Polypeptide for catalytic synthesis of nano-silica, nano-silica and method
By designing the reaction of silicide No. 1 and silicide No. 2 polypeptides with silicic acid solutions, the problems of high temperature and high pressure and serious pollution in traditional nanosilicon synthesis are solved, and rapid and green nanosilicon synthesis is achieved. The products are suitable for drug carriers and biosensors and other fields.
Patent Information
- Application Number
- CN202211164708.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-23
- Publication Date
- 2025-05-30
- Estimated Expiration
- 2042-09-23
AI Technical Summary
The prior art requires high temperature and high pressure when synthesising nano silica, which is highly contaminated, expensive, and involves toxic materials, and lacks effective alternatives to natural products.
A polypeptide called Silicide No. 1 and Silicide No. 2 were designed, and nanosilicon dioxide was obtained by dissolving it in a buffer and mixing it with a silica acid solution, leaving it stand, and centrifugation, washing and drying.
The nanosilica reaction conditions of the catalytic synthesis of this polypeptide are suitable, the reaction speed is fast, and there is no toxic and harmful substances are produced. It provides a green synthesis method, with uniform product shape and few residues, and has huge potential for biotechnology application.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a polypeptide for catalytic synthesis of nano-silica, nano-silica and a method thereof. Background Art
[0002] Due to its unique optical properties, high specific surface area and low toxicity, nano-silica is applied in various fields such as catalysts, coatings, food additives, drug carriers, biosensors, etc. However, the traditional method for synthesizing nano-silica requires reactions under high temperature and high pressure, with high pollution, expensive synthesis and involving toxic materials, and there is an urgent need to replace it with effective natural products.
[0003] Diatoms are a very abundant single-celled alga in nature, with a unique silica cell wall, which is composed of nano-silica and organic molecules, has a unique micro-nano structure, consists of an upper shell and a lower shell, and forms a structure similar to a petri dish. The diatom shell has a large specific surface area and strong adsorption ability, and can be used as an adsorbent and molecular sieve. Its biocompatibility is strong and can be used as a drug carrier, which can solve the defect that some drugs have low solubility in water, and can also make the drugs slowly release in the body to obtain a better therapeutic effect. Diatoms have a high utilization rate of sunlight and are also used in new energy batteries.
[0004] The diatom shell has excellent properties, which are determined by its nano-structure and unique morphology. Diatomite is the natural diatom shell obtained after the deposition and corrosion of diatoms, is an important mineral resource, and is also the main source of diatom shells at present. However, as a mineral resource, diatomite has limited reserves, and multiple processes are required to remove impurities during the development and utilization process, which often leads to pollution. Diatomite on the market often has an incomplete structure, small and uneven morphology, and substances such as acids used for impurity removal may also remain in the processed diatomite, restricting the use of diatomite.
[0005] A variety of proteins extracted from diatom shells have been proven to be related to the formation of diatom shells, and a variety of polypeptides designed therefrom have also been proven to be able to synthesize nano-silica under certain conditions. However, currently only a few polypeptides have been studied and have not been used in actual production. Therefore, there is an urgent need for a new polypeptide for synthesizing nano-silica to provide a new direction for the catalytic synthesis of nano-silica. Summary of the Invention
[0006] The purpose of the present invention is to solve the deficiencies in the prior art, and provide a polypeptide for catalytic synthesis of nano-silica, nano-silica and a method thereof.
[0007] The specific technical solutions adopted by the present invention are as follows:
[0008] In the first aspect, the present invention provides a polypeptide for catalytic synthesis of nano-silica, including polypeptide silicification No. 1 or silicification No. 2. The amino acid sequence structure of the above-mentioned silicification No. 1 is CSSKK-n(GSK)-RRIL, where n≥1; the amino acid sequence structure of the above-mentioned silicification No. 2 is as shown in SEQ ID No. 2.
