CN104876228A - Solid-phase synthesis method for nano silicon dioxide particles based on histidine tag - Google Patents
Solid-phase synthesis method for nano silicon dioxide particles based on histidine tag Download PDFInfo
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- CN104876228A CN104876228A CN201510174320.3A CN201510174320A CN104876228A CN 104876228 A CN104876228 A CN 104876228A CN 201510174320 A CN201510174320 A CN 201510174320A CN 104876228 A CN104876228 A CN 104876228A
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Abstract
The invention discloses a solid-phase synthesis method for nano silicon dioxide particles based on a histidine tag. The method comprises the following steps: firstly complexing with nickel ions on resin particle surfaces by virtue of the histidine tag of histidine tag protein; adsorbing the histidine tag protein on the resin particle surfaces; and growing the nano silicon dioxide particles employing immobilized protein as nucleus. The method has significant characteristics and advantages that nano particles are always kept in non-covalent link through the histidine tag and resin in the overall synthesis process of the nano silicon dioxide particles and the subsequent surface functionalization processes; the purification process after the reaction in each step is greatly simplified into a simple leaching process; the non-covalent link is reversible; after synthesis and functionalization are finished, the nano particles can be eluted from the resin particle surfaces though an eluant; and meanwhile, the eluted nano particles with the histidine tag can also be recovered through adsorption of the resin particles after being applied.
Description
Technical field
The present invention relates to a kind of solid phase synthesis process of nanometer silicon dioxide particle, be specifically related to a kind of solid phase synthesis process based on histidine-tagged nanometer silicon dioxide particle.
Background technology
Nano particle refers to the microscopic particles of nanometer scale, there are surface effects, small-size effect and quantum effect etc., show as many new different qualities, namely the character of its optics, mechanics, calorifics and other chemical aspects will have obvious difference with comparing during bulk solid.The excellent potential qualities such as the particle diameter that nano particle has due to itself is large compared with little, specific surface area, favorable dispersity, are all widely used in fields such as comprising 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 is a lot of advantage: 1) nontoxic as one, tasteless, the free of contamination non-metallic material of nano silicon, have good biological safety; 2) silicon-dioxide is as a kind of chemical inert material, stable chemical nature; 3) nano SiO 2 particle composition principle is simple, pattern is regular, size tunable, is easy to application.But, traditional method preparation process preparing nano SiO 2 particle is generally complicated, particularly prepare particle diameter nano particle that is smaller, good dispersity in the solution, separating-purifying in the middle of the multistep manufacture of sample and after all having prepared is more difficult, is the major reason causing nano particle production cost to rise.Meanwhile, the nanometer silicon dioxide particle of the good dispersity in the solution that traditional method obtains is difficult to be recovered and is re-used after use, and therefore utilization ratio is very low.
Summary of the invention
It is difficult to the object of the invention is for existing traditional preparation methods separating-purifying, and sample is difficult to the deficiency reclaimed after using, invent a kind of solid phase synthesis process of the nanometer silicon dioxide particle based on histidine-tagged protein matter.The method enormously simplify the process that nanometer silicon dioxide particle is purified, and can reclaim by adopting resin particle absorption after nanometer silicon dioxide particle uses.
For achieving the above object, should the mechanism of employing of method be: first by histidine-tagged protein matter and the mixed with resin of surface containing nickel ion, make it to be adsorbed on resin surface, stir and add in reaction vessel after making it fully mixing; The histidine-tagged protein matter being adsorbed on resin surface activates its surface with (3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).Then functionalization is carried out with 3-aminopropyl triethoxysilane.Last with the histidine-tagged protein matter of this functionalization for core, add silane coupling agent and make it react to grow and the nanometer silicon dioxide particle obtaining being adsorbed on resin surface; After nanometer silicon dioxide particle has synthesized, further solid functional can be proceeded; With imidazoles as elutriant, can to make with histidine-tagged nanometer silicon dioxide particle, from wash-out resin, to enter application process; With histidine-tagged nano particle after application, can also again be adsorbed by resin particle and reclaim.
According to above-mentioned mechanism, the present invention adopts following technical scheme:
Based on a solid phase synthesis process for histidine-tagged nanometer silicon dioxide particle, it is characterized in that the method concrete steps be:
A. surface contained the resin particle of nickel ion and join in the phosphate buffer soln of pH=7.0 ~ 8.0 with histidine-tagged fluorescin according to 0.05 ~ 0.25mg protein/mg resin particle, stirring adds in the gravity settling post with filter membrane after making it fully mixing;
B. by (3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in mass ratio 1:2 ~ 5:2 be dissolved in deionized water, then according to 0.2 ~ 0.5mg EDC/mg resin particle, mixing solutions is joined in the gravity settling post of step a, concussion 30 ~ 60min, completes the activation to fluorescin surface; Add 5 ~ 10 μ L 3-aminopropyl triethoxysilanes again, fully concussion makes it abundant dissolving, and reaction 12 ~ 16h, uses deionized water rinsing pillar, remove linking agent and the byproduct of reaction of non-complete reaction;
C. tetraethoxysilane (TEOS) and hexanaphthene (Cyc) are mixed according to volume ratio 1:1 ~ 1:1.5, by mixing solutions in adding in gravity settling post with resin according to the ratio of 0.25 ~ 0.5 μ L/mg, concussion makes it abundant dissolving, reaction 8 ~ 12h, obtain the nanometer silicon dioxide particle being adsorbed on resin surface, with deionized water wash;
D. the imidazole solution by phosphate buffer soln and concentration being 1 ~ 2M is joined in gravity settling post according to the elutriant that volume ratio 2:1 ~ 4:1 mixes, fully concussion 15 ~ 30min after mixing; Open gravity settling post bottom stopper, by filter membrane, resin particle is retained, be that super filter tube ultrafiltration under the centrifugal action of 3000g of 10KD is washed 2 ~ 3 times with molecular weight, remove the imidazoles in solution, obtain required nanometer silicon dioxide particle.
