CN106115792B - One kind preparing Fe by biomineralization template of collagen2O3The method of nano-particle - Google Patents
One kind preparing Fe by biomineralization template of collagen2O3The method of nano-particle Download PDFInfo
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- CN106115792B CN106115792B CN201610458525.9A CN201610458525A CN106115792B CN 106115792 B CN106115792 B CN 106115792B CN 201610458525 A CN201610458525 A CN 201610458525A CN 106115792 B CN106115792 B CN 106115792B
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- 238000000034 method Methods 0.000 title claims abstract description 28
- 230000033558 biomineral tissue development Effects 0.000 title claims abstract description 18
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 18
- 102000008186 Collagen Human genes 0.000 claims abstract description 58
- 108010035532 Collagen Proteins 0.000 claims abstract description 58
- 229920001436 collagen Polymers 0.000 claims abstract description 57
- 239000002086 nanomaterial Substances 0.000 claims abstract description 20
- 238000002360 preparation method Methods 0.000 claims abstract description 20
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims abstract description 15
- 238000001027 hydrothermal synthesis Methods 0.000 claims abstract description 13
- 229910003145 α-Fe2O3 Inorganic materials 0.000 claims abstract description 11
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 claims abstract description 10
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 10
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 10
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 10
- 238000000746 purification Methods 0.000 claims abstract description 10
- 239000002131 composite material Substances 0.000 claims abstract description 8
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000005516 engineering process Methods 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 5
- 239000008240 homogeneous mixture Substances 0.000 claims abstract description 4
- 239000007787 solid Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 241000894006 Bacteria Species 0.000 claims description 12
- 229910000859 α-Fe Inorganic materials 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 239000000725 suspension Substances 0.000 claims description 9
- 239000013612 plasmid Substances 0.000 claims description 8
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 6
- 235000019750 Crude protein Nutrition 0.000 claims description 6
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 230000003115 biocidal effect Effects 0.000 claims description 6
- 229960002897 heparin Drugs 0.000 claims description 6
- 229920000669 heparin Polymers 0.000 claims description 6
- 239000012460 protein solution Substances 0.000 claims description 6
- 238000010792 warming Methods 0.000 claims description 6
- 241000588724 Escherichia coli Species 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 239000006227 byproduct Substances 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 230000007850 degeneration Effects 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 239000012634 fragment Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 230000014509 gene expression Effects 0.000 claims description 3
- 239000008187 granular material Substances 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 150000002460 imidazoles Chemical class 0.000 claims description 3
- 230000008676 import Effects 0.000 claims description 3
- 102000006495 integrins Human genes 0.000 claims description 3
- 108010044426 integrins Proteins 0.000 claims description 3
- 238000011068 loading method Methods 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000001488 sodium phosphate Substances 0.000 claims description 3
- 235000011008 sodium phosphates Nutrition 0.000 claims description 3
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical class [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 3
- 241000193830 Bacillus <bacterium> Species 0.000 claims 1
- 239000004744 fabric Substances 0.000 claims 1
- 210000002429 large intestine Anatomy 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 4
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 11
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000004833 X-ray photoelectron spectroscopy Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000012805 post-processing Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000002411 thermogravimetry Methods 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000003519 biomedical and dental material Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000012776 electronic material Substances 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000383 hazardous chemical Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000003837 high-temperature calcination Methods 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N iron oxide Inorganic materials [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 1
- YOBAEOGBNPPUQV-UHFFFAOYSA-N iron;trihydrate Chemical compound O.O.O.[Fe].[Fe] YOBAEOGBNPPUQV-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
- 150000004706 metal oxides Chemical class 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 230000006911 nucleation Effects 0.000 description 1
- 235000006408 oxalic acid Nutrition 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
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000013759 synthetic iron oxide Nutrition 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G49/00—Compounds of iron
- C01G49/02—Oxides; Hydroxides
- C01G49/06—Ferric oxide [Fe2O3]
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/70—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data
- C01P2002/72—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data by d-values or two theta-values, e.g. as X-ray diagram
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/80—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70
- C01P2002/85—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70 by XPS, EDX or EDAX data
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/80—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70
- C01P2002/88—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70 by thermal analysis data, e.g. TGA, DTA, DSC
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
- C01P2004/03—Particle morphology depicted by an image obtained by SEM
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
- C01P2004/04—Particle morphology depicted by an image obtained by TEM, STEM, STM or AFM
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/60—Particles characterised by their size
- C01P2004/61—Micrometer sized, i.e. from 1-100 micrometer
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses one kind preparing Fe by biomineralization template of collagen2O3The method of nano-particle, includes the following steps：(1) biology gene engineering technology Prepare restructuring collagen is utilized：1., determine the sequence of recombined collagen；2., the nucleic acid of composite coding recombined collagen；3., the preparation and purification of recombined collagen；(2)α‑Fe2O3The preparation of nano material：1., the preparation of recombined collagen and Iron(III) chloride hexahydrate homogeneous mixture solotion；2., nanometer alpha Fe is prepared using hydrothermal reaction kettle2O3；3., purifying and kept dry prepare nano material.For the present invention using recombined collagen as biological template, Iron(III) chloride hexahydrate is prepared for the α Fe of size and morphology controllable as raw material by hydrothermal method2O3Nano material.The present invention is not necessarily to add any other chemical reagent or post-process in entire building-up process, simple and convenient, easily operated, has great application prospect, can be large-scale production α Fe2O3Nano material provides basis.
