CN104437560A - Nano black selenium peroxide analogue enzyme and preparation method thereof - Google Patents
Nano black selenium peroxide analogue enzyme and preparation method thereof Download PDFInfo
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- CN104437560A CN104437560A CN201410559673.0A CN201410559673A CN104437560A CN 104437560 A CN104437560 A CN 104437560A CN 201410559673 A CN201410559673 A CN 201410559673A CN 104437560 A CN104437560 A CN 104437560A
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- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 47
- 239000011669 selenium Substances 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 title abstract description 24
- 108090000790 Enzymes Proteins 0.000 title abstract description 24
- -1 selenium peroxide Chemical class 0.000 title 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims abstract description 54
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 39
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 39
- JPJALAQPGMAKDF-UHFFFAOYSA-N selenium dioxide Chemical compound O=[Se]=O JPJALAQPGMAKDF-UHFFFAOYSA-N 0.000 claims abstract description 22
- 229920001940 conductive polymer Polymers 0.000 claims abstract description 21
- 238000006243 chemical reaction Methods 0.000 claims abstract description 19
- 230000003197 catalytic effect Effects 0.000 claims abstract description 13
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 10
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000002904 solvent Substances 0.000 claims abstract description 6
- 239000000758 substrate Substances 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 17
- 238000003756 stirring Methods 0.000 claims description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 239000008367 deionised water Substances 0.000 claims description 14
- 229910021641 deionized water Inorganic materials 0.000 claims description 14
- 238000006555 catalytic reaction Methods 0.000 claims description 12
- 235000011187 glycerol Nutrition 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 7
- 238000001291 vacuum drying Methods 0.000 claims description 7
- 238000004088 simulation Methods 0.000 claims description 3
- 239000012046 mixed solvent Substances 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 239000000126 substance Substances 0.000 abstract description 13
- 238000003786 synthesis reaction Methods 0.000 abstract description 10
- 230000015572 biosynthetic process Effects 0.000 abstract description 9
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- 239000007795 chemical reaction product Substances 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 239000002086 nanomaterial Substances 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 238000004925 denaturation Methods 0.000 abstract description 2
- 230000036425 denaturation Effects 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 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 abstract 1
- 239000003638 chemical reducing agent Substances 0.000 abstract 1
- 230000007547 defect Effects 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 22
- 239000002322 conducting polymer Substances 0.000 description 16
- 239000000178 monomer Substances 0.000 description 11
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 10
- 230000002255 enzymatic effect Effects 0.000 description 9
- 229920000767 polyaniline Polymers 0.000 description 9
- 239000003054 catalyst Substances 0.000 description 7
- 230000002779 inactivation Effects 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 229920000128 polypyrrole Polymers 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000006116 polymerization reaction Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- GKWLILHTTGWKLQ-UHFFFAOYSA-N 2,3-dihydrothieno[3,4-b][1,4]dioxine Chemical compound O1CCOC2=CSC=C21 GKWLILHTTGWKLQ-UHFFFAOYSA-N 0.000 description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 4
- YTPLMLYBLZKORZ-UHFFFAOYSA-N Thiophene Chemical compound C=1C=CSC=1 YTPLMLYBLZKORZ-UHFFFAOYSA-N 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- 230000007812 deficiency Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 239000007800 oxidant agent Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 229920001609 Poly(3,4-ethylenedioxythiophene) Polymers 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 108010015428 Bilirubin oxidase Proteins 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 229940059939 kayexalate Drugs 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 229930192474 thiophene Natural products 0.000 description 2
- WUPHOULIZUERAE-UHFFFAOYSA-N 3-(oxolan-2-yl)propanoic acid Chemical compound OC(=O)CCC1CCCO1 WUPHOULIZUERAE-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229910003321 CoFe Inorganic materials 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 239000005955 Ferric phosphate Substances 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010029541 Laccase Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- HSFWRNGVRCDJHI-UHFFFAOYSA-N alpha-acetylene Natural products C#C HSFWRNGVRCDJHI-UHFFFAOYSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229910052980 cadmium sulfide Inorganic materials 0.000 description 1
