CN104876228A - Solid-phase synthesis method for nano silicon dioxide particles based on histidine tag - Google Patents

Abstract

The invention discloses a method for solid-phase synthesis of nano silicon dioxide based on histidine tagged protein. In this method, the histidine tag of the histidine-tagged protein is complexed with the nickel ions on the surface of the resin particle, so that the histidine-tagged protein is first adsorbed on the surface of the resin particle, and the immobilized protein is used as the nucleus to grow nanometer particles. Silicon oxide particles. The salient feature and advantage of this method is that throughout the synthesis of silica nanoparticles and the subsequent surface functionalization process, the nanoparticles remain non-covalently linked to the resin through the histidine tag, enabling purification after each step The process is greatly simplified into a simple elution process; and this non-covalent link is reversible. After the synthesis and functionalization are completed, the nanoparticles can be eluted from the surface of the resin particles through the eluent, and at the same time, the elution contains histamine After the acid-tagged nanoparticles are applied, they can also be recovered by adsorption to the resin particles again.

Description

Solid Phase Synthesis of Nano-Silica Particles Based on Histidine Tag

technical field

The invention relates to a solid phase synthesis method of nano silicon dioxide particles, in particular to a solid phase synthesis method of nano silicon dioxide particles based on histidine tags.

Background technique

Nanoparticles refer to microscopic particles of the nanometer scale, which have surface effects, small size effects, and quantum effects, and exhibit many new and different properties, that is, their optical, mechanical, thermal, and other chemical properties and bulk solids. There will be a noticeable difference. Nanoparticles are widely used in fields including energy, materials, biomedicine, and the environment due to their excellent potentials such as small particle size, large specific surface area, and good dispersion.

As a special nanoparticle, nano-silica particles have many advantages while having the universal characteristics of nanoparticles: 1) As a non-toxic, odorless, and pollution-free non-metallic material, nano-silica particles have good 2) As a chemically inert material, silica has stable chemical properties; 3) The synthesis principle of silica nanoparticles is simple, the shape is regular, the particle size is controllable, and it is easy to apply. However, the preparation process of the traditional method for preparing silica nanoparticles is generally complicated, especially for the preparation of nanoparticles with relatively small particle size and good dispersion in the solution. It is more difficult, which is an important reason for the increase in the production cost of nanoparticles. At the same time, the nano-silica particles with good dispersion in the solution obtained by traditional methods are difficult to be recycled for reuse after use, so the utilization rate is very low.

Contents of the invention

The purpose of the present invention is to invent a method for solid-phase synthesis of nano-silica particles based on histidine-tagged proteins, aiming at the shortcomings of existing traditional preparation methods that are difficult to separate and purify, and samples are difficult to recover after use. The method greatly simplifies the process of purifying the nano-silica particles, and the nano-silica particles can be recovered by adsorption of resin particles after use.

In order to achieve the above purpose, the mechanism adopted in this method is: first mix the histidine-labeled protein with the resin containing nickel ions on the surface, make it adsorbed on the surface of the resin, stir to make it fully mixed and then add it to the reaction container; Histidine-tagged proteins on the resin surface were activated with (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) . It was then functionalized with 3-aminopropyltriethoxysilane. Finally, the functionalized histidine-tagged protein is used as the core, and a silane coupling agent is added to allow it to react and grow and obtain nano-silica particles adsorbed on the surface of the resin; after the synthesis of nano-silica particles is completed, further steps can be carried out Solid-phase functionalization; using imidazole as the eluent, the nano-silica particles with histidine tags can be eluted from the resin and enter the application process; after the application of the nanoparticles with histidine tags, It can also be recovered by adsorption of resin particles again.