Description
Technical field
The present invention relates to Fe2O3Nano-particle preparation method, and in particular to one kind is using collagen as biomineralization template system
Standby Fe2O3The method of nano-particle belongs to Inorganic biomatetials preparing technical field.
Background technology
Biomineralization refers to the process of that organism generates inorganic mineral by the regulating and controlling effect of large biological molecule, it is chain
Connect it is inorganic with it is biological between bridge.Be with the maximum difference of general mineralising, it be biology at specific position, certain
Under physical and chemical condition, biological organic substance participation control or under the influence of, by the ion transit in solution be solid phase mineral
Process.As bone, scale, the formation of tooth etc. is all biomineralization process relatively common in nature.With industrial production
Condition is different, and biomineralization does not need exacting terms, it is a mild condition, low power consuming and free of contamination physical chemistry mistake
Journey.Biomineralization uses component fairly simple and common in nature, and may be implemented to sample from nucleation to crystallization
The regulation and control of process.Therefore, mineralising mechanism of the inorganic matter under biomolecule regulation and control in simultaneously mimic biology mineralising is explored, can be to prepare
Composite material with unique texture and performance provides new visual angle.
Alpha-type ferric oxide (α-Fe2O3) it is a kind of critically important metal oxide, since its is nontoxic, non-environmental-pollution,
The features such as of low cost, is all widely used in flash coating material, plastics, electronic material and biomedical engineering etc..
It has now been established α-Fe2O3A variety of synthetic methods of nano material, including：(1) it is raw material to use ferric trichloride and oxalic acid
Then the primary structure of synthetic iron oxide first synthesizes hollow fusiformis and spherical structure α-Fe by high-temperature calcination again2O3；(2)
Under alkaline condition using ferric trichloride and tetrabutyl desert money, flower-shaped α-Fe2O3 nanostructures are prepared by post-processing.These
Method some need adds poisonous and hazardous chemical reagent in the synthesis process, and some need is by heating removal solvent or template
Equal post-processings can just obtain product, all relatively complicated, and there are time consumption and energy consumptions and the higher disadvantage of cost.Meanwhile by
In at present to α-Fe2O3The still inadequate system of basic research of nano particle morphology and size controllability and fully, therefore for meet not
Same demand regulates and controls α-Fe using simple, easily-controllable, environmentally protective method2O3The morphology and size of nano particle has very heavy
The meaning wanted.
Collagen is the main constituents of extracellular matrix, is almost distributed in all histoorgans, it is feeding
Content is up to a quarter of total protein concentration in newborn animal.There is collagen good biocompatibility, biological degradability to inhale
The property received and promotion cell form equal various functions, therefore, in fields such as bio-medical material, organizational project, cosmetics, food
It has a wide range of applications.Using recombined collagen as biological template, Iron(III) chloride hexahydrate passes through the present invention as raw material
Hydrothermal method is prepared for the α-Fe of size and morphology controllable2O3Nano material.The present invention is in entire building-up process without addition
Any other chemical reagent is post-processed, simple and convenient, easily operated, has comparable feasibility and application value, can
For large-scale production α-Fe2O3Nano material provides basis.
Invention content
The purpose of the present invention is being directed to deficiency in the prior art, one kind is provided using collagen as biomineralization template system
Standby Fe2O3The method of nano-particle, using the most protein collagen of animal in-vivo content as biological template, six hydrations
Ferric trichloride is prepared for the α-Fe of size and morphology controllable as raw material by hydrothermal method2O3Nano material.
