CN104876228B - Solid phase synthesis process based on histidine-tagged nanometer silicon dioxide particle - Google Patents
Solid phase synthesis process based on histidine-tagged nanometer silicon dioxide particle Download PDFInfo
- Publication number
- CN104876228B CN104876228B CN201510174320.3A CN201510174320A CN104876228B CN 104876228 B CN104876228 B CN 104876228B CN 201510174320 A CN201510174320 A CN 201510174320A CN 104876228 B CN104876228 B CN 104876228B
- Authority
- CN
- China
- Prior art keywords
- histidine
- tagged
- particle
- silicon dioxide
- nanometer silicon
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Abstract
The invention discloses a kind of solid phase synthesis process of the nano silicon based on histidine-tagged protein matter.This method is that the nickel ion of histidine-tagged and resin particle surfaces first by histidine-tagged protein matter is complexed so that histidine-tagged protein is first adsorbed in resin particle surfaces, and using the protein of this immobilization as nucleus growth nanometer silicon dioxide particle.The notable feature and advantage of this method are during the whole synthesis of Nano particles of silicon dioxide and subsequently surface-functionalized, nano-particle all keeps non-covalent link by histidine-tagged and resin always, the purge process after every step reaction is greatly simplified into simple lessivation；And this non-covalent link is reversible, after the completion of synthesis and functionalization, nano particle can be eluted from resin particle surfaces by eluent, while after elution with histidine-tagged nano particle after application, can also adsorb and reclaim again by resin particle.
Description
Technical field
The present invention relates to a kind of solid phase synthesis process of nanometer silicon dioxide particle, and in particular to one kind is based on histidine mark
The solid phase synthesis process of the nanometer silicon dioxide particle of label.
Background technology
Nano particle refers to the microscopic particles of nanometer scale, with skin effect, small-size effect and quantum effect etc., table
Be now many new different qualities, i.e. its optics, mechanics, property and bulk solid phase in terms of calorifics and other chemistry
Than obvious difference will be had.The particle diameter that nano particle has by itself is compared with big, favorable dispersibility of small, specific surface area etc.
Excellent potential quality, is all widely used including fields such as the energy, material, biomedicine and environment.
Nanometer silicon dioxide particle is as a kind of special nano particle, while possessing the universal feature of nano particle
Also there are many advantages：1）Nano silicon is as nontoxic, tasteless, the free of contamination nonmetallic materials of one kind, with good
Biological safety；2）Silica is as a kind of chemical inert material, and chemical property is stable；3）Nano SiO 2 particle is synthesized
Principle is simple, pattern is regular, size tunable, it is easy to apply.But, prepared by traditional method for preparing nano SiO 2 particle
Process is generally complicated, particularly prepares that particle diameter is smaller, good dispersion in the solution nano particle, is prepared in the multistep of sample
Middle and all after the completion of preparation separating-purifying is relatively difficult, is the major reason for causing nano particle production cost to rise.
Meanwhile, the nanometer silicon dioxide particle for the good dispersion in the solution that conventional method is obtained is difficult to be recovered to be subject to after use
Re-use, therefore utilization rate is very low.
The content of the invention
The purpose of the present invention is difficult for existing traditional preparation methods separating-purifying, is difficult to reclaim not after sample use
Foot, invents a kind of solid phase synthesis process of the nanometer silicon dioxide particle based on histidine-tagged protein matter.This method is significantly simple
Change the process of nanometer silicon dioxide particle purification, and can be by using resin after nanometer silicon dioxide particle use
Granular absorption is reclaimed.
To reach above-mentioned purpose, should method the mechanism used for：First histidine-tagged protein matter and surface are contained
The mixed with resin of nickel ion, is allowed to absorption in resin surface, and stirring is allowed to add in reaction vessel after being sufficiently mixed；Absorption is in tree
The histidine-tagged protein matter on fat surface is used（3- dimethylamino-propyls）- 3- ethyl-carbodiimide hydrochlorides（EDC）With N- hydroxyls
Succimide（NHS）Its surface is activated.Then functionalization is carried out with 3- aminopropyl triethoxysilanes.Finally with this
The histidine-tagged protein matter of functionalization is core, and adding silane coupler makes its reaction grow and obtain adsorbing in resin surface
Nanometer silicon dioxide particle；After the completion of nanometer silicon dioxide particle synthesis, further solid functional can be proceeded；
With imidazoles as eluent, it can make to elute from resin with histidine-tagged nanometer silicon dioxide particle, into application
Process；With histidine-tagged nano particle after application, it can also adsorb and reclaim again by resin particle.