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, yellow fluorescence protein YFP or cyan fluorescent protein CFP with histidine-tagged fluorescin.
The present invention, compared with the existing method preparing nanometer silicon dioxide particle, has following beneficial effect:
1, due to the inventive method in the process preparing nanometer silicon dioxide particle using as the protein adsorption of core at resin surface, with silicon-dioxide, it is wrapped up again, whole nanoparticle preparation all completes on a solid-phase resin surface with the polystep reaction process of functionalization, adopts simple flushing just can reach the object of purifying polystep reaction intermediate product and the finished product.And prior synthesizing method often walk in building-up process reacted after often need to apply complicated Method and Technology purifying carried out to the sample obtained, process is complicated and easily cause sample loss.Preparation process of the present invention simplifies more, and purifying is convenient and be not easy the loss causing sample.
2, the nanometer silicon dioxide particle surface prepared due to the inventive method has and only has a Histidine, therefore, easily can again be adsorbed by resin particle and then be recovered after use, again can come into operation after simple purge process, substantially increase the utilization ratio of nanometer silicon dioxide particle itself.
3, the nanometer silicon dioxide particle prepared due to the inventive method has callable good nature, add the excellent specific property that silicon-dioxide is easy to surface-functionalized, nanometer silicon dioxide particle after recovery can carry out further surface-functionalized according to applicable cases before, and then can strengthen the effect of nanometer silicon dioxide particle and widen its range of application.
Accompanying drawing explanation
Fig. 1 is the schema that embodiment one prepares His-RFPSilica nanometer silicon dioxide particle
Fig. 2 is (a) TEM figure and (b) particle diameter statistical graph that embodiment one prepares His-RFPSilica nanometer silicon dioxide particle.
Fig. 3 is the TEM figure that embodiment one preparation is adsorbed on the His-RFPSilica nanometer silicon dioxide particle of resin particle surfaces.
Fig. 4 is the SEM figure that embodiment one preparation is adsorbed on the His-RFPSilica nanometer silicon dioxide particle of resin particle surfaces.
Fig. 5 is that embodiment two prepares His-RFPSilica-FITC(particle surface functionalization FITC) (a) of nanometer silicon dioxide particle red and (b) yellow-green fluorescence spectrogram.
Embodiment
Provide embodiment below so that the invention will be further described.What be necessary to herein means out is that following examples can not be interpreted as limiting the scope of the invention; if the person skilled in the art in this field makes some nonessential improvement and adjustment according to the invention described above content to the present invention, still belong to scope.
embodiment one:
prepare based on histidine-tagged red fluorescent protein matter (RFP) nanometer silicon dioxide particle
First surface is contained nickel ion resin particle and based on histidine-tagged red fluorescent protein (can replace with green fluorescent protein GFP, near-infrared fluorescent protein I FP, yellow fluorescence protein YFP, cyan fluorescent protein CFP and other with histidine-tagged protein etc.) add in the phosphate buffer soln of pH=7.0-8.0 according to 0.05-0.25mg protein/mg resin particle, stir in the gravity settling post added after making it fully mixing with filter membrane.By (3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in mass ratio 1:2-5:2 add in deionized water, of short duration concussion mixing adds in gravity post, concussion 30-60min, completes the activation to red fluorescent protein surface.Add 5-10 μ L 3-aminopropyl triethoxysilane, fully concussion makes it abundant dissolving, and reaction 12-16h, uses deionized water rinsing pillar, remove linking agent and the byproduct of reaction of non-complete reaction.TEOS and hexanaphthene are added gravity post according to volume ratio 1:1-1:1.5, and concussion makes it abundant dissolving, and reaction 8-12h, obtains the nanometer silicon dioxide particle being adsorbed on resin surface, use deionized water wash.The elutriant be made up of phosphate buffer soln and imidazole solution is added gravity post, and wherein imidazole solution concentration is 1-2M, and fully shake 15-30min after mixing, imidazoles can reduce the absorption of His-tagged albumen, and albumen is come off from resin surface.Open gravity post bottom stopper, by filter membrane, resin particle is retained, it is ultrafiltration washing 2-3 time under the centrifugal action of 3000g of the super filter tube of 10KD with molecular weight, remove the imidazoles in solution, obtain required nanometer silicon dioxide particle, uniform particle sizes and nanoparticulate dispersed is good, has good fluorescence property.