To achieve the above object, the invention discloses following technical solutions：
One kind preparing Fe by biomineralization template of collagen2O3The method of nano-particle, includes the following steps：
(1) biology gene engineering technology Prepare restructuring collagen is utilized
1., determine the sequence of recombined collagen；
The sequence of recombined collagen is：
GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRG
LQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAG
AQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKD
GERGFPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDG
LPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKYGPPGPPGPP GPPGPPGPPGPPGPPGPPGPP, this is heavy
Group collagen has good triple helices structure, and thermal change temperature is close to 37 DEG C；Binding site with integrin
GERGFPGERGVE can well be adhered to cell；Binding site GRPGKRGKQGQK with heparin, can be with heparin knot
It closes；
2., the nucleic acid of composite coding recombined collagen；
The nucleic acid of composite coding step 1. recombined collagen, structure import the plasmid of above-mentioned nucleic acid, and plasmid is converted
E. coli bl21-DE3 bacterial strains；
3., the preparation and purification of recombined collagen；
Frost is placed in -80 DEG C of competent escherichia coli cell in ice bath and is melted, by competent cell and 3-5 μ L
Plasmid mixes, and mixture places into 90s in 42 DEG C of water-baths in ice bath, then places into 2min in ice bath after 30min；Above-mentioned
The not antibiotic LB culture mediums of 1ml are added in mixture, then are positioned over 1hrs in 37 DEG C of constant-temperature tables, then at 4 DEG C,
20min is centrifuged under the conditions of 4000rpm, discards supernatant liquor；It is after so that bacterium is suspended again in remaining culture liq, culture solution is equal
On the even AMP culture plates for being applied to 37 DEG C, it is placed in 37 DEG C of constant-temperature tables and is incubated overnight；The next day preferable bacterium of picking growing way
It puts off in the antibiotic LB liquid mediums of 100 ml, constant-temperature table carries out Zengjing Granule overnight；100ml is incubated overnight
Bacterium pour into 1L LB culture mediums, continue amplification cultivation in 37 DEG C of constant-temperature tables；It waits for that OD values reach 0.8-1 ranges, will shake
Bed tempertaure is adjusted to 25 DEG C, and 1mM IPTG induced expressions are added, and constant temperature is incubated overnight；
The bacterium that above-mentioned albumen has been expressed is centrifuged in refrigerated centrifuge, so that thalline is detached with culture medium, centrifugal condition：
12000rpm, centrifuges 1.0-3.0min by 4 DEG C；By the A buffer solutions of the thalline after centrifugation, A buffer solutions are 20mM imidazoles,
20mM sodium phosphates, 0.5M sodium chloride, pH 7.4；Bacterial suspension is put into ultrasonic cell disruption instrument and carries out clasmatosis,
It can release albumen and albumen can be dissolved in A buffer solutions；Bacterial suspension need to be put in ice bath when ultrasonic, to prevent temperature
Spending height leads to albuminous degeneration；The suspension being crushed is centrifuged again, cell fragment is made to be detached with protein solution, centrifuges item
Part：14000rpm, 4 DEG C, 30-50min；Supernatant is collected, this is crude protein solution；After crude protein is filtered, pass through liquid phase
Chromatography is further purified；By freeze-drying, white fluffy solid is obtained；This solid is put in -20 DEG C of refrigerators and preserves, and when use is logical
Cross weight method calibration concentration.
(2)α-Fe2O3The preparation of nano material
1., the preparation of recombined collagen and Iron(III) chloride hexahydrate homogeneous mixture solotion；
1-50mg Iron(III) chloride hexahydrates and 0-10mg collagen solids are added in 1ml water, is uniformly mixed, slowly stirs
After mixing 5-90min, pale yellow transparent and uniform liquid are obtained；
2., nanometer alpha-Fe is prepared using hydrothermal reaction kettle2O3；
Gained mixed liquor is poured into 5ml hydrothermal reaction kettles, is put into Muffle furnace and is warming up to the speed of 3-18 DEG C/min
120-200 DEG C, and 1-15hrs is reacted at such a temperature；
3., purifying and kept dry prepare nano material；
After hydrothermal reaction kettle is cooled to room temperature, by product by centrifuging, centrifugal condition 1200r is discarded supernatant
Liquid leaves solid；It is used in combination deionized water dispersing solid, then centrifugal purification 3-5 times, it is dry in 50-80 DEG C of thermostatic drying chamber.