- MIOPJNTWMNEORI-UHFFFAOYSA-N camphorsulfonic acid Chemical compound C1CC2(CS(O)(=O)=O)C(=O)CC1C2(C)C MIOPJNTWMNEORI-UHFFFAOYSA-N 0.000 description 1
- 229910000420 cerium oxide Inorganic materials 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000006872 enzymatic polymerization reaction Methods 0.000 description 1
- 229940032958 ferric phosphate Drugs 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 229910021389 graphene Inorganic materials 0.000 description 1
- 238000011090 industrial biotechnology method and process Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N iron oxide Inorganic materials [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 1
- WBJZTOZJJYAKHQ-UHFFFAOYSA-K iron(3+) phosphate Chemical compound [Fe+3].[O-]P([O-])([O-])=O WBJZTOZJJYAKHQ-UHFFFAOYSA-K 0.000 description 1
- 229910000399 iron(III) phosphate Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000002122 magnetic nanoparticle Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000002159 nanocrystal Substances 0.000 description 1
- 239000002070 nanowire Substances 0.000 description 1
- 239000011368 organic material Substances 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- BMMGVYCKOGBVEV-UHFFFAOYSA-N oxo(oxoceriooxy)cerium Chemical compound [Ce]=O.O=[Ce]=O BMMGVYCKOGBVEV-UHFFFAOYSA-N 0.000 description 1
- NDLPOXTZKUMGOV-UHFFFAOYSA-N oxo(oxoferriooxy)iron hydrate Chemical compound O.O=[Fe]O[Fe]=O NDLPOXTZKUMGOV-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920001197 polyacetylene Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 235000019394 potassium persulphate Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 150000003233 pyrroles Chemical class 0.000 description 1
- 150000005839 radical cations Chemical class 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
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- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses nano black selenium with peroxidase simulating activity, and a preparation method of the nano black selenium, and belongs to the fields of preparation and application of nano materials. The preparation method is as follows: nano-sized black elemental selenium is prepared from selenium dioxide as a raw material and concentrated sulfuric acid as a reducing agent under a solvent thermal condition. The substance has catalytic activity of similar peroxidase; and chromogenic reaction of a peroxidase substrate 3,3',5,5'-tetramethyl benzidine and hydrogen peroxide can be catalyzed, so that the preparation method is simple; the reaction product is easy to separate and purify; the used reaction apparatus and reagent are low in price; the defects that a natural enzyme is difficult to purify and high in price are overcome; and the nano black selenium can be applied to a working environment with easy occurrence of denaturation, and has a good application prospect in the field of green synthesis of a water-soluble conductive polymer.
Description
Technical field
The present invention relates to a kind of nano black selenium Mimetic Peroxidase and preparation method thereof, metal nanometer material preparation and applied technical field thereof.
Background technology
Along with the enhancing of mankind's environmental consciousness and social sustainable development theory, enzymatic technique has become the focus of investigation and application in current chemical industrial field as a kind of synthetic technology of green.As the core of industrial biotechnology, enzyme technology is described as the most promising technology of industrial sustainable development.Living things catalysis and conversion technology will be that China's biochemical lines realizes mode of production change, the strong guarantee that product restructuring and clean and effective manufacture.In recent years, along with the rise of Green Chemistry, enzymatic is as an important component part of Green Chemistry, become one of most active research field in modern biology and chemical crossing domain, in the production of much enzymatic technique for the fine chemicals such as chiral drug, agricultural chemicals, and have and grow steadily, fast-developing trend.
Adulterated by polyacetylene with 1973 and find that high conductivity organic material is for starting point, conducting polymer receives the concern of each side day by day as new function material, especially Intrinsical grips conducting polymer altogether, has broad application prospects in electrochmical power source, electromagnetic shielding, antistatic, the field such as information storage and process, electrochromic material, sensor, stealth material.In many conducting polymers, polyaniline (being called for short PANI), polypyrrole (being called for short PPy) and poly-(3,4-ethylenedioxy thiophene) (be called for short PEDOT) because of its cheaper starting materials be easy to get, excellent chemical property and chemical stability, be considered to the conducting polymer being hopeful most to be applied in practice, thus become the study hotspot of conducting polymer.
