enzyme expression contents is in vivo relevant to various diseases, as sacroiliitis, bacterium and virus infection, cancer etc.In recent years, people have developed fluorescence imaging, Magnetic resonance imaging and radioactive element imaging method and have studied this enzyme activity in vivo and in vitro, but these methods all need expensive instrument as support, and complex operation step, cost is high, is unfavorable for enzymic activity research and involved enzyme inhibitor screening.Therefore, exploitation simply, fast method to analyze this enzymic activity still very necessary.
Summary of the invention
In order to realize the real-time analysis to enzymic activity, the object of the invention is to, a kind of poly-diacetylene variable color vesica and the application for GRD beta-glucuronidase activation analysis thereof are provided.
In order to realize above-mentioned task, the present invention takes following technical solution:
A kind of poly-diacetylene variable color vesica, is characterized in that, formed by two kinds of diacetylene monomer polymerizations, described two kinds of diacetylene monomers are respectively:
1) structure is as 10 of formula I, 12-25 diine carboxylic acids (PCDA);
2) structure as the carboxyl terminal to 10,12-, 25 diine carboxylic acids of formula II carry out obtaining after covalent modification with β-D-Glucose aldehydic acid glucosides part and can there is the new monomer (PCDA-GlcA) of 3-nitro-4-hydroxy-benzyl alcohol connection chain of self-decomposition reaction:
The preparation method of above-mentioned poly-diacetylene variable color vesica is: the diacetylene monomer shown in formula I and formula II is dissolved in chloroform according to the amount of mol ratio 7:3, in Rotary Evaporators, remove all solvents, then add 80 DEG C of deionized waters ultrasonic 20 minutes, after membrane filtration with 0.4 μ m, 4 DEG C keep in Dark Place, within 15 minutes, carry out photopolymerization with the ultra violet lamp of 254nm before use, obtain poly-diacetylene variable color vesica.
Adopt polyacetylene variable color vesica of the present invention, for analyzing GRD beta-glucuronidase activity and building high flux screening enzyme inhibitors, wherein:
In the time analyzing GRD beta-glucuronidase activity, utilize β-D-Glucose aldehydic acid glycosides that enzyme can specific hydrolysis vesica surface, reaction is eliminated in self-decomposition along 3-nitro-4-hydroxy-benzyl alcohol connection chain in trigger molecule, and make the amino on connection chain exposed, cause that the static between micro-capsule attracts each other, thereby cause the variation of micro-capsule conjugated structure conformation, produce variable color and fluorescence and strengthen, by real-time analysis colour-change degree, realize the real-time detection to GRD beta-glucuronidase.
Prepared poly-diacetylene vesica analyze GRD beta-glucuronidase when active can with the feature of high-throughout microwell plate pattern compatibility, can be configured to high flux screening enzyme inhibitors.
In the time building inhibitor sifting, after the GRD beta-glucuronidase inhibitor of different concns is mixed with GRD beta-glucuronidase, join in poly-diacetylene vesica solution, due to the inhibition difference of different concns inhibitor to enzymic activity, cause the variable color degree of the poly-diacetylene vesica causing different with fluorescence enhancing degree, after the relation of analyzing between inhibitor concentration and colour-change degree and fluorescence enhancing degree, can obtain the concentration of the inhibitor required at the suppressed half of enzymic activity, i.e. IC
50value.
The poly-diacetylene variable color vesica that the present invention builds is in the time carrying out activation analysis to GRD beta-glucuronidase, its detection signal can directly be converted to colour-change, and is attended by Enhancement of Fluorescence, and method is simple and quick, highly sensitive, and intuitively reaction enzymes catalyzed reaction process of energy.Meanwhile, the invention provides a kind of method of screening GRD beta-glucuronidase inhibitor, can in microwell plate, carry out the potential inhibitor screening of enzyme by high-throughput, there is certain application value.
Brief description of the drawings
Fig. 1 is the principle schematic that the poly-diacetylene variable color vesica of preparation detects GRD beta-glucuronidase activity.
Fig. 2 is that add GRD beta-glucuronidase in the poly-diacetylene vesica of preparation after, absorption spectrum is schemed over time.
Fig. 3 is that add GRD beta-glucuronidase in the poly-diacetylene vesica of preparation after, fluorescence spectrum is schemed over time.