As a preferred technical solution of the present invention, the purity of 3. recombined collagen that step obtains after purification reaches
95% or more.
As a preferred technical solution of the present invention, the collagen solids addition described in step (2) is 0.1-
5mg, collagen quality score are 0.01-0.5wt%.
As a preferred technical solution of the present invention, the Iron(III) chloride hexahydrate solids loading content described in step (2)
For 2.7-27mg, the concentration distribution of iron (III) is from 0.01 to 0.1mol/L.
As a preferred technical solution of the present invention, Iron(III) chloride hexahydrate and collagen described in step (2)
Mixed liquor is slowly stirred the time as 20-50min.
As a preferred technical solution of the present invention, the mixed liquor described in step (2) is in Muffle furnace with 3-10
DEG C/speed of min is warming up to 140-180 DEG C, and reacts 6-12hrs at such a temperature.
It is disclosed by the invention a kind of using collagen as biomineralization template preparation Fe2O3The method of nano-particle, have with
Lower advantage：It is simple and convenient without adding any other chemical reagent or being post-processed in entire building-up process, it is easy to grasp
Make, there is comparable feasibility and application value, can be large-scale production α-Fe2O3Nano material provides basis.
Description of the drawings
Fig. 1 is the α-Fe prepared2O3X-ray powder polycrystalline diffraction (XRD) figure of nano material；
Fig. 2 is the α-Fe prepared2O3X-ray photoelectron spectroscopy (XPS) figure of nano material；
Fig. 3 is the α-Fe prepared2O3Thermogravimetric analysis (TGA) figure of nano material；
Fig. 4 is the α-Fe prepared2O3The scanning electron microscope of nano material, transmission electron microscope, electronic diffraction and energy dispersive X are penetrated
Line analysis figure；
Fig. 5 is the recombined collagen of various concentration to α-Fe2O3The influence of nanoparticle structure；
Specific implementation mode
To make the technical means, the creative features, the aims and the efficiencies achieved by the present invention be easy to understand, with reference to
Specific implementation mode, the present invention is further explained.
As Figure 1-Figure 5, a kind of to prepare Fe by biomineralization template of collagen2O3The method of nano-particle, including
Following steps：
(1) biology gene engineering technology Prepare restructuring collagen is utilized
1., determine the sequence of recombined collagen；
The sequence of recombined collagen is：
GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRG
LQGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGKDGEAG
AQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKD
GERGFPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDG
LPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGKYGPPGPPGPP GPPGPPGPPGPPGPPGPPGPP, this is heavy
Group collagen has good triple helices structure, and thermal change temperature is close to 37 DEG C；Binding site with integrin
GERGFPGERGVE can well be adhered to cell；Binding site GRPGKRGKQGQK with heparin, can be with heparin knot
It closes；
2., the nucleic acid of composite coding recombined collagen；
The nucleic acid of composite coding step 1. recombined collagen, structure import the plasmid of above-mentioned nucleic acid, and plasmid is converted
E. coli bl21-DE3 bacterial strains；
3., the preparation and purification of recombined collagen；
Frost is placed in -80 DEG C of competent escherichia coli cell in ice bath and is melted, by competent cell and 3-5 μ L matter
Grain mixing, mixture place into 90s in 42 DEG C of water-baths in ice bath, then place into 2min in ice bath after 30min；Above-mentioned mixed
The not antibiotic LB culture mediums of 1ml are added in zoarium, then are positioned over 1hrs in 37 DEG C of constant-temperature tables, then at 4 DEG C,
20min is centrifuged under the conditions of 4000rpm, discards supernatant liquor；It is after so that bacterium is suspended again in remaining culture liq, culture solution is equal
On the even AMP culture plates for being applied to 37 DEG C, it is placed in 37 DEG C of constant-temperature tables and is incubated overnight；The next day preferable bacterium of picking growing way
It puts off in the antibiotic LB liquid mediums of 100 ml, constant-temperature table carries out Zengjing Granule overnight；100ml is incubated overnight
Bacterium pour into 1L LB culture mediums, continue amplification cultivation in 37 DEG C of constant-temperature tables；It waits for that OD values reach 0.8-1 ranges, will shake
Bed tempertaure is adjusted to 25 DEG C, and 1mM IPTG induced expressions are added, and constant temperature is incubated overnight；
The bacterium that above-mentioned albumen has been expressed is centrifuged in refrigerated centrifuge, so that thalline is detached with culture medium, centrifugal condition：
12000rpm, centrifuges 1.0-3.0min by 4 DEG C；By the A buffer solutions of the thalline after centrifugation, A buffer solutions are 20mM imidazoles,
20mM sodium phosphates, 0.5M sodium chloride, pH 7.4；Bacterial suspension is put into ultrasonic cell disruption instrument and carries out clasmatosis,
It can release albumen and albumen can be dissolved in A buffer solutions；Bacterial suspension need to be put in ice bath when ultrasonic, to prevent temperature
Spending height leads to albuminous degeneration；The suspension being crushed is centrifuged again, cell fragment is made to be detached with protein solution, centrifuges item
Part：14000rpm, 4 DEG C, 30-50min；Supernatant is collected, this is crude protein solution；After crude protein is filtered, pass through liquid phase
Chromatography is further purified；By freeze-drying, white fluffy solid is obtained；This solid is put in -20 DEG C of refrigerators and preserves, and when use is logical
Cross weight method calibration concentration.