At present, business-like conducting polymer is generally obtained by conventional chemical methods or electrochemical method, and the first step of reaction is all the oxidized generation radical cation of monomer.The advantage of conventional chemical methods is that productive rate is higher; can accomplish scale production; but the organic solvent of hostile environment must be used or use strong oxidizer just can complete first step reaction; these strong oxidizers can produce a large amount of accessory substances simultaneously; such as make oxidant with potassium peroxydisulfate; often obtain the polymer of 1 kilogram, the ammonium sulfate accessory substance of 1 kilogram will be produced, and need loaded down with trivial details purification and separation process after reaction.Electrochemical process relative environmental protection, but productive rate is too low, is difficult to accomplish scale production.In addition, current business-like conducting polymer is almost insoluble not to be melted, and is difficult to processing and forming.These deficiencies are all the huge obstacles expanding conductive polymer applications, and enzymatic polymerization exactly can overcome these deficiencies.
Enzyme is a kind of effective catalyst existed in organism, and enzymatic has oneself unique distinction.First, enzymatic has high efficiency and very high regioselectivity and stereoselectivity, and reacts great majority and can carry out in water, avoids the use of the organic solvent of contaminated environment; Secondly, enzyme catalyst reaction condition is gentle, and large quantity research confirms, enzyme can at environmentally friendly oxidant (such as molecular oxygen or H
2o
2) assistance under, the polymerization of the monomers such as catalysed aniline, pyrroles, thiophene, prepares conducting polymer, thus overcomes conventional chemical methods and adopt that accessory substance existing for strong oxidizer is many, the deficiency of separating-purifying complex steps; 3rd, the conducting polymer that enzymatic generates mostly has water-soluble, and this is for the processability improving conducting polymer, and expanding its range of application is highly significant.
The report that relevant native enzyme catalyzes and synthesizes conducting polymer the earliest sees the Journal of Biotechnology magazine of nineteen ninety, author adopts bilirubin oxidase (bilirubin oxidase) the catalysis polymerization of aniline, prepare polyaniline film (J.Biotechnol.1990,14,3-4:301-309).Since then, the report of enzymatic aniline polymerization emerges in multitude out, and wherein studying enzyme is the most widely horseradish peroxidase (HRP) (Macromolecules, 2004,37,4130-4138).Afterwards, people find again successively, palm peroxidase (the Enzyme Microb.Technol.2003 extracted from imperial palm, 33 (5): 661-667) laccase (J.Appl.Polym.Sci.2009, extracted from whiterot fungi bacterial strain, 114:928-934), the soybean peroxidase (Eur.Polym.J.2005 extracted from soybean, 41:1129-1135) and glucose oxidase (Polymer2009,50:1846-1851) also can the polymerization of catalysed aniline.
Regrettably, due to native enzyme to heat and pH very responsive, therefore there is poor stability, mutability inactivation, the storage shortcoming such as difficult, expensive, thus limit scale development and the utilization of enzymatic technique.There is bibliographical information, horseradish peroxidase HRP only can the synthesis of catalysis water-soluble conducting polyaniline within the scope of the very narrow pH of 4.0 ~ 4.65, pH is higher than 4.65, nonconducting polyaniline can only be obtained, and pH just loses catalytic activity (J.Am.Chem.Soc., 1999 lower than 4.0, HRP, 121,71).Under this research background, a class can simulate the material of the catalytic activity of native enzyme---analogue enztme, developed by people gradually.Analogue enztme is the mimetic enzyme catalyst of Prof. Du Yucang, and it has the catalysis of similar enzyme, but structural rate native enzyme is simple, stable chemical nature, also has efficient, high selectivity and the advantage such as cheap and easy to get.The research of analogue enztme is not only significant to biochemistry, and has important researching value to the exploitation of Green Chemistry and social sustainable development.