According to above-mentioned mechanism, the present invention is adopted the following technical scheme that：
A kind of solid phase synthesis process based on histidine-tagged nanometer silicon dioxide particle, it is characterised in that this method
Concretely comprise the following steps：
A. by surface contain nickel ion resin particle and with histidine-tagged fluorescin according to 0.05~
0.25mg protein/mg resin particles are added in the phosphate buffer solution of pH=7.0~8.0, and stirring is allowed to after being sufficiently mixed
Add in the gravitational settling post with filter membrane；
B. will（3- dimethylamino-propyls）- 3- ethyl-carbodiimide hydrochlorides（EDC）With N- hydroxysuccinimides（NHS）
In mass ratio 1:2~5:2 are dissolved in deionized water, then add mixed solution according to 0.2~0.5mg EDC/mg resin particles
Enter into step a gravitational settling post, shake 30~60min, complete the activation to fluorescin surface；Add 5~10 μ L
3- aminopropyl triethoxysilanes, fully shaking is allowed to abundant dissolving, reacts 12~16h, uses deionized water rinsing pillar, removes
The crosslinking agent and byproduct of reaction not reacted completely；
C. by tetraethoxysilane（TEOS）And hexamethylene（Cyc）According to volume ratio 1:1~1:1.5 mixing, will be mixed molten
Liquid is added in gravitational settling post in resin according to 0.25~0.5 μ L/mg ratio, and concussion is allowed to fully dissolve, and reaction 8~
12h, obtains adsorbing the nanometer silicon dioxide particle in resin surface, is washed with deionized；
D. by by phosphate buffer solution and concentration for 1~2M imidazole solution according to volume ratio 2:1~4:1 mixes
Eluent be added in gravitational settling post, after being sufficiently mixed shake 15~30min；Gravitational settling post bottom plug is opened, is led to
Filter membrane is retained to resin particle, and the super filter tube for being 10KD with molecular weight ultrafiltration under 3000g centrifugal action washs 2
~3 times, the imidazoles in solution is removed, required nanometer silicon dioxide particle is obtained.
The resin particle that nickel ion is contained on above-mentioned surface is：Ni Sepharose Fast Flow(GE)；Ni
Aogarose 6 FF(AOGMA)。
Above-mentioned has green fluorescent protein GFP, near-infrared fluorescent protein I FP, Huang with histidine-tagged fluorescin
Color fluorescin YFP or cyan fluorescent protein CFP.
It is of the invention with it is existing prepare the method for nanometer silicon dioxide particle compared with, have the advantages that：
1st, because the inventive method will set during nanometer silicon dioxide particle is prepared as the protein adsorption of core
Fat surface, then it is wrapped up with silica, the multistep reaction process of whole nano-particle preparation and functionalization is all one
Individual solid-phase resin surface is completed, and the mesh of purifying multistep reaction intermediate product and final products can be just reached using simple flushing
's.And prior synthesizing method is generally required in building-up process using complicated methods and techniques after often step reaction to obtaining
Sample is purified, and process is complicated and easily causes sample loss.The preparation process of the present invention more simplifies, and purifying more facilitates
And cannot be easily caused the loss of sample.
2nd, because the nanometer silicon dioxide particle surface that the inventive method is prepared has and only one of which histidine, because
This, easily can be adsorbed and then be recovered by resin particle again after use, can be weighed after simple purge process
Newly come into operation, substantially increase the utilization rate of nanometer silicon dioxide particle in itself.
3rd, because nanometer silicon dioxide particle prepared by the inventive method has recyclable good nature, along with dioxy
SiClx is easy to surface-functionalized excellent specific property, and the applicable cases that the nanometer silicon dioxide particle after recovery can be before are entered
Row is further surface-functionalized, and then can strengthen the application effect of nanometer silicon dioxide particle and widen it and apply model
Enclose.