Fig. 4 be different concns GRD beta-glucuronidase while adding in the poly-diacetylene vesica of preparation CR response value scheme over time.
Fig. 5 be different concns GRD beta-glucuronidase while adding in the poly-diacetylene vesica of preparation 560nm place fluorescence intensity scheme over time.
Fig. 6 adds to hatch 1 result schematic diagram (wherein, A: colour-change figure as a child after GRD beta-glucuronidase and different concns inhibitor in the poly-diacetylene variable color micro-capsule of preparation; B: change in fluorescence figure).
Fig. 7 is by measuring the inhibitor concentration that calculates of Change of absorption and the graph of a relation of suppression efficiency.
Fig. 8 is by measuring the inhibitor concentration that calculates of fluorescence intensity change and the graph of a relation of suppression efficiency.
Below in conjunction with drawings and Examples, the invention will be further described.
Embodiment
Referring to accompanying drawing, the present embodiment provides a kind of poly-diacetylene variable color vesica, is formed by two kinds of diacetylene monomer polymerizations, and described two kinds of diacetylene monomers are respectively:
1) structure is as 10 of formula I, 12-25 diine carboxylic acids (PCDA);
2) structure as the carboxyl terminal to 10,12-, 25 diine carboxylic acids of formula II carry out obtaining after covalent modification with β-D-Glucose aldehydic acid glucosides part and can there is the new monomer (PCDA-GlcA) of 3-nitro-4-hydroxy-benzyl alcohol connection chain of self-decomposition reaction;
Concrete preparation process is as follows:
One, synthetic with β-D-Glucose aldehydic acid glucosides part diacetylene monomer
Synthesize and be divided into three parts, be respectively:
(1) the glucosides part with self-decomposition chain is synthetic
Taking three-O-ethanoyl-α-D-acetylbromoglycose aldehydic acid methyl esters as raw material and the glucosides part becoming with self-decomposition chain, concrete steps are as follows:
1) by three-O-ethanoyl-α-D-acetylbromoglycose aldehydic acid methyl esters (1.53g; 3.84mmol) be dissolved in the middle of 50mL anhydrous acetonitrile, under nitrogen protection, in solution, add 3-nitro-4-hydroxy benzaldehyde (1.29g; 7.70mmol) and silver suboxide (1.8g; 7.76mmol), stirring at room temperature 4 hours, reaction solution after filtration, concentrated after; resistates is dissolved in to 50mL ethyl acetate; wash 6 times with saturated sodium bicarbonate aqueous solution, anhydrous magnesium sulfate drying, concentrates to obtain beige solid.
2) beige solid (1.12g step 1) being obtained, 2.33mmol) be dissolved in the mixed solvent containing 10mL Virahol and 50mL chloroform, in solution, add silica gel (5g) and sodium borohydride (0.12g, 3.17mmol), mixed solution stirring reaction after 45 minutes at 0 DEG C, pour in the middle of 100mL frozen water, solids removed by filtration, organic phase obtains white solid after anhydrous magnesium sulfate drying.
3) by step 2) white solid (0.5g, 1.03mmol) that obtains is dissolved in the middle of 10mL anhydrous methylene chloride, under nitrogen protection; add wherein N; N '-carbonyl dimidazoles (0.34g, 2.07mmol) and DMAP (0.025g, 0.21mmol); mixed solution at room temperature stirred after 3 hours; water, saturated sodium bicarbonate aqueous solution respectively, and salt water washing, organic phase is after anhydrous magnesium sulfate drying; concentrate to obtain white solid, synthesized the glucosides part with self-decomposition chain.
(2) amido modified diacetylene molecule synthesis
Concrete steps are as follows:
1) by 10,12-25 diine carboxylic acid (PCDA, 0.5g, 1.33mmol) be dissolved in the middle of 20mL anhydrous methylene chloride, in the middle of this solution, add N-hydroxy-succinamide (0.3g, 1.56mmol) and 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) (0.17g, 1.5mmol), mixed solution at room temperature stirs after 2 hours and concentrates, 20mL ethyl acetate extraction for concentrated solution, and with deionized water wash, organic phase after anhydrous sodium sulfate drying, concentrated white solid.
2) by the white solid (0.5g in step 1), 1.06mmol) be dissolved in the middle of 20mL methylene dichloride, and slowly add wherein 2,2 '-(ethylidene dioxy) two ethamine (0.8mL, 5.4mmol), the mixed solution obtaining at room temperature stirred after 3 hours, and except desolventizing, resistates is dissolved in 30mL methylene dichloride, with deionized water wash, anhydrous magnesium sulfate drying, and purify (methylene chloride-methanol that eluent is volume ratio 9:1) with silicagel column, molecular formula obtained for [CH
3-(CH
2)
11-C ≡ C-C ≡-(CH
2)
8-CONH-(CH
2o)
2-NH
2] amination product, product is white solid.
(3) synthesizing with β-D-Glucose aldehydic acid glucosides part diacetylene monomer
Concrete steps are as follows:
1) by the end product obtaining in (1) (0.5g; 0.86mmol) be dissolved in 7mL anhydrous methylene chloride; under nitrogen protection, slowly drip wherein trifluoromethane sulfonic acid methyl esters (90 μ L, 0.8mmol); gained mixed solution stirring reaction 30 minutes at 0 DEG C; then add wherein 4mL ether, and at-20 DEG C, make the product precipitation generating, gained precipitates after filtration; after ether washing, dry.Gained solid is dissolved in 5mL anhydrous methylene chloride again, under nitrogen, add wherein the amidized diacetylene (0.4g obtaining in (2), 0.79mmol), under ice bath, add wherein 110 μ L triethylamines (0.79mmol), mixed solution reacted after 4 hours at 0 DEG C, make it restore to room temperature, then react 2 hours.Saturated common salt water washing for mixed solution, anhydrous magnesium sulfate drying, concentrated.Gained is silicagel column (eluent is the methylene chloride-methanol of volume ratio 9:1) purifying for solid, obtains light yellow solid.
2) by gained solid (0.2g in step 1), 0.2mmol) be dissolved in 5mL anhydrous methanol, under ice bath, add wherein the methyl alcohol of 28 μ L containing 30% sodium methylate (mass percent), this solution at room temperature reacted stirring reaction after 2 hours, 5 μ L acetic acid cancellation for reaction.After concentrated, silicagel column for product (eluent is methylene chloride-methanol-water of volume ratio 60:35:5) purifying, obtains glassy yellow solid, is target diacetylene monomer PCDA-GlcA.
Two, the poly-diacetylene variable color vesica of preparation
PCDA and PCDA-GlcA are dissolved in chloroform according to mol ratio 7:3, after mixing, on Rotary Evaporators, all solvents are removed in decompression, add 10mL deionized water, making mixture concentration is finally 1mmol/L, ultrasonic dispersion 20 minutes at 80 DEG C, obtain translucent settled solution, with after 0.4 μ m membrane filtration, 4 DEG C keep in Dark Place.When use, taken out, under room temperature, penetrated 15 minutes by the UV illumination of 254nm, obtained required variable color vesica.
Three, the poly-diacetylene variable color vesica of preparation detects GRD beta-glucuronidase activity
Detecting step is as follows: get variable color vesica solution prepared by 0.6mL and insert in 5mL centrifuge tube, adding 0.1mL concentration is the GRD beta-glucuronidase solution of 1mg/mL, the phosphate buffered that is 7.4 with pH (PBS) is diluted to 2.4mL, under the condition of 35 DEG C, hatch, observe vesica colour-change in time.Meanwhile, recording absorption curve every ten minutes with uv-visible absorption spectra and fluorescence spectrum changes and fluorescence growth degree.
As shown in Figure 2, along with incubation time increases, absorption curve blueing absorbs (λ ≈ 645nm) mutually to be reduced gradually, and red absorb mutually (λ ≈ 545nm) strengthens gradually.Meanwhile, this process follows fluorescence to strengthen gradually phenomenon, and its maximum emission wavelength is more 560nm.
In order to react more intuitively this enzymatic reaction process, use colorimetric response value (Colorimetric Response, CR%) and 560nm place fluorescence intensity to map to incubation time here, obtain the curvilinear motion process of enzymatic reaction.CR value can be used for weighing the colour-change degree of variable color vesica, and its calculation formula is: CR=[PB
0-PB
1]/PB
0× 100%, wherein PB=A
645/ [A
645+ A
545], A
645the absorption of vesica at blue phase 645nm place, A
545the absorption of vesica at red phase 545nm place, PB
0the ratio value in the time not adding GRD beta-glucuronidase, and PB
1to add the enzyme ratio value of different time points afterwards.