At present, domestic and international multiple seminar all in the research being devoted to analogue enztme energetically, and has carried out the research and development in a large number with the enzyme mimics of catalytic activity.
Before 2007, the research of Mimetic Peroxidase mainly focuses on Metal-organic complex analogue enztme, although the cost of these manual simulation's things declines to some extent than native enzyme, but still there is the limitation that organic synthesis technique is complicated, purification & isolation is difficult, particularly as organic matter, the deficiency of its poor heat stability, easy in inactivation, compared with native enzyme, not be improved significantly.
Nano particle analogue enztme be in recent years in the class novel analog enzyme that emerges.2007, Chinese Academy of Sciences biophysics research institute Yan Xiyun researcher Late Cambrian ferric oxide nanometer particle has the catalytic activity of similar peroxidase, and propose the concept (Nat.Nanotechnol.2007,2,577-583) of nano particle analogue enztme.Since then, the catalytic activity of nano material Yin Qigao, unique small-size effect and skin effect, non-easy in inactivation, easily the feature such as to preserve and the focus that becomes that analogue enztme studies gradually.The result of study display in this field subsequently: be in the graphene oxide (Adv.Mater.2010,22,2206-2210) of nanoscale, cadmium sulfide nano-particles (Angew.Chem.Int.Edit.2008,47:5335-5339), CoFe
2o
4magnetic nanoparticle (Chem.Commun.2011,47:10785-10787), SWCN (Chem.-Eur.J.2010,16:3617-3621), FeS nanometer sheet (Chem.-Eur.J.2009,15:4321-4326), cerium oxide nano particle (Chem.Commun.2010,46,2736-2738), V
2o
5nano wire (Adv.Funct.Mater.2011,21:501-509), Pt nanocrystal (Colloid.Surface, A 2011,373:6-10), cerous phosphate nano particle (Chem.Commun.2012,48:6839-6841), ferric phosphate micron particles (Chem.Commun.2012,48:7289-7291) etc. also all show similar enzymatic activity.But employing nano material analogue enztme is the synthesis of catalyst, catalysis conducting polymer, has not yet to see report.
Summary of the invention
The present invention is directed to the problems such as native enzyme easy in inactivation, difficult purification, price height, provide a kind of not easy in inactivation, easily purification, cheap nano black selenium Mimetic Peroxidase and preparation method thereof.
For achieving the above object, the present invention is achieved through the following technical solutions:
The simulation that nano black selenium Mimetic Peroxidase of the present invention has similar peroxidase is active, can catalysis peroxidase substrate 3,3', 5, the chromogenic reaction of 5'-tetramethyl benzidine and hydrogen peroxide, can the green syt of catalytic water soluble conductive polymer; Its preparation method is, is dissolved in by selenium dioxide in the concentrated sulfuric acid, adds solvent, is transferred in autoclave and adopts solvent-thermal method to prepare nano black selenium Mimetic Peroxidase.
Wherein, described solvent is the mixed solvent of water and glycerine, and the volume ratio of water and glycerine is 1:6 ~ 6:1, is preferably 1:3.
Described solvent-thermal method reaction temperature is 120 ~ 200 DEG C, is preferably 180 DEG C.
The described solvent-thermal method reaction time is 6 ~ 18 hours, is preferably 12 hours.
The concrete preparation process of described nano black selenium Mimetic Peroxidase:
Weigh 0.022 ~ 0.44g selenium dioxide (SeO
2), be dissolved under room temperature in 0.5 ~ 10mL deionized water, add the concentrated sulfuric acid that 0.08 ~ 1.6mL mass concentration is 98%, stir after 5 minutes, add 10 ~ 30mL glycerine, continue stirring 30 minutes, be transferred to by mixture in autoclave, 120 ~ 180 DEG C are reacted 6 ~ 18 hours; Product is centrifugal, after the precipitate with deionized water after centrifugal and ethanol respectively wash 3 times, and 60 DEG C of vacuum drying, obtained nano black selenium powder end.