(3)α-Fe2O3The preparation of nano material
1., the preparation of recombined collagen and Iron(III) chloride hexahydrate homogeneous mixture solotion；
1-50mg Iron(III) chloride hexahydrates and 0-10mg collagen solids are added in 1ml water, is uniformly mixed, slowly stirs
After mixing 5-90min, pale yellow transparent and uniform liquid are obtained；
2., nanometer alpha-Fe is prepared using hydrothermal reaction kettle2O3；
Gained mixed liquor is poured into 5ml hydrothermal reaction kettles, is put into Muffle furnace and is warming up to the speed of 3-18 DEG C/min
120-200 DEG C, and 1-15hrs is reacted at such a temperature；
3., purifying and kept dry prepare nano material；
After hydrothermal reaction kettle is cooled to room temperature, by product by centrifuging, centrifugal condition 1200r is discarded supernatant
Liquid leaves solid；It is used in combination deionized water dispersing solid, then centrifugal purification 3-5 times, it is dry in 50-80 DEG C of thermostatic drying chamber.
Wherein, the purity for the recombined collagen that 3. step obtains after purification reaches 95%.
Wherein, the collagen solids addition described in step (2) is 1mg, and collagen quality score is
0.1wt%.
Wherein, the Iron(III) chloride hexahydrate solids loading content described in step (2) is 16mg, the concentration distribution of iron (III)
From 0.06mol/L.
Wherein, the Iron(III) chloride hexahydrate described in step (2) and collagen mixed liquor are slowly stirred the time and are
30min。
Wherein, the mixed liquor described in step (2) is warming up to 160 DEG C in Muffle furnace with the speed of 5 DEG C/min, and
10hrs is reacted at this temperature.
The above is the details of the exemplary implementation case of the present invention.For those skilled in the art,
The present invention can have various modifications and variations according to specific preparation condition in actual application, be not limited to this hair
It is bright.All within the spirits and principles of the present invention, it should all be included in the protection scope of the present invention, it should not will be in claim
Any reference numeral be considered as and limit the claims involved.
Claims (6)
1. a kind of preparing Fe by biomineralization template of collagen2O3The method of nano-particle, it is characterised in that including walking as follows
Suddenly：
(1) biology gene engineering technology Prepare restructuring collagen is utilized
1., determine the sequence of recombined collagen；
The sequence of recombined collagen is：
GSPGLPGPRGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGLQGERGEQGPTGLAGKAGEAGAKG
ETGPAGPQGPRGEQGPQGLPGKDGEAGAQGRPGKRGKQGQKGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKD
GERGFPGERGVEGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGK
DGKDGQPGKPGKYGPPGPPGPPGPPGPPGPPGPPGPPGPPGPP, the recombined collagen have good triple helices
Structure, thermal change temperature is close to 37 DEG C；Binding site GERGFPGERGVE with integrin can well be adhered to cell；
Binding site GRPGKRGKQGQK with heparin, can be with Heparin-binding；
2., the nucleic acid of composite coding recombined collagen；
The nucleic acid of composite coding step 1. recombined collagen, structure import the plasmid of above-mentioned nucleic acid, and plasmid is converted large intestine
Bacillus BL21-DE3 bacterial strains；
3., the preparation and purification of recombined collagen；
Frost is placed in -80 DEG C of competent escherichia coli cell in ice bath and is melted, competent cell and 3-5 μ L plasmids are mixed
It closes, mixture places into 90s in 42 DEG C of water-baths in ice bath, then places into 2min in ice bath after 30min；In above-mentioned mixture
Middle not antibiotic LB culture mediums of addition 1mL, then be positioned over 1hrs in 37 DEG C of constant-temperature tables, then at 4 DEG C, 4000rpm items
20min is centrifuged under part, discards supernatant liquor；After so that bacterium is suspended again in remaining culture liq, culture solution even spread is arrived
On 37 DEG C of AMP culture plates, it is placed in 37 DEG C of constant-temperature tables and is incubated overnight；The next day preferable bacterium colony of picking growing way is put into