Brief description of the drawings
Fig. 1 is the flow chart that embodiment one prepares His-RFP@Silica nanometer silicon dioxide particles
Fig. 2 is (a) TEM figures and (b) particle diameter system that embodiment one prepares His-RFP@Silica nanometer silicon dioxide particles
Meter figure.
Fig. 3 is that embodiment one prepares His-RFP@Silica nanometer silicon dioxide particle of the absorption in resin particle surfaces
TEM schemes.
Fig. 4 is that embodiment one prepares His-RFP@Silica nanometer silicon dioxide particle of the absorption in resin particle surfaces
SEM schemes.
Fig. 5 is that embodiment two prepares His-RFP@Silica-FITC（Particle surface functionalization FITC）Nano silicon
(a) red and (b) yellow-green fluorescence spectrogram of particle.
Embodiment
Embodiment is given below so that the invention will be further described.It is necessarily pointed out that following examples can not
Be interpreted as limiting the scope of the invention, if the person skilled in the art in the field according to the invention described above content to this hair
It is bright to make some nonessential modifications and adaptations, still fall within the scope of the present invention.
Embodiment one：Based on histidine-tagged red fluorescent protein matter（RFP）It is prepared by nanometer silicon dioxide particle
The resin particle of nickel ion is first contained into and based on histidine-tagged red fluorescent protein in surface（It could alternatively be
Green fluorescent protein GFP, near-infrared fluorescent protein I FP, yellow fluorescence protein YFP, cyan fluorescent protein CFP and other band groups
Protein of His tag etc.）PH=7.0-8.0 phosphate-buffered is added according to 0.05-0.25mg protein/mg resin particles
In solution, stirring is allowed to add in the gravitational settling post with filter membrane after being sufficiently mixed.Will（3- dimethylamino-propyls）- 3- ethyls
Carbodiimide hydrochloride（EDC）With N- hydroxysuccinimides（NHS）In mass ratio 1:2-5:2 add in deionized water, of short duration
Concussion, which is mixed, to be added in gravity post, is shaken 30-60min, is completed the activation to red fluorescent protein surface.Add 5-10 μ L 3-
Aminopropyl triethoxysilane, fully shaking is allowed to abundant dissolving, reacts 12-16h, uses deionized water rinsing pillar, removes not
The crosslinking agent and byproduct of reaction reacted completely.By TEOS and hexamethylene according to volume ratio 1:1-1:1.5 add gravity post, shake
Swing and be allowed to fully dissolving, react 8-12h, obtain adsorbing the nanometer silicon dioxide particle in resin surface, be washed with deionized.
The eluent being made up of phosphate buffer solution and imidazole solution is added into gravity post, wherein imidazole solution concentration is 1-2M, is filled
15-30min is shaken after dividing mixing, imidazoles can reduce the absorption of His-tagged albumen so that albumen comes off from resin surface.
Gravity post bottom plug is opened, resin particle is retained by filter membrane, the super filter tube for being 10KD with molecular weight is 3000g's
Ultrafiltration is washed 2-3 times under centrifugal action, is removed the imidazoles in solution, is obtained required nanometer silicon dioxide particle, uniform particle sizes
And nano particle good dispersion, with good fluorescence property.
With 20% ethanol and deionized water rinsing gravity post, the nanometer silicon dioxide particle obtained before is rejoined into post
Son, fully shaking, nanometer silicon dioxide particle can again be adsorbed by resin particle, can be weighed after simple purge process
Newly come into operation,（This measure can be applied equally to isolate and purify used nanometer silicon dioxide particle）Substantially increase nanometer
The utilization rate of silica dioxide granule in itself.