As shown from the above technical solution, the present invention take selenium dioxide as primary raw material, and water and glycerine are solvent, adopts solvent-thermal method to prepare the nano black selenium with mimetic peroxidase activity.
Compared with native enzyme, the invention has the advantages that:
(1) preparation method of nano black selenium Mimetic Peroxidase is simple, the easy separating-purifying of product, reaction kit used and reagent cheap, overcome that native enzyme difficulty is purified, expensive shortcoming.
(2) nano black selenium Mimetic Peroxidase stable in properties, can, in the wider pH range of 3.0 ~ 6.0, keep analogue enztme active, catalysis TMB and H
2o
2chromogenic reaction, overcome native enzyme pH lower than 4.0 time and the shortcoming of inactivation.
(3) nano black selenium Mimetic Peroxidase can the synthesis of catalytic water soluble conductive polymer in the wider pH range of 2.2 ~ 4.2, and catalytic activity is the strongest when pH=2.2, overcoming native enzyme catalyzes and synthesizes in the technique of water-soluble conducting polymer, native enzyme easy in inactivation, mutability, only can in the shortcoming compared with catalytic water soluble conductive high molecular polymerization in narrow pH range of 4.0 ~ 4.65.
(4) nano black selenium Mimetic Peroxidase of the present invention can be used for the working environment easily causing enzyme denaturation, and has good application prospect in the green syt field of water-soluble conducting polymer.
Accompanying drawing explanation
Fig. 1: the XRD collection of illustrative plates of nano black selenium.
Fig. 2: the SEM photo of nano black selenium.
TMB-H under Fig. 3: pH=1.0 ~ 9.0 conditions
2o
2the UV-Visible absorption collection of illustrative plates of mixed solution.
Fig. 4: the photo of the water-soluble conducting polymer of nano black selenium Catalyzed Synthesis By Peroxidase synthesis and uv-visible absorption spectra.
Detailed description of the invention
Embodiment 1
1, the preparation of nano black selenium Mimetic Peroxidase:
Step:
Weigh 0.222g selenium dioxide (SeO
2), be dissolved under room temperature in 5mL deionized water, add the concentrated sulfuric acid that 0.8mL mass concentration is 98%, stir after 5 minutes, add 30mL glycerine, continue stirring 30 minutes, be transferred to by mixture in autoclave, 180 DEG C are reacted 6 hours, product is centrifugal, after centrifugation deionized water and ethanol respectively wash 3 times, 60 DEG C of vacuum drying, obtained nano black selenium powder end.
Result:
From accompanying drawing 1, the product prepared is the selenium simple substance that purity is very high.
From accompanying drawing 2, the black selenium prepared is spheric granules, and size is between 500nm ~ 10 μm.
2, the test of nano black selenium mimetic peroxidase activity:
Step:
1. get 10mg nano black selenium, be scattered in 10mL ultra-pure water, obtain the aqueous dispersions that mass concentration is the nano black selenium of 1mg/mL.
2. the TMB aqueous solution that 500 μ L molar concentrations are 1mM is accurately measured and 100 μ L molar concentrations are the H of 1M
2o
2the aqueous solution, then add 1.8mL natrium citricum-disodium hydrogen phosphate buffer solution (pH is changed to 9.0 by 1.0), in 200 ~ 800nm wave-length coverage, record TMB-H
2o
2the uv-visible absorption spectra of mixed solution.
3. accurately measure the aqueous dispersions that the above-mentioned mass concentration of 100 μ L is the nano black selenium of 1mg/mL, add TMB-H
2o
2in mixed solution, observe the color change of mixed solution, when discovery color is almost constant, the uv-visible absorption spectra of record mixed solution.
Result:
From accompanying drawing 3, between pH=3.0 ~ 6.0, after adding nano black selenium, TMB-H
2o
2all having there is obvious absworption peak at 653nm place in mixed solution, illustrates that nano black selenium is within the scope of the pH of 3.0 ~ 6.0, all has good mimetic peroxidase active.