According to above-mentioned mechanism, the present invention adopts following technical scheme:

A method for solid-phase synthesis of nano-silica particles based on histidine tags, characterized in that the specific steps of the method are:

a. Add the resin particles containing nickel ions on the surface and the fluorescent protein with histidine tag to the phosphate buffer solution with pH=7.0-8.0 according to 0.05-0.25 mg protein/mg resin particles, stir to make them fully mixed Add to the gravity settling column with filter membrane;

b. Dissolve (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at a mass ratio of 1:2 to 5:2 In deionized water, then add the mixed solution into the gravity settling column in step a according to 0.2-0.5mg EDC/mg resin particles, shake for 30-60min to complete the activation of the fluorescent protein surface; then add 5-10μL 3-ammonia Propyltriethoxysilane, fully shake to dissolve it, react for 12-16 hours, wash the column with deionized water, remove incompletely reacted cross-linking agent and reaction by-products;

c. Mix tetraethoxysilane (TEOS) and cyclohexane (Cyc) at a volume ratio of 1:1 to 1:1.5, and add the mixed solution to the resin particles at a ratio of 0.25 to 0.5 μL/mg to the gravity settling column In the process, oscillate to fully dissolve it, react for 8-12 hours to obtain nano-silica particles adsorbed on the surface of the resin, and wash with deionized water;

d. Add the eluent obtained by mixing phosphate buffer solution and imidazole solution with a concentration of 1-2M according to the volume ratio of 2:1-4:1 into the gravity settling column, mix well and shake for 15-30min; turn on the gravity The bottom of the settling column is plugged, and the resin particles are intercepted by the filter membrane, and the ultrafiltration tube with a molecular weight of 10KD is used for ultrafiltration and washing under the centrifugal force of 3000g for 2 to 3 times to remove the imidazole in the solution and obtain the required nano silica particles.

The above-mentioned resin particles containing nickel ions on the surface are: Ni Sepharose Fast Flow (GE); Ni Aogarose 6 FF (AOGMA).

The above-mentioned fluorescent proteins with histidine tags include green fluorescent protein GFP, near-infrared fluorescent protein IFP, yellow fluorescent protein YFP or cyan fluorescent protein CFP.

Compared with the existing method for preparing nano silicon dioxide particles, the present invention has the following beneficial effects:

1. Since the method of the present invention absorbs the protein as the nucleus on the surface of the resin in the process of preparing nano-silica particles, and then wraps it with silica, the multi-step reaction process of the whole nano-particle preparation and functionalization is within The surface of a solid-phase resin is completed, and the purpose of purifying the intermediate products and final products of multi-step reactions can be achieved by simple washing. However, in the traditional synthesis method, complex methods and techniques are often required to purify the obtained samples after each step of the reaction in the synthesis process, which is complicated and easily causes sample loss. The preparation process of the invention is more simplified, the purification is more convenient and the sample loss is not easy to be caused.

2. Since the surface of the nano-silica particles prepared by the method of the present invention has only one histidine, it is easy to be re-adsorbed by the resin particles after use and then recovered, and can be put into use again after a simple purification process , greatly improving the utilization rate of the nano-silica particles themselves.

3. Because the nano-silica particles prepared by the method of the present invention have the good properties of recyclability, and the excellent characteristics that silicon dioxide is easy to surface functionalize, the nano-silica particles after recovery can be further processed according to the previous application conditions. The surface functionalization of nano-silica particles can enhance the application effect and broaden its application range.

Description of drawings

Fig. 1 is the flow chart that embodiment one prepares His-RFPSilica nano silicon dioxide particle

Fig. 2 is (a) TEM image and (b) particle size statistics image of His-RFPSilica nano-silica particles prepared in Example 1.

3 is a TEM image of His-RFP Silica nano-silica particles adsorbed on the surface of resin particles prepared in Example 1.

4 is a SEM image of His-RFP Silica nano-silica particles adsorbed on the surface of resin particles prepared in Example 1.

Fig. 5 is (a) red and (b) yellow-green fluorescence spectra of His-RFPSilica-FITC (particle surface functionalized FITC) nano-silica particles prepared in Example 2.