With 20% ethanol and deionized water rinsing gravity post, the nanometer silicon dioxide particle obtained before is rejoined pillar, abundant concussion, nanometer silicon dioxide particle can be adsorbed again by resin particle, can again come into operation after simple purge process, (this measure can be applied to the used nanometer silicon dioxide particle of separation and purification equally) substantially increases the utilization ratio of nanometer silicon dioxide particle itself.
embodiment two: based on the histidine-tagged further functionalization of red fluorescent protein matter (RFP) nanometer silicon dioxide particle
Functionalization can be carried out further according to demand by the nanometer silicon dioxide particle surface that resin particle adsorbs again: add 5-10 μ L 3-aminopropyl triethoxysilane in embodiment one, abundant concussion makes it abundant dissolving, reaction 12-16h, with deionized water drip washing gravity post, remove linking agent and the byproduct of reaction of non-complete reaction.Add 0.01-0.02mM FITC afterwards, fully after concussion, put into shaking table and at the uniform velocity shake 8-12h.Again use deionized water drip washing pillar, remove the FITC of non-complete reaction.Just the nanometer silicon dioxide particle of flag F ITC is obtained after removing the imidazoles in solution through simple elution process.
embodiment three: prepare based on histidine-tagged catalase nanometer silicon dioxide particle
First surface contained the resin particle of nickel ion and add in the phosphate buffer solution of pH=7.0-8.0 based on histidine-tagged catalase according to 0.05-0.5mg enzyme/mg resin particle, stirring joins after making it fully mixing in the gravity settling post with filter membrane.Be prepared according to the preparation method in embodiment one, the catalatic nano particle of Silica-coated that median size is 20nm, uniform particle sizes, favorable dispersity can be obtained, activity of catalase through Silica-coated still can remain on more than 90% of original vigor, and be provided with more excellent stability, histidine-tagged the making it of nano grain surface can repeatedly recycle simultaneously.
Claims (3)
1. based on a solid phase synthesis process for histidine-tagged nanometer silicon dioxide particle, it is characterized in that the method concrete steps be:
A. surface contained the resin particle of nickel ion and join in the phosphate buffer soln of pH=7.0 ~ 8.0 with histidine-tagged fluorescin according to 0.05 ~ 0.25mg protein/mg resin particle, stirring adds in the gravity settling post with filter membrane after making it fully mixing;
B. by (3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in mass ratio 1:2 ~ 5:2 be dissolved in deionized water, then according to 0.2 ~ 0.5mg EDC/mg resin particle, mixing solutions is joined in the gravity settling post of step a, concussion 30 ~ 60min, completes the activation to fluorescin surface; Add 5 ~ 10 μ L 3-aminopropyl triethoxysilanes again, fully concussion makes it abundant dissolving, and reaction 12 ~ 16h, uses deionized water rinsing pillar, remove linking agent and the byproduct of reaction of non-complete reaction;
C. tetraethoxysilane (TEOS) and hexanaphthene (Cyc) are mixed according to volume ratio 1:1 ~ 1:1.5, by mixing solutions in adding in gravity settling post with resin according to the ratio of 0.25 ~ 0.5 μ L/mg, concussion makes it abundant dissolving, reaction 8 ~ 12h, obtain the nanometer silicon dioxide particle being adsorbed on resin surface, with deionized water wash;
D. the imidazole solution by phosphate buffer soln and concentration being 1 ~ 2M is joined in gravity settling post according to the elutriant that volume ratio 2:1 ~ 4:1 mixes, fully concussion 15 ~ 30min after mixing; Open gravity settling post bottom stopper, by filter membrane, resin particle is retained, be that super filter tube ultrafiltration under the centrifugal action of 3000g of 10KD is washed 2 ~ 3 times with molecular weight, remove the imidazoles in solution, obtain required nanometer silicon dioxide particle.
2. the solid phase synthesis process based on histidine-tagged nanometer silicon dioxide particle according to claim 1, is characterized in that the resin particle that nickel ion is contained on described surface is: Ni Sepharose Fast Flow (GE); Ni Aogarose 6 FF (AOGMA).
3. the solid phase synthesis process based on histidine-tagged nanometer silicon dioxide particle according to claim 1, is characterized in that described having green fluorescent protein GFP, near-infrared fluorescent protein I FP, yellow fluorescence protein YFP or cyan fluorescent protein CFP with histidine-tagged fluorescin.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN113030057A (en) * | 2021-04-12 | 2021-06-25 | 国家纳米科学中心 | Method for detecting affinity between nano material and protein and application thereof |
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2015
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Cited By (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 |
| CN113030057A (en) * | 2021-04-12 | 2021-06-25 | 国家纳米科学中心 | Method for detecting affinity between nano material and protein and application thereof |
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