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

[0024] Example one : Preparation of red fluorescent protein (RFP) nano-silica particles based on histidine tag

[0025] First, the resin particles containing nickel ions on the surface and the red fluorescent protein based on histidine tag (can be replaced with green fluorescent protein GFP, near-infrared fluorescent protein IFP, yellow fluorescent protein YFP, cyan fluorescent protein CFP, and other histidine tags Proteins, etc.) according to 0.05-0.25mg protein/mg resin particles into the phosphate buffer solution with pH=7.0-8.0, stir to make it fully mixed, and then add to the gravity sedimentation column with filter membrane. Add (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in a mass ratio of 1:2-5:2 to deionize In water, briefly shake and mix evenly and add it to the gravity column, shake for 30-60 minutes to complete the activation of the red fluorescent protein surface. Add 5-10μL of 3-aminopropyltriethoxysilane, shake well to dissolve it, react for 12-16h, rinse the column with deionized water to remove incompletely reacted crosslinking agent and reaction by-products. Add TEOS and cyclohexane into a gravity column in a volume ratio of 1:1-1:1.5, shake to make them fully dissolved, and react for 8-12 hours to obtain nano-silica particles adsorbed on the resin surface, and wash with deionized water. Add the eluent composed of phosphate buffer solution and imidazole solution to the gravity column, where 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 the resin surface . Open the stopper at the bottom of the gravity column, trap the resin particles through the filter membrane, use an ultrafiltration tube with a molecular weight of 10KD under the action of 3000g centrifugal force to ultrafiltration and wash 2-3 times to remove the imidazole in the solution to obtain the desired nano-dioxide Silicon particles, with uniform particle size and good nanoparticle dispersion, have good fluorescent properties.

[0026] Rinse the gravity column with 20% ethanol and deionized water, add the previously obtained nano-silica particles to the column again, and shake it sufficiently. The nano-silica particles can be re-adsorbed by the resin particles and can be reused after a simple purification process , (This can also be applied to the separation and purification of used nano silica particles) greatly improving the utilization rate of the nano silica particles themselves.

[0027] Example 2: Further functionalization of red fluorescent protein (RFP) nano-silica particles based on histidine tag

[0028] The surface of the nano-silica particles re-adsorbed by the resin particles in Example 1 can be further functionalized according to requirements: add 5-10μL of 3-aminopropyltriethoxysilane, shake sufficiently to make it fully dissolved, and react 12- 16h, rinse the gravity column with deionized water to remove the incompletely reacted crosslinker and reaction by-products. Then add 0.01-0.02mM FITC, shake well, put it in a shaker and shake at a constant speed for 8-12h. Rinse the column again with deionized water to remove FITC that has not completely reacted. After a simple elution process and removal of imidazole in the solution, FITC-labeled nano silica particles were obtained.

[0029] Example 3: Preparation of catalase nano-silica particles based on histidine tag

[0030] First, the resin particles containing nickel ions on the surface and the histidine tag-based catalase are added to the phosphate buffer solution with pH=7.0-8.0 according to 0.05-0.5mg enzyme/mg resin particles, and then added to the phosphate buffer solution. In a gravity settling column with a 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 dispersibility, and silica-coated catalase activity It can still maintain more than 90% of the original vitality, and has more excellent stability. At the same time, the histidine label on the surface of the nanoparticle allows it to be recycled many times.