Embodiment two：Based on histidine-tagged red fluorescent protein matter（RFP）The further work(of nanometer silicon dioxide particle
Energyization
The nanometer silicon dioxide particle surface adsorbed again by resin particle in embodiment one can be further according to demand
Carry out functionalization：5-10 μ L 3- aminopropyl triethoxysilanes are added, fully shaking is allowed to abundant dissolving, reacts 12-
16h, gravity post is eluted with deionized water, removes the crosslinking agent and byproduct of reaction not reacted completely.0.01- is added afterwards
After 0.02mM FITC, fully shaking, it is put into shaking table and at the uniform velocity shakes 8-12h.Pillar is eluted with deionized water again, is removed not
The FITC reacted completely.Flag F ITC nanometer has just been obtained after the imidazoles in solution by simple elution process and removing
Silica dioxide granule.
Embodiment three：Prepared based on histidine-tagged catalase nanometer silicon dioxide particle
The resin particle of nickel ion is first contained into and based on histidine-tagged catalase according to 0.05- in surface
0.5mg enzymes/mg resin particles are added in pH=7.0-8.0 phosphate buffer solution, and stirring, which is allowed to be added to after being sufficiently mixed, to be carried
In the gravitational settling post of filter membrane.Prepared according to the preparation method in embodiment one, average grain diameter can be obtained for 20nm, grain
Footpath is uniform, the nano particle of the Silica-coated catalase of favorable dispersibility, by the peroxidating of Silica-coated
Hydrogen enzyme activity is maintained to more than the 90% of original vigor, and is provided with more excellent stability, while nanometer
The histidine-tagged of particle surface makes it possible to be recycled for multiple times.
Claims (3)
1. a kind of solid phase synthesis process based on histidine-tagged nanometer silicon dioxide particle, it is characterised in that the tool of this method
Body step is：
A. the resin particle of nickel ion is contained into and with histidine-tagged fluorescin according to 0.05~0.25mg eggs in surface
White matter/mg resin particles are added in the phosphate buffer solution of pH=7.0~8.0, and stirring is allowed to add after being sufficiently mixed and carried
In the gravitational settling post of filter membrane；
B. will（3- dimethylamino-propyls）- 3- ethyl-carbodiimide hydrochlorides and N- hydroxysuccinimides in mass ratio 1:2~
5:2 are dissolved in deionized water, then according to 0.2~0.5mg（3- dimethylamino-propyls）- 3- ethyl-carbodiimide hydrochlorides/mg
Mixed solution is added in step a gravitational settling post by resin particle, shakes 30~60min, is completed to fluorescin surface
Activation；5~10 μ L 3- aminopropyl triethoxysilanes are added, fully shaking is allowed to abundant dissolving, reacts 12~16h, uses
Deionized water rinsing pillar, removes the crosslinking agent and byproduct of reaction not reacted completely；
C. by tetraethoxysilane and hexamethylene according to volume ratio 1:1~1:1.5 mixing, mixed solution is pressed by with resin particle
Ratio according to 0.25~0.5 μ L/mg is added in gravitational settling post, and concussion is allowed to fully dissolving, reacts 8~12h, obtains adsorbing
The nanometer silicon dioxide particle of resin surface, is washed with deionized；
D. by by phosphate buffer solution and concentration for 1~2M imidazole solution according to volume ratio 2:1~4:1 mix wash
De- liquid is added in gravitational settling post, and 15~30min is shaken after being sufficiently mixed；Gravitational settling post bottom plug is opened, passes through filter
Film is retained to resin particle, and the super filter tube for being 10KD with molecular weight ultrafiltration under 3000g centrifugal action washs 2~3
It is secondary, the imidazoles in solution is removed, required nanometer silicon dioxide particle is obtained.