Detailed ways

Examples are given below to further illustrate the present invention. It is necessary to point out that the following examples cannot be interpreted as limiting the protection scope of the present invention, if those skilled in the art make some non-essential improvements and adjustments to the present invention according to the above-mentioned content of the present invention, they still belong to the protection scope of the present invention .

Example 1 : Preparation of red fluorescent protein (RFP) nano-silica particles based on histidine tag

First, resin particles containing nickel ions on the surface and red fluorescent protein based on histidine tag (can be replaced by green fluorescent protein GFP, near-infrared fluorescent protein IFP, yellow fluorescent protein YFP, cyan fluorescent protein CFP and other histidine-labeled protein, etc.) according to 0.05-0.25mg protein/mg resin particles into the phosphate buffer solution of pH = 7.0-8.0, stirred to make it fully mixed and then added to the gravity settling column with filter membrane. Add (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in a mass ratio of 1:2-5:2 for deionization In water, shake briefly and add to the gravity column, shake for 30-60min to complete the activation of the red fluorescent protein surface. Add 5-10 μL of 3-aminopropyltriethoxysilane, shake fully to dissolve it, react for 12-16 hours, wash the column with deionized water, and remove the incompletely reacted cross-linking agent and reaction by-products. Add TEOS and cyclohexane into the gravity column according to the volume ratio of 1:1-1:1.5, shake to dissolve them fully, and react for 8-12 hours to obtain nano-silica particles adsorbed on the surface of the resin, which are washed with deionized water. Add the eluent composed of phosphate buffer solution and imidazole solution to the gravity column, in which the concentration of imidazole solution is 1-2M, mix well and shake for 15-30min. Imidazole can reduce the adsorption of His-tagged protein and make the protein fall off from the resin surface . Open the plug at the bottom of the gravity column, intercept the resin particles through the filter membrane, use an ultrafiltration tube with a molecular weight of 10KD to wash 2-3 times under the centrifugal force of 3000g, remove the imidazole in the solution, and obtain the required nanometer dioxide Silicon particles, with uniform particle size and good dispersion of nanoparticles, have good fluorescence properties.

Rinse the gravity column with 20% ethanol and deionized water, re-add the previously obtained nano-silica particles into the column, shake fully, the nano-silica particles can be re-adsorbed by the resin particles, and can be put into use again after a simple purification process , (This can also be applied to the separation and purification of used nano-silica particles) greatly improving the utilization of nano-silica particles themselves.

Example 2: Further functionalization of red fluorescent protein (RFP) nano-silica particles based on histidine tag

The surface of the nano-silica particles re-adsorbed by the resin particles in Example 1 can be further functionalized according to requirements: add 5-10 μL of 3-aminopropyltriethoxysilane, fully shake it to dissolve it, and react 12- After 16 hours, rinse the gravity column with deionized water to remove incompletely reacted cross-linking agent and reaction by-products. Then add 0.01-0.02mM FITC, shake fully, put it in a shaker and shake it at a constant speed for 8-12h. Rinse the column again with deionized water to remove incompletely reacted FITC. After a simple elution process and removal of imidazole in the solution, the FITC-labeled nano-silica particles were obtained.

Example 3: Preparation of catalase nano-silica particles based on histidine tag

First, add the resin particles containing nickel ions on the surface and the catalase based on histidine tag into the phosphate buffer solution with pH=7.0-8.0 according to 0.05-0.5 mg enzyme/mg resin particles, stir to make them fully mixed and then add to Gravity settling column with filter membrane. According to the preparation method in Example 1, it is possible to obtain silica-coated catalase nanoparticles with an average particle size of 20nm, uniform particle size, and good dispersion, and the activity of catalase encapsulated by silica It can still maintain more than 90% of the original activity, and has more excellent stability. At the same time, the histidine tag on the surface of the nanoparticles enables it to be recycled many times.

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.