3, the synthesis of nano black selenium Mimetic Peroxidase catalysis water-soluble conducting polyaniline PANI:
Step:
1. take 0.093 gram of pyrrole monomer and 0.067 gram of kayexalate, join in the citrate-phosphate disodium hydrogen cushioning liquid of 20 milliliters of pH=2.2, stir 4 hours under ice bath.
2. add the nano black selenium Mimetic Peroxidase prepared by 5 milligrams of embodiments 1,40 DEG C of water-baths, make aniline monomer be polymerized under the catalysis of nano black selenium.
3. polymerisation terminates reaction after 12 hours, by reaction system under the rotating speed of 3000 revs/min centrifugal 10 minutes, to remove nano black selenium catalyst.
4. upper strata centrifugate proceeds to molecular cut off is in the bag filter of 3500KDa, dialyses 24 hours, remove the oligomer of unreacted monomer and molecular weight, obtain end product water-soluble polyaniline in the hydrochloric acid solution of pH=5.
4, the synthesis of nano black selenium Mimetic Peroxidase catalytic water soluble conductive polypyrrole PPy:
Step:
1. take 0.029 gram of pyrrole monomer and 0.089 gram of kayexalate, join in the citrate-phosphate disodium hydrogen cushioning liquid of 20 milliliters of pH=2.2, stir 4 hours under ice bath.
2. add the nano black selenium Mimetic Peroxidase prepared by 10 milligrams of embodiments 1,60 DEG C of water-baths, make pyrrole monomer be polymerized under the catalysis of nano black selenium.
3. polymerisation terminates reaction after 12 hours, by reaction system under the rotating speed of 3000 revs/min centrifugal 10 minutes, to remove nano black selenium catalyst.
4. upper strata centrifugate proceeds to molecular cut off is in the bag filter of 3500KDa, dialyses 24 hours, remove the oligomer of unreacted monomer and molecular weight, obtain end product water-soluble poly pyrroles in the hydrochloric acid solution of pH=5.
5, nano black selenium Mimetic Peroxidase catalytic water soluble conductive gathers the synthesis of 3,4-ethylene dioxythiophene PEDOT:
Step:
1. take 0.282 gram of 3,4-ethylene dioxythiophene monomer and 0.464 gram of D-camphor-10-sulfonic acid, join in the citrate-phosphate disodium hydrogen cushioning liquid of 20 milliliters of pH=3.2, stir 4 hours under ice bath.
2. add the nano black selenium Mimetic Peroxidase prepared by 20 milligrams of embodiments 1,60 DEG C of water-baths, make thiophene monomer be polymerized under the catalysis of nano black selenium.
3. polymerisation terminates reaction after 12 hours, by reaction system under the rotating speed of 3000 revs/min centrifugal 10 minutes, to remove nano black selenium catalyst.
4. upper strata centrifugate proceeds to molecular cut off is in the bag filter of 3500KDa, dialyses 24 hours, remove the oligomer of unreacted monomer and molecular weight, obtain end product water-soluble poly 3,4-ethylene dioxythiophene in the hydrochloric acid solution of pH=5.Result:
From accompanying drawing 4A, 3 kinds of conducting polymers that nano black selenium catalyzes and synthesizes all can be dissolved in water, form the uniform aqueous solution, from accompanying drawing 4B, their uv-visible absorption spectra meets the feature of polyaniline, polypyrrole, poly-3,4-ethylene dioxythiophene respectively.
Embodiment 2
Weigh 0.111g selenium dioxide (SeO
2), be dissolved under room temperature in 5mL deionized water, add the concentrated sulfuric acid that 0.4mL mass concentration is 98%, stir after 5 minutes, add 30mL glycerine, continue stirring 30 minutes, be transferred to by mixture in autoclave, 120 DEG C are reacted 10 hours, product is centrifugal, after centrifugation deionized water and ethanol respectively wash 3 times, 60 DEG C of vacuum drying, obtained nano black selenium powder end.