2. the solid phase synthesis process according to claim 1 based on histidine-tagged nanometer silicon dioxide particle, it is special
Levy and be that the resin particle that nickel ion is contained on described surface is：Ni Sepharose Fast Flow；Ni Aogarose 6
FF。
3. the solid phase synthesis process according to claim 1 based on histidine-tagged nanometer silicon dioxide particle, it is special
Levy and be that the described histidine-tagged fluorescin that carries has green fluorescent protein GFP, near-infrared fluorescent protein I FP, yellow
Fluorescin YFP or cyan fluorescent protein CFP.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510174320.3A CN104876228B (en) | 2015-04-14 | 2015-04-14 | Solid phase synthesis process based on histidine-tagged nanometer silicon dioxide particle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510174320.3A CN104876228B (en) | 2015-04-14 | 2015-04-14 | Solid phase synthesis process based on histidine-tagged nanometer silicon dioxide particle |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104876228A CN104876228A (en) | 2015-09-02 |
| CN104876228B true CN104876228B (en) | 2017-08-11 |
Family
ID=53943956
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510174320.3A Expired - Fee Related CN104876228B (en) | 2015-04-14 | 2015-04-14 | Solid phase synthesis process based on histidine-tagged nanometer silicon dioxide particle |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104876228B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109870516A (en) * | 2017-12-05 | 2019-06-11 | 中国科学院大连化学物理研究所 | A kind of screening of metabolite-protein Interaction System and characterizing method |
| CN110498419A (en) * | 2018-05-16 | 2019-11-26 | 南京理工大学 | Nanometer silicon dioxide particle and preparation method thereof for histidine-tagged protein purifying |
| CN111777051A (en) * | 2020-07-03 | 2020-10-16 | 南京理工大学 | Preparation method of sheet lithium iron phosphate electrode material |
| CN113030057B (en) * | 2021-04-12 | 2023-06-16 | 国家纳米科学中心 | Method for detecting affinity between nano material and protein and application thereof |
-
2015
- 2015-04-14 CN CN201510174320.3A patent/CN104876228B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN104876228A (en) | 2015-09-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104876228B (en) | Solid phase synthesis process based on histidine-tagged nanometer silicon dioxide particle | |
| CN105195111B (en) | Preparation method of polyethyleneimine-modified shitosan coated magnetic nano-cellulose adsorbent and products thereof | |
| CN102641702B (en) | Ionic liquid functionalized magnetic nanoparticle and preparation method and application thereof | |
| CN104801262B (en) | Preparation method and application of magnetic composite uranium adsorbent | |
| CN104607231B (en) | There is carbon nitride photocatalyst of three-dimensional ordered macroporous structure and preparation method thereof | |
| CN105771908B (en) | A kind of magnetic silica core-shell composite material and preparation method thereof for heavy metal adsorption | |
| CN110508249B (en) | Amidoxime modified silicon dioxide nano microsphere composite material and preparation method thereof | |
| CN109092254A (en) | A kind of preparation of double virtual template phthalic acid ester molecular engram magnetic materials and application method | |
| CN108745321B (en) | For separating the preparation method of the virtual template molecular blotting magnetic microsphere of anthocyanin | |
| CN106519301B (en) | A kind of preparation of SiO2/ spiral nanometer carbon fibers dual phase filler and its application in reinforcing rubber | |
| CN101815507A (en) | Micro-particles, blood-substitute and method for forming same | |
| CN104383548B (en) | A kind of preparation method for the controlled release nano material that can be recycled | |
| CN105817267B (en) | A kind of aminopropyl type solid base complex microsphere and preparation method thereof | |
| CN107970878A (en) | A kind of preparation method of phosphate group functionalization hollow mesoporous silica microsphere | |
| CN111151218A (en) | Preparation method and application of mesoporous geopolymer adsorbent | |
| JP2013542157A (en) | Production of irregular porous silicon dioxide material and application of fatty alcohol polyoxyethylene ether in its production | |
| JP2003525191A (en) | Amorphous silica particles containing boron | |
| CN107999019B (en) | Amphiphilic magnetic nanosphere and preparation method and adsorption application thereof | |
| CN110177617A (en) | The alkaline-resisting albumin A magnetic bead of high carrying capacity and its application method | |
| CN105056890B (en) | A kind of magnetic Nano disk and the method using its heavy metal-polluted water process of progress | |
| CN107275023B (en) | Golden shell magnetic bead and its preparation method and application | |
| CN110771623A (en) | Preparation method of mesoporous silica long-acting antibacterial nanomaterial with high selenium loading | |
| WO2016043470A1 (en) | Organic-inorganic hybrid nanoporous silica material having high selectivity to metal ion, and method for preparing same | |
| CN104826601B (en) | A kind of diatomaceous preparation method of magnetism | |
| CN100448455C (en) | Method for preparing coating particles for lowering moisture-absorption of Chinese medicine extract |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170811 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |