阅读说明：本技术 一种生物模板法构筑多级结构硫化铜纳米酶的方法 (Method for constructing multilevel-structure copper sulfide nanoenzyme by biological template method ) 是由 刘燕 靳海佳 郭荣 于 2018-08-16 设计创作，主要内容包括：本发明公开了一种生物模板法构筑多级结构硫化铜纳米酶的方法,以硫酸铜作为铜源反应物,以硫脲作为硫源反应物,利用生物大分子-蛋白质诱导硫化铜自组装成具有精细结构的微纳米材料。制得的线团状多级结构硫化铜纳米酶兼具类半胱氨酸氧化酶和类过氧化物酶活性；所述方法只需常温常压,反应条件温和,安全性和能耗得到显著改善；生产工艺简单、设备投资少、环境友好、原料价廉易得；所得产物具有无毒、生物相容性较好、类酶催化活性高、稳定性好等特点,可以在食品、环境和生物医学等领域有很好的应用前景。(The invention discloses a method for constructing a copper sulfide nanoenzyme with a multilevel structure by a biological template method. The prepared copper sulfide nanoenzyme with the coil-shaped multilevel structure has the activity of both cysteine-like oxidase and peroxidase-like enzyme; the method only needs normal temperature and normal pressure, the reaction condition is mild, and the safety and the energy consumption are obviously improved; the production process is simple, the equipment investment is low, the environment is friendly, and the raw materials are cheap and easy to obtain; the obtained product has the characteristics of no toxicity, good biocompatibility, high enzyme-like catalytic activity, good stability and the like, and can have good application prospects in the fields of food, environment, biomedicine and the like.)

1. A method for constructing a multilevel-structure copper sulfide nanoenzyme by a biological template method is characterized by comprising the following steps:

slowly adding the protein solution into the copper sulfate solution, and stirring to obtain a protein-copper sulfate mixed solution; and adding the thiourea solution into the mixed solution of copper sulfate and protein, stirring for reaction at high temperature, and centrifuging and cleaning after the reaction is finished to obtain the copper sulfide nanoenzyme with the coil-shaped multilevel structure.

2. The method of claim 1, wherein the protein solution is prepared by adding the protein to water at a temperature of 60-80 ℃, adjusting the pH of the system to 10.0-12.0, stirring the system until the protein is completely dissolved, cooling to room temperature, and storing at 3 ~ 8 ℃ for further use.

3. The method of claim 1, wherein the concentration of copper sulfate in the protein-copper sulfate mixed solution is 0.05M, and the concentration of protein is 1.0-4.0 mg/mL.

4. The method as claimed in claim 1, wherein the protein solution is slowly added to the copper sulfate solution and stirred at a speed of 800 to 1200 r/min for 20 to 40 min to obtain the protein-copper sulfate mixed solution.

5. The method of claim 1, wherein the molar ratio of copper sulfate to thiourea is from 1:1 to 1: 3.

6. The method of claim 1, wherein the thiourea solution is added to the copper sulfate and protein mixed solution and stirred to react at 75-90 ℃ for 12-24 hours.

7. The method of claim 1, wherein the product obtained after the reaction is centrifugally precipitated at a rotation speed of 2000 to 4000 r/min for 1 to 3 min.

Technical Field

The invention relates to the technical field of nano biology, in particular to a method for constructing copper sulfide with a multilevel structure by a biological template method.

Background

The nano-enzyme is a nano-material with the catalytic capability similar to that of protease, and the magnetic Fe is reported for the first time from the Onychii academyelinator topic group in 2007₃O₄Since nanoparticles have peroxidase activity similar to that of natural horseradish peroxidase (HRP), efforts have been made to develop nanoenzymes having various natural enzyme activities. Compared with natural enzymes, the nano-enzyme has the following advantages: 1) different from natural enzymes which are very sensitive to environmental conditions, the nano-enzyme can still keep certain enzyme-like catalytic activity in more extreme environments, which enables the nano-enzyme to have high stability and long service life, 2) compared with the natural enzymes, the nano-enzyme has relatively low cost, which enables the nano-enzyme to be easily produced and used in large scale, 3) the catalytic performance of the nano-enzyme is closely related to factors such as composition, structure and size, and the like, so that the performance of the nano-enzyme can be flexibly adjusted, and 4) some nano-enzymes can be recycled. In view of these characteristics, nanoenzymes have attracted increasing attention in the fields of analysis, environment, and biology in recent years. In order to further widen the application of the nano-enzyme in the related fields, the preparation of the nano-enzyme with low price, good biocompatibility, high activity, high stability and various enzyme activities has certain theoretical and practical significance.

Copper sulfide (CuS) is a cheap and environmentally friendly semiconductor due to its abundant, low toxicity. Copper sulfide nanomaterials are drawing attention in the fields of sensors, catalytic reactions, battery devices and the like due to their unique photoelectric physical properties. Because the catalytic performance of the nano-enzyme is closely related to factors such as the composition, the structure and the size of the nano-enzyme, the copper sulfide with a special micro-nano structure has very important application prospect in the field of nano-enzyme.

Disclosure of Invention

The invention aims to provide a method for constructing a copper sulfide nanoenzyme with a multilevel structure by a biological template method, the method is used for obtaining the clew-shaped copper sulfide nanoenzyme with the multilevel structure, and the nanoenzyme has the activities of cysteine-like oxidase and peroxidase-like enzyme.

The technical solution for realizing the purpose of the invention is as follows: a method for constructing a multilevel-structure copper sulfide nanoenzyme by a biological template method comprises the steps of taking copper sulfate as a copper source reactant, taking thiourea as a sulfur source reactant, and utilizing biomacromolecule-protein to induce copper sulfide to self-assemble a micro-nano material with a fine structure. The method comprises the following specific steps:

1. in the process of preparing the protein solution, 0.1-0.5M sodium hydroxide solution is used for adjusting the pH value of the system to 10.0-12.0, the dissolving temperature is 60-80 ℃, the stirring is continuously carried out until the protein is completely dissolved, and the protein solution is placed into a refrigerator for being stored overnight for later use after the temperature is reduced to room temperature.

2. Slowly adding the protein solution into the copper sulfate solution under the conditions of room temperature and magnetic stirring to obtain a protein-copper sulfate mixed solution, wherein the concentration of copper sulfate is 0.05M, and the concentration of protein is 1.0-4.0 mg/mL; and then transferring the mixed solution into a water bath, and stirring for 20-40 min at a stirring speed of 800-1200 r/min to combine protein and copper ions to form a compound.

3. Adding a thiourea solution into a copper sulfate and protein mixed solution, ensuring that the molar ratio of copper sulfate to thiourea in a reaction system is 1: 1-1: 3, and heating to 75-90 ℃ for reaction for 12-24 hours.

4. And centrifuging the product for 1-3 min at 2000-4000 r/min, and washing the product for three times by using deionized water and absolute ethyl alcohol respectively to obtain the copper sulfide nanoenzyme with the coil-shaped multilevel structure.

The preparation method is carried out at normal temperature and normal pressure, the reaction condition is mild, and the safety and the energy consumption are obviously improved; the method has the advantages of simple production process, less equipment investment, environmental protection, and cheap and easily-obtained raw materials. The prepared material has the characteristics of no toxicity, good biocompatibility, high enzyme-like catalytic activity, good stability and the like, and can have good application prospects in the fields of food, environment, biomedicine and the like.

Drawings

FIG. 1 is an SEM photograph of a copper sulfide nanomaterial prepared in example 1 at a copper sulfate concentration of 0.05M, a protein concentration of 2mg/mL, and a thiourea concentration of 0.1M.

FIG. 2 is an XRD pattern of the coil-shaped copper sulfide nanomaterial prepared in example 1.

FIG. 3 is a view of a wire-ball shaped CuS nano material catalytic H₂O₂Ultraviolet-visible spectrum of color developed by oxidation of TMB.

FIG. 4 is a fluorescence spectrum diagram of a linear CuS nano material catalyzing L-cysteine/TA cascade reaction.

Detailed Description