[0009] Preferably, the amino acid sequence structure of the above-mentioned silicification No. 1 is as shown in SEQ ID No. 1.
[0010] In the second aspect, the present invention provides a method for preparing nano-silica using the polypeptide described in the first aspect, specifically as follows:
[0011] S1: Dissolve the above-mentioned polypeptide in a buffer solution, mix evenly to obtain a polypeptide solution;
[0012] S2: Dissolve the silicon source in water, add a hydrochloric acid solution, mix evenly and then stand still to prepare a silicic acid solution;
[0013] S3: Mix the silicic acid solution and the polypeptide solution evenly, stand still until the reaction is complete, and obtain nano-silica through centrifugation, washing and drying.
[0014] Preferably, the buffer solution in S1 is a phosphate buffer solution or a Tris-HCl buffer solution; the concentration of the phosphate buffer solution is 50-200 mM, and the concentration of the Tris-HCl buffer solution is 25-100 mM.
[0015] Preferably, the above-mentioned phosphate buffer solution is prepared by mixing 200 mM sodium dihydrogen phosphate and 200 mM disodium hydrogen phosphate, adjusting the pH to 6-8, and diluting to the required concentration.
[0016] Preferably, the concentration of the above-mentioned polypeptide solution is 0.5 mM.
[0017] Preferably, the silicon source in S2 is tetramethoxysilane or tetraethoxysilane; the concentration of the silicon source in the silicic acid solution is 0.25-2 M, and the concentration of hydrochloric acid in the silicic acid solution is 1 mM.
[0018] Preferably, in S3, the silicic acid solution and the polypeptide solution are mixed in a volume ratio of 1:9.
[0019] Preferably, in S3, the centrifugation speed is 10000-15000 rpm, the time is 2-5 min, the drying temperature is 70 °C, and the drying time is 6 h.
[0020] In the third aspect, there is provided nano-silica prepared by using the method for preparing nano-silica described in the second aspect.
[0021] Compared with the prior art, the present invention has the following beneficial effects:
[0022] The polypeptide silicification No. 1 provided by the present invention can rapidly catalyze the synthesis of nano-silica under certain conditions, and the polypeptide silicification No. 1 has catalytic activity within the range of pH = 6 - 8. When the reaction conditions are appropriate, the reaction can be basically completed within 5 minutes. Compared with chemical synthesis, the nano-silica synthesized by this polypeptide has simple reaction steps, high rate, and does not produce toxic and harmful substances during the reaction process, which is of great significance for the green synthesis of nano-silica. The synthesized nano-silica has relatively uniform morphological size and less residual toxic and harmful substances, making it have great application potential in the field of biotechnology, such as drug carriers, biosensors, etc. Description of the Drawings
[0023] Figure 1 It is a comparison chart of the yields of nano-silica synthesized by R5, silicification No. 1 and silicification No. 2 in Example 1;
[0024] Figure 2 It is a scanning electron microscope image of the nano-silica prepared in Example 1, where (a) is the scanning electron microscope image of the nano-silica synthesized by silicification No. 2, (b) is the scanning electron microscope image of the nano-silica synthesized by silicification No. 1, and (c)(d) are the energy spectrum analysis diagrams of the nano-silica synthesized by silicification No. 1;
[0025] Figure 3 It is a comparison chart of the yields of nano-silica synthesized by R5, silicification No. 1 and silicification No. 2 in Example 2;
[0026] Figure 4 It is a semi-quantitative analysis of the synthesis rates of nano-silica by R5 (a), silicification No. 1 (b) and silicification No. 2 (c) in Example 2;
[0027] Figure 5 It is a comparison chart of the yield of nano-silica synthesized by silicification No. 1 in Example 3 (a) and an electron microscope image of spherical nano-silica synthesized in the Tris-HCl system (b). Detailed Embodiments
[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
[0029] Example 1
[0030] This example provides a method for catalytically synthesizing nano-silica using a phosphate buffer with pH = 8, polypeptide R5, siliconized one (zju101) as shown in SEQ ID No.1, and siliconized two (zju102) as shown in SEQ ID No.2, specifically as follows:
[0031] (1) Mix a disodium hydrogen phosphate solution with a concentration of 200 mM and a sodium dihydrogen phosphate solution, adjust the pH to 8, and dilute to a concentration of 50 mM to obtain a phosphate buffer;
[0032] (2) Dissolve the polypeptide in the above phosphate buffer and mix evenly to obtain a polypeptide solution with a concentration of 0.5 mM;
[0033] (3) Dissolve 0.25 mmol of trimethoxysilane in 1 mL of water, and add 10 μL of 100 mM hydrochloric acid to prepare a 0.25 M silicic acid solution, and let it stand for 4 min for later use;
[0034] (4) Take 50 μL of the silicic acid solution and add it to a 1.5 mL centrifuge tube, mix evenly with 0.45 mL of the polypeptide solution, and react fully for 2 h to obtain a white precipitate;
[0035] (5) Centrifuge the above white precipitate at 12000 rpm for 5 min, remove the supernatant, wash it twice with deionized water, then wash it once with 95% ethanol, and dry it at 70 °C for 6 h to obtain nano-silica powder.
[0036] The yield m of nano-silica 二氧化硅 is determined by the mass difference between the mass m 总 of the product after drying and the centrifuge tube and the mass m 离心管 of the centrifuge tube before the experiment:
[0037] m 二氧化硅 = m 总 - m 离心管
[0038] Figure 1 is a comparison chart of the yields of nano-silica catalytically synthesized by different polypeptides in a phosphate buffer with pH = 8. As Figure 1As shown, the yields of nano-silica of Silication No. 1 and Silication No. 2 are significantly higher than that of polypeptide R5. Polypeptide R5 is derived from the protein related to the synthesis of silica in the frustule of Cylindrotheca fusiformis. Through research, it is found that the KXXK (X = S / G / A) sequence plays an important role in the catalytic synthesis of nano-silica by polypeptides. Therefore, based on polypeptide R5, Silication No. 1 retains the starting SSKK and ending RRIL structures, inserts 3 groups of GSK structures in the middle, so that the polypeptide contains three groups of KGSK structures, and adds C at the beginning to obtain the polypeptide sequence of Silication No. 1, and its amino acid sequence structure is as shown in SEQ ID No.1, specifically CSSKKGSKGSKGSKRRIL. Since amino acids S and T have a high structural similarity, based on R5, Silication No. 2 changes the S in it to T, and also obtains a polypeptide with the function of catalyzing the synthesis of nano-silica, and its amino acid sequence structure is as shown in SEQ ID No.2, specifically TTKKTGTYTGTKGTKRRIL.
[0039] Figure 2 Scanning electron micrographs of the synthesis of nano-silica by Silication No. 2 (a) and Silication No. 1 (b) in a phosphate buffer solution with pH = 8. According to Figure 2 the results, Silication No. 1 and Silication No. 2 have catalytic activity under the condition of pH = 8, can catalyze the synthesis of spherical nano-silica, and according to Figure 2 the energy spectrum analysis diagrams of (c) and (d), it is confirmed that the reaction product is nano-silica.
[0040] It should be noted that in this embodiment, there are 3 groups of GSK structures in the polypeptide sequence of Silication No. 1 as shown in SEQ ID No.1, but the specific number of groups can be adjusted according to the actual situation. The minimum is 1 group, and similar effects can also be achieved.