In the antibiotic LB liquid mediums of 100mL, constant-temperature table carries out Zengjing Granule overnight；The bacterium that 100mL is incubated overnight
It pours into 1LLB culture mediums, continues amplification cultivation in 37 DEG C of constant-temperature tables；Wait for that OD values reach 0.8-1 ranges, by shaking table temperature
25 DEG C are adjusted to, 1mMIPTG induced expressions are added, constant temperature is incubated overnight；
The bacterium that above-mentioned albumen has been expressed is centrifuged in refrigerated centrifuge, so that thalline is detached with culture medium, centrifugal condition：
12000rpm, centrifuges 1.0-3.0min by 4 DEG C；By the A buffer solutions of the thalline after centrifugation, A buffer solutions are 20mM imidazoles,
20mM sodium phosphates, 0.5M sodium chloride, pH 7.4；Bacterial suspension is put into ultrasonic cell disruption instrument and carries out clasmatosis,
It can release albumen and albumen can be dissolved in A buffer solutions；Bacterial suspension need to be put in ice bath when ultrasonic, to prevent temperature
It is excessively high to lead to albuminous degeneration；The suspension being crushed is centrifuged again, cell fragment is made to be detached with protein solution, centrifugal condition：
14000rpm, 4 DEG C, 30-50min；Supernatant is collected, this is crude protein solution；After crude protein is filtered, pass through liquid chromatogram
It is further purified；By freeze-drying, white fluffy solid is obtained；This solid is put in -20 DEG C of refrigerators and preserves, and when use passes through title
Weight method demarcates concentration；
(2)α-Fe2O3The preparation of nano material
1., the preparation of recombined collagen and Iron(III) chloride hexahydrate homogeneous mixture solotion；
1-50mg Iron(III) chloride hexahydrates and 0-10mg collagen solids are added in 1mL water, is uniformly mixed, is slowly stirred 5-
After 90min, pale yellow transparent and uniform liquid are obtained；
2., nanometer alpha-Fe is prepared using hydrothermal reaction kettle2O3；
Gained mixed liquor is poured into 5mL hydrothermal reaction kettles, is put into Muffle furnace and 120- is warming up to the speed of 3-18 DEG C/min
200 DEG C, and 1-15hrs is reacted at such a temperature；
3., purifying and kept dry prepare nano material；
After hydrothermal reaction kettle is cooled to room temperature, by product by centrifuging, centrifugal condition 1200rpm discards supernatant liquid,
Leave solid；It is used in combination deionized water dispersing solid, then centrifugal purification 3-5 times, it is dry in 50-80 DEG C of thermostatic drying chamber.
2. according to claim 1 a kind of using collagen as biomineralization template preparation Fe2O3The method of nano-particle,
It is characterized in that：The purity of 3. recombined collagen that step obtains after purification reaches 95% or more.
3. according to claim 1 a kind of using collagen as biomineralization template preparation Fe2O3The method of nano-particle,
It is characterized in that：Collagen solids addition described in step (2) is 0.1-5mg, and collagen quality score is 0.01-
0.5wt%.
4. according to claim 1 a kind of using collagen as biomineralization template preparation Fe2O3The method of nano-particle,
It is characterized in that：Iron(III) chloride hexahydrate solids loading content described in step (2) is 2.7-27mg, the concentration point of iron (III)
Cloth is from 0.01 to 0.1mol/L.
5. according to claim 1 a kind of using collagen as biomineralization template preparation Fe2O3The method of nano-particle,
It is characterized in that：Iron(III) chloride hexahydrate and collagen mixed liquor described in step (2) are slowly stirred the time as 20-
50min。
6. according to claim 1 a kind of using collagen as biomineralization template preparation Fe2O3The method of nano-particle,
It is characterized in that：Mixed liquor described in step (2) is warming up to 140-180 DEG C in Muffle furnace with the speed of 3-10 DEG C/min,
And 6-12hrs is reacted at such a temperature.
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