As shown in Figure 3, adding the fluorescence intensity at the CR value done after the GRD beta-glucuronidase of different concns and 560nm place with the changing conditions of incubation time in vesica solution, can find out, in the time that enzyme concn is lower (as 0.3 and 0.6 μ mmol), CR value and fluorescence intensity all present lasting slow propagation process, and in the time that enzyme concn is higher (as 1.2 and 2.4 μ mmol), CR value and fluorescence intensity change present first quick and back slow, finally reach the process of balance, this phenomenon meets the rule of general enzymatic reaction, the present invention success is described and has realized real-time detection enzymatic reaction process.
Four, the inhibitor screening of the poly-diacetylene variable color vesica of preparation to enzyme
Testing inhibitor solution preparation: choose saccharic acid-Isosorbide-5-Nitrae-lactone here as screening object, this material is known GRD beta-glucuronidase to be had to one of active inhibitor of better inhibition.Get saccharic acid-Isosorbide-5-Nitrae-lactone and be dissolved in the solution that is mixed with 1mmol/L in PBS, and be diluted to desired concn by the requirement of following experiment.
Screening step is as follows: the inhibition solution of 1mmol/L is diluted to respectively to 4 μ mol/L with PBS, 20 μ mol/L, 40 μ mol/L, 120 μ mol/L, 320 μ mol/L, the solution of 800 μ mol/L, get the solution of the each dilution of 50 μ L in 96 orifice plates, add respectively the GRD beta-glucuronidase solution described in 20 μ L examples 1, at 35 DEG C, hatch after 30 minutes, the poly-diacetylene vesica solution that adds respectively 50 μ L to prepare, and be diluted to 200 μ L with PBS, in the mixed solution finally obtaining, each inhibitor concentration is respectively 1 μ mol/L, 5 μ mol/L, 10 μ mol/L, 30 μ mol/L, 80 μ mol/L, 200 μ mol/L.In addition, control group is set to: 1) 50 μ L vesica solution dilution to 200 μ L, not containing enzyme solution and inhibitor solution; 2) 50 μ L vesica solution and 20 μ L enzyme solution are diluted to 200 μ L after mixing, not containing inhibitor.Above-mentioned each mixed solution is hatched after 1 hour at 35 DEG C, utilize digital camera and fluorescent microscope to gather respectively colour-change figure and change in fluorescence figure, its result as shown in Figure 4.As can be seen from the figure, along with inhibitor concentration raises, poly-diacetylene vesica solution colour changes less, a little less than following fluorescence more and more, in the time that inhibitor concentration reaches 200 μ mol/L, it is blue constant that solution colour almost keeps simultaneously, almost can't see fluorescence, the inhibitor inhibitory enzyme work more fully that this absolutely proves in high density, causes enzymic hydrolysis speed to decline, and cannot cause vesica variable color.
Meanwhile, utilize microplate reader to measure the suppression efficiency (IE) that each mixed solution can quantitative Analysis different concns inhibitor after the absorption at 645nm and 545nm place, its calculation formula is: IE=(R
a-R
b)/(R
0-R
b) × 100%, wherein R=A
545/ A
645, A is the absorption of each mixed solution at 645nm or 545nm place, R
afor containing its 545nm of mixed solution of inhibitor and the ratio that 645nm goes out to absorb, R
bfor the ratio that its 545nm of control group and the 645nm containing inhibitor do not go out to absorb, and R
0the ratio containing the control group of inhibitor and enzyme solution not, as shown in Figure 5, for the logarithm of inhibitor concentration to utilize above-mentioned formula calculate the mapping curve of IE value, the IC50 value that can estimate inhibitor from figure is 33 μ mol/L.
Fig. 6 be inhibitor concentration logarithm to utilize fluorescence intensity calculate the mapping curve of IE value, its calculation formula is: IE=(F
a-F
b)/(F
0-F
b) × 100%, wherein F
afor the fluorescence intensity of the mixed solution containing inhibitor, F
bfor not containing the fluorescence intensity of the control group of inhibitor, F
0for not containing the fluorescence intensity of the control group of inhibitor and enzyme solution.
