CN105108137B - A kind of preparation method of the nano particle of strong catalase activity - Google Patents
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- MPNGTXCDSYEMHI-UHFFFAOYSA-K Cl[Au](Cl)(Cl)[Cl][Au] Chemical compound Cl[Au](Cl)(Cl)[Cl][Au] MPNGTXCDSYEMHI-UHFFFAOYSA-K 0.000 claims 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
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Abstract
Description
技术领域technical field
本发明属于生物活性纳米材料技术领域，具体涉及一种强过氧化氢酶活性的纳米颗粒的制备方法。The invention belongs to the technical field of bioactive nanometer materials, and in particular relates to a preparation method of nanoparticles with strong catalase activity.
背景技术Background technique
过氧化氢酶是一种生物体内普遍存在能够催化分解过氧化氢产生氧气和水的蛋白酶。(George,P.Reaction between Catalase and Hydrogen Peroxide[J].Nature1947,160,41-43；Lemberg,R.；Foulkes,E.C.Reaction between Catalase and HydrogenPeroxide[J].Nature 1948,161,131-132)目前，过氧化氢酶由于具有强过氧化氢酶活性已被广泛用于多种靶标的分析检测中。(Zhu,Z.；Guan,Z.；Jia,S.；Lei,Z.；Lin,S.；Zhang,H.；Ma,Y.；Tian,Z.-Q.；Yang,C.J.Au@Pt Nanoparticle Encapsulated Target-ResponsiveHydrogel with Volumetric Bar-Chart Chip Readout for Quantitative Point-of-Care Testing[J].Angew.Chem.,Int.Ed.2014,53,12503-12507；Song,Y.；Zhang,Y.；Bernard,P.E.；Reuben,J.M.；Ueno,N.T.；Arlinghaus,R.B.；Zu,Y.；Qin,L.MultiplexedVolumetric Bar-Chart Chip for Point-of-Care Diagnostics[J].Nat.Commun.2012,3,1-9；Song,Y.；Xia,X.；Wu,X.；Wang,P.；Qin,L.Integration of Platinum Nanoparticleswith a Volumetric Bar-Chart Chip for Biomarker Assays[J].Angew.Chem.,Int.Ed.2014,53,12451-12455；Song,Y.；Wang,Y.；Qin,L.A Multistage Volumetric BarChart Chip for Visualized Quantification of DNA[J].J.Am.Chem.Soc.2013,135,16785-16788)但是，过氧化氢酶也存在一些不足：首先，作为一种蛋白，过氧化氢酶在细胞裂解液或血清等复杂体系中易被降解；其次，过氧化氢酶的二级结构易受极端环境如高温、强酸和强碱等因素的影响，使催化效率发生不可逆的下降；(Song,Y.；Wang,Y.；Qin,L.AMultistage Volumetric Bar Chart Chip for Visualized Quantification of DNA[J].J.Am.Chem.Soc.2013,135,16785-16788；Melov,S.；Ravenscroft,J.；Malik,S.；Gill,M.S.；Walker,D.W.；Clayton,P.E.；Wallace,D.C.；Malfroy,B.；Doctrow,S.R.；Lithgow,G.J.Extension of Life-Span with Superoxide Dismutase/Catalase Mimetics[J].Science 2000,289,1567-1569)最后，利用生物工程制备、纯化和保存过氧化氢酶使它保持较高纯度和催化活性成本较高。因此，发展强过氧化氢酶活性纳米颗粒的合成方法有重要的意义。Catalase is a protease ubiquitous in organisms that can catalyze the decomposition of hydrogen peroxide to produce oxygen and water. (George, P.Reaction between Catalase and Hydrogen Peroxide[J].Nature 1947,160,41-43; Lemberg,R.; Foulkes,E.C.Reaction between Catalase and Hydrogen Peroxide[J].Nature 1948,161,131-132) Catalase has been widely used in the analysis and detection of various targets due to its strong catalase activity. (Zhu, Z.; Guan, Z.; Jia, S.; Lei, Z.; Lin, S.; Zhang, H.; Ma, Y.; Tian, Z.-Q.; Yang, C.J.Au@Pt Nanoparticle Encapsulated Target-Responsive Hydrogel with Volumetric Bar-Chart Chip Readout for Quantitative Point-of-Care Testing[J].Angew.Chem.,Int.Ed.2014,53,12503-12507;Song,Y.;Zhang,Y. ; Bernard, P.E.; Reuben, J.M.; Ueno, N.T.; Arlinghaus, R.B.; 1-9; Song, Y.; Xia, X.; Wu, X.; Wang, P.; Qin, L. Integration of Platinum Nanoparticles with a Volumetric Bar-Chart Chip for Biomarker Assays[J].Angew.Chem., Int.Ed.2014,53,12451-12455; Song,Y.;Wang,Y.;Qin,L.A Multistage Volumetric BarChart Chip for Visualized Quantification of DNA[J].J.Am.Chem.Soc.2013,135, 16785-16788) However, catalase also has some shortcomings: first, as a protein, catalase is easily degraded in complex systems such as cell lysate or serum; secondly, the secondary structure of catalase Susceptible to extreme environments such as high temperature, strong acid and strong alkali, etc., which will cause irreversible decline in catalytic efficiency; (Song, Y.; Wang, Y.; Qin, L. A Multistage Volumetric Bar Chart Chip for Visualized Quantification of DNA[ J]. J. Am. Chem. Soc. 2013, 135, 16785-16788; Melov, S.; Ravenscroft, J.; Malik, S.; .S.; Walker, D.W.; Clayton, P.E.; Wallace, D.C.; Malfroy, B.; Doctrow, S.R.; Lithgow, G.J.Extension of Life-Span with Superoxide Dismutase/Catalase Mimetics[J]. 1569) Finally, the cost of using bioengineering to prepare, purify and preserve catalase to keep it higher in purity and catalytic activity is higher. Therefore, it is of great significance to develop synthetic methods for nanoparticles with strong catalase activity.