[0041] Example 2
[0042] This embodiment provides a method for catalytically synthesizing nano-silica by using phosphate buffer solutions with different pH values, polypeptide R5, Silication No. 1 (zju101) as shown in SEQ ID No.1, and Silication No. 2 (zju101) as shown in SEQ ID No.2, which is specifically as follows:
[0043] (1) Mix the disodium hydrogen phosphate solution with a concentration of 200 mM and the sodium dihydrogen phosphate solution with a concentration of 200 mM, adjust the pH respectively, and dilute to 50 mM to obtain 5 kinds of phosphate buffer solutions with pH values of 6, 6.5, 7, 7.5, and 8;
[0044] (2) Dissolve the polypeptides in the above phosphate buffer solutions respectively, mix them evenly to obtain polypeptide solutions with a concentration of 0.5 mM;
[0045] (3) Dissolve 0.25 mmol of trimethoxysilane in 1 mL of water, and add 10 μL of 100 mM hydrochloric acid thereto to prepare a 0.25 M silicic acid solution. Let it stand for 4 min and then set aside for use.
[0046] (4) Take 50 μL of the silicic acid solution and add it to a 1.5 mL centrifuge tube. Mix it evenly with 0.45 mL of the polypeptide solution and react fully for 2 h to obtain a white precipitate.
[0047] (5) Centrifuge the above white precipitate at 12000 rpm for 5 min, remove the supernatant, wash it twice with deionized water, then wash it once with 95% ethanol, and dry it at 70 °C for 6 h to obtain nano-silica powder.
[0048] The yield m of nano-silica 二氧化硅 is determined by the mass difference between the mass m 总 of the product after drying and the centrifuge tube and the mass m 离心管 of the centrifuge tube before the experiment:
[0049] m 二氧化硅 = m 总 - m 离心管
[0050] Figure 3 Figure for comparing the yields of nano-silica synthesized by R5, Silicification No. 1, and Silicification No. 2 in phosphate buffer solutions with different pH values. As Figure 3 shown, R5 and Silicification No. 2 can only catalyze the synthesis of nano-silica in the range of pH = 6.5 - 8, while Silicification No. 1 has catalytic activity in the range of pH = 6 - 8, and the reaction range is relatively wide.
[0051] In order to semi-quantitatively analyze the catalytic reaction rate of the polypeptide at different pH values, according to the characteristic that the reaction system becomes turbid due to the continuous generation of nano-silica particles during the reaction, the change in absorbance during the reaction process is tracked with a UV spectrophotometer to semi-quantitatively analyze the rate of this reaction. The specific implementation process is the same as the application method of the polypeptide for catalytic synthesis of nano-silica described above, except that the reaction is carried out in a disposable cuvette and the solution volume is twice that in the above method. During this reaction process, a UV spectrophotometer is used to scan at 290 nm, starting from the addition of the silicic acid solution until the absorbance basically remains unchanged.
[0052] Figure 4 Semi-quantitative analysis of the synthesis rates of nano-silica by R5 (a), Silicification No. 1 (b), and Silicification No. 2 (c) in phosphate buffer solutions with different pH values. As Figure 5 can be seen, at the same pH value, the catalytic rates of Silicification No. 1 and Silicification No. 2 are both higher than that of R5, and among them, Silicification No. 1 can basically react when the solution is just mixed.
[0053] Example 3
[0054] This example provides a method for catalytic synthesis of nano-silica using different types of buffer solutions and polypeptides R5, such as siliconized one (zju101) shown in SEQ ID No.1 and siliconized two (zju102) shown in SEQ ID No.2, as follows:
[0055] (1) Prepare phosphate buffer solutions with concentrations of 50 mM, 100 mM, and 200 mM and 50 mM Tris-HCl buffer solution respectively;
[0056] (2) Dissolve the polypeptides in the above buffer solutions respectively, mix them evenly to obtain polypeptide solutions with a concentration of 0.5 mM;
[0057] (3) Dissolve 0.25 mmol of trimethoxysilane in 1 mL of water, and add 10 μL of 100 mM hydrochloric acid thereto to prepare a 0.25 M silicic acid solution, and let it stand for 4 min for later use;
[0058] (4) Take 50 μL of the silicic acid solution and add it to a 1.5 mL centrifuge tube, mix it evenly with 0.45 mL of the polypeptide solution, and react fully for 2 h to obtain a white precipitate;
[0059] (5) Centrifuge the above white precipitate at 12000 rpm for 5 min, remove the supernatant, wash it twice with deionized water, then wash it once with 95% ethanol, and dry it at 70 °C for 6 h to obtain nano-silica powder.