Embodiment 3
Weigh 0.022g selenium dioxide (SeO
2), be dissolved under room temperature in 7.5mL deionized water, add the concentrated sulfuric acid that 0.08mL mass concentration is 98%, stir after 5 minutes, add 30mL glycerine, continue stirring 30 minutes, be transferred to by mixture in autoclave, 150 DEG C are reacted 8 hours, product is centrifugal, after centrifugation deionized water and ethanol respectively wash 3 times, 60 DEG C of vacuum drying, obtained nano black selenium powder end.
Embodiment 4
Weigh 0.35g selenium dioxide (SeO
2), be dissolved under room temperature in 8mL deionized water, add the concentrated sulfuric acid that 1.3mL mass concentration is 98%, stir after 5 minutes, add 32mL glycerine, continue stirring 30 minutes, be transferred to by mixture in autoclave, 160 DEG C are reacted 15 hours, product is centrifugal, after centrifugation deionized water and ethanol respectively wash 3 times, 60 DEG C of vacuum drying, obtained nano black selenium powder end.
Embodiment 5
Weigh 0.44g selenium dioxide (SeO
2), be dissolved under room temperature in 20mL deionized water, add the concentrated sulfuric acid that 1.6mL mass concentration is 98%, stir after 5 minutes, add 20mL glycerine, continue stirring 30 minutes, be transferred to by mixture in autoclave, 140 DEG C are reacted 18 hours, product is centrifugal, after centrifugation deionized water and ethanol respectively wash 3 times, 60 DEG C of vacuum drying, obtained nano black selenium powder end.
Claims (4)
1. a nano black selenium Mimetic Peroxidase, it is characterized in that, the simulation that this nano black selenium Mimetic Peroxidase has similar peroxidase is active, can catalysis peroxidase substrate 3,3', the chromogenic reaction of 5,5'-tetramethyl benzidine and hydrogen peroxide, can the green syt of catalytic water soluble conductive polymer;
Its preparation method is, is dissolved in by selenium dioxide in the concentrated sulfuric acid, adds solvent, is transferred in autoclave and adopts solvent-thermal method to prepare nano black selenium Mimetic Peroxidase.
2. nano black selenium Mimetic Peroxidase according to claim 1, is characterized in that, described solvent is the mixed solvent of water and glycerine, and the volume ratio of water and glycerine is 1:6 ~ 6:1.
3. nano black selenium Mimetic Peroxidase according to claim 1, is characterized in that, described solvent-thermal method reaction temperature is 120 ~ 200 DEG C; The described solvent-thermal method reaction time is 6 ~ 18 hours.
4. nano black selenium Mimetic Peroxidase according to claim 1, is characterized in that, the preparation process of described nano black selenium Mimetic Peroxidase:
Weigh 0.022 ~ 0.44g selenium dioxide (SeO
2), be dissolved under room temperature in 0.5 ~ 10mL deionized water, add the concentrated sulfuric acid that 0.08 ~ 1.6mL mass concentration is 98%, stir after 5 minutes, add 10 ~ 30mL glycerine, continue stirring 30 minutes, be transferred to by mixture in autoclave, 120 ~ 180 DEG C are reacted 6 ~ 18 hours; Product is centrifugal, after the precipitate with deionized water after centrifugal and ethanol respectively wash 3 times, and 60 DEG C of vacuum drying, obtained nano black selenium powder end.
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| CN104897846A (en) * | 2015-06-23 | 2015-09-09 | 江南大学 | Alkaline phosphatase activity assay method based on in-situ formation of optical active nanometer material mimic enzyme |
| CN111579548A (en) * | 2020-05-20 | 2020-08-25 | 重庆师范大学 | Luminol-gallium nano assembly and preparation method and application thereof |
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Cited By (2)
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| CN104897846A (en) * | 2015-06-23 | 2015-09-09 | 江南大学 | Alkaline phosphatase activity assay method based on in-situ formation of optical active nanometer material mimic enzyme |
| CN111579548A (en) * | 2020-05-20 | 2020-08-25 | 重庆师范大学 | Luminol-gallium nano assembly and preparation method and application thereof |
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