Claims (5)
1. a poly-diacetylene variable color vesica, is characterized in that, is formed by two kinds of diacetylene monomer polymerizations, and described two kinds of diacetylene monomers are respectively:
1) structure is as 10 of formula I, 12-25 diine carboxylic acids (PCDA);
2) structure as the carboxyl terminal to 10,12-, 25 diine carboxylic acids of formula II carry out obtaining after covalent modification with β-D-Glucose aldehydic acid glucosides part and can there is the new monomer (PCDA-GlcA) of 3-nitro-4-hydroxy-benzyl alcohol connection chain of self-decomposition reaction;
2. gather as described in claim 1 the preparation method of diacetylene variable color vesica, it is characterized in that, diacetylene monomer shown in formula I and formula II is dissolved in chloroform according to the amount of mol ratio 7:3, in Rotary Evaporators, remove all solvents, then add 80 DEG C of deionized waters ultrasonic 20 minutes, after the membrane filtration with 0.4 μ m, 4 DEG C keep in Dark Place, within 15 minutes, carry out photopolymerization with the ultra violet lamp of 254nm before use, obtain poly-diacetylene variable color vesica.
3. poly-diacetylene variable color vesica claimed in claim 1 is for analyzing the active application with building high flux screening enzyme inhibitors of GRD beta-glucuronidase.
4. application as claimed in claim 3, it is characterized in that, in the time carrying out inhibitor screening, after the GRD beta-glucuronidase inhibitor of different concns is mixed with GRD beta-glucuronidase, join in poly-diacetylene vesica solution, due to the inhibition difference of different concns inhibitor to enzymic activity, cause the variable color degree of the poly-diacetylene vesica causing different with fluorescence enhancing degree, after the relation of analyzing between inhibitor concentration and colour-change degree and fluorescence enhancing degree, can obtain the concentration of the inhibitor required at the suppressed half of enzymic activity, i.e. IC
50value.
5. application as claimed in claim 3, it is characterized in that, in the time carrying out GRD beta-glucuronidase activation analysis, utilize β-D-Glucose aldehydic acid glycosides that enzyme can specific hydrolysis vesica surface, spontaneous elimination along 3-nitro-4-hydroxy-benzyl alcohol connection chain in trigger molecule is reacted, and make the amino on connection chain exposed, cause that the static between micro-capsule attracts each other, thereby cause the variation of micro-capsule conjugated structure conformation, producing variable color and fluorescence strengthens, by real-time analysis colour-change degree, realize the real-time detection to GRD beta-glucuronidase.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106634942A (en) * | 2016-12-04 | 2017-05-10 | 苏州大学 | Sunlight-driven thermochromic material and preparation method thereof |
| CN108844911A (en) * | 2018-09-20 | 2018-11-20 | 中国药科大学 | Application of the poly- diacetylene vesica of phosphatide as optical sensor in the detection of electropositive substance film affinity |
| CN113402646A (en) * | 2021-05-21 | 2021-09-17 | 西北工业大学 | Method for detecting silver ions |
| CN114957549A (en) * | 2022-05-17 | 2022-08-30 | 西北工业大学 | Polydiacetylene color-changing vesicle and method for preparing and detecting alkaline phosphatase activity |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102435572A (en) * | 2011-09-20 | 2012-05-02 | 中国药科大学 | Drug-film affinity measuring method based on polydiacetylene sensor |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102435572A (en) * | 2011-09-20 | 2012-05-02 | 中国药科大学 | Drug-film affinity measuring method based on polydiacetylene sensor |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106634942A (en) * | 2016-12-04 | 2017-05-10 | 苏州大学 | Sunlight-driven thermochromic material and preparation method thereof |
| CN106634942B (en) * | 2016-12-04 | 2019-09-10 | 苏州大学 | A kind of thermochromic material and preparation method thereof of sun optical drive |
| CN108844911A (en) * | 2018-09-20 | 2018-11-20 | 中国药科大学 | Application of the poly- diacetylene vesica of phosphatide as optical sensor in the detection of electropositive substance film affinity |
| CN113402646A (en) * | 2021-05-21 | 2021-09-17 | 西北工业大学 | Method for detecting silver ions |
| CN113402646B (en) * | 2021-05-21 | 2022-04-15 | 西北工业大学 | Method for detecting silver ions |
| CN114957549A (en) * | 2022-05-17 | 2022-08-30 | 西北工业大学 | Polydiacetylene color-changing vesicle and method for preparing and detecting alkaline phosphatase activity |
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