[0060] The yield m of nano-silica 二氧化硅 is determined by the mass difference between the mass m 总 of the dried product and the centrifuge tube and the mass m 离心管 of the centrifuge tube before the experiment:
[0061] m 二氧化硅 = m 总 - m 离心管
[0062] Figure 5 Figure (a) shows the yield comparison diagram of siliconized one catalyzing the synthesis of nano-silica in different buffer systems and the electron microscopy image (b) of spherical nano-silica synthesized in the Tris-HCl system. According to Figure 5 (a), it can be seen that in the Tris-HCl buffer solution, siliconized one can also catalyze the synthesis of nano-silica and the yield is higher. In the prior art, it is generally considered that phosphate is the key factor for R5 to catalyze the synthesis of nano-silica. However, it is proved in this example that the polypeptide also has catalytic activity in the Tris-HCl buffer solution, and the scanning electron microscopy image of the catalytic synthesis of spherical nano-silica is as shown in Figure 5 (b).
[0063] The embodiments described above are only a preferred solution of the present invention, but they are not intended to limit the present invention. Those of ordinary skill in the relevant technical field can still make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.
Claims
1. A polypeptide for catalytic synthesis of nano-silica, characterized in that, the polypeptide is Silicified No. 1 or Silicified No.
2. The amino acid sequence structure of Silicified No. 1 is CSSKK-n(GSK)-RRIL, where n≥1; the amino acid sequence structure of Silicified No. 2 is shown as SEQ ID No.2; the amino acid sequence structure of Silicified No. 1 shown as SEQ ID No.1 is CSSKKGSKGSKGSKRRIL.
2. A method for preparing nano-silica using the polypeptide according to claim 1, characterized in that, specifically as follows: S1: Dissolve the polypeptide in a buffer solution, mix evenly to obtain a polypeptide solution; S2: Dissolve the silicon source in water, add hydrochloric acid solution, mix evenly and then stand still to prepare a silicic acid solution; S3: Mix the silicic acid solution and the polypeptide solution evenly, stand still until the reaction is complete, and obtain nano-silica through centrifugation, washing and drying.
3. The method for preparing nano-silica according to claim 2, characterized in that, the buffer solution in S1 is phosphate buffer solution or Tris-HCl buffer solution; the concentration of the phosphate buffer solution is 50~200 mM; the concentration of the Tris-HCl buffer solution is 25~100 mM.
4. The method for preparing nano-silica according to claim 2, characterized in that, the phosphate buffer solution is prepared by mixing 200 mM sodium dihydrogen phosphate and 200 mM disodium hydrogen phosphate, adjusting the pH to 6-8, and diluting to the required concentration.
5. The method for preparing nano-silica according to claim 2, characterized in that, the concentration of the polypeptide solution is 0.5 mM.
6. The method for preparing nano-silica according to claim 2, characterized in that, the silicon source in S2 is tetramethoxysilane or tetraethoxysilane; the concentration of the silicon source in the silicic acid solution is 0.25-2 M, and the concentration of hydrochloric acid in the silicic acid solution is 1 mM.
7. The method for preparing nano-silica according to claim 6, characterized in that, the silicic acid solution and the polypeptide solution in S3 are mixed at a volume ratio of 1:
9.
8. The method for preparing nano-silica according to claim 2, characterized in that, the centrifugation speed in S3 is 10000~15000 rpm, and the time is 2~5 min; the drying temperature is 70℃, and the drying time is 6 h.