发明内容Contents of the invention
本发明的目的在于克服现有技术缺陷，提供一种强过氧化氢酶活性的纳米颗粒的制备方法。The purpose of the present invention is to overcome the defects of the prior art and provide a method for preparing nanoparticles with strong catalase activity.
本发明的具体技术方案如下：Concrete technical scheme of the present invention is as follows:
一种强过氧化氢酶活性的纳米颗粒的制备方法，包括如下步骤：A method for preparing nanoparticles with strong catalase activity, comprising the steps of:
(1)将氯金酸溶液在连续搅拌下煮沸回流，同时匀速加入柠檬酸钠溶液以还原氯金酸形成粒径12～14nm金纳米颗粒，其中氯金酸溶液与柠檬酸钠溶液的体积比为100:1～1.5，且氯金酸溶液中含有0.01～0.02wt％的氯金酸，柠檬酸钠溶液中含有2.8～3.5wt％的柠檬酸钠；(1) The chloroauric acid solution is boiled and refluxed under continuous stirring, and at the same time, the sodium citrate solution is added at a constant speed to form 12-14nm gold nanoparticles with a particle size of chloroauric acid, wherein the volume ratio of the chloroauric acid solution to the sodium citrate solution is It is 100:1～1.5, and the chloroauric acid solution contains 0.01～0.02wt% chloroauric acid, and the sodium citrate solution contains 2.8～3.5wt% sodium citrate;
(2)向步骤(1)制得的物料中加入氯金酸和氯铂酸的混合溶液，然后缓慢加入抗坏血酸溶液，10～50℃的温度下以100～800rpm的速度搅拌反应2～12h，以使上述金纳米颗粒上包裹金铂双金属壳层，即得所述强过氧化氢酶活性的纳米颗粒，其中在该步骤的反应体系中，氯铂酸的浓度为0.04～0.064mM，氯金酸的浓度为0.2～3.2mM，抗坏血酸的浓度为1～16mM，且上述氯铂酸和氯金酸的质量比为10～40:100，氯铂酸和氯金酸的总质量与抗坏血酸的质量比为13.4～53.5:100，金纳米颗粒与氯铂酸和氯金酸总质量的质量比为2.7～11.2:100。(2) Add a mixed solution of chloroauric acid and chloroplatinic acid to the material obtained in step (1), then slowly add ascorbic acid solution, stir and react at a speed of 100-800 rpm at a temperature of 10-50°C for 2-12 hours, In order to wrap the gold-platinum bimetallic shell on the above-mentioned gold nanoparticles, the nanoparticles with strong catalase activity are obtained, wherein in the reaction system of this step, the concentration of chloroplatinic acid is 0.04-0.064mM, and the chloride The concentration of gold acid is 0.2～3.2mM, the concentration of ascorbic acid is 1～16mM, and the mass ratio of above-mentioned chloroplatinic acid and chloroauric acid is 10～40:100, the total mass of chloroplatinic acid and chloroauric acid and ascorbic acid The mass ratio is 13.4-53.5:100, and the mass ratio of the gold nanoparticles to the total mass of chloroplatinic acid and chloroauric acid is 2.7-11.2:100.
在本发明的一个优选实施方案中，所述步骤(1)为：将氯金酸溶液在连续搅拌下煮沸回流，同时匀速加入柠檬酸钠溶液以还原氯金酸形成粒径13nm金纳米颗粒，其中氯金酸溶液与柠檬酸钠溶液的体积比为100:1，且氯金酸溶液中含有0.01wt％的氯金酸，柠檬酸钠溶液中含有3wt％的柠檬酸钠。In a preferred embodiment of the present invention, the step (1) is: the chloroauric acid solution is boiled and refluxed under continuous stirring, and at the same time, a sodium citrate solution is added at a constant speed to form 13nm gold nanoparticles with a particle size of 13nm to reduce the chloroauric acid, Wherein the volume ratio of the chloroauric acid solution and the sodium citrate solution is 100:1, and the chloroauric acid solution contains 0.01wt% chloroauric acid, and the sodium citrate solution contains 3wt% sodium citrate.
进一步优选的，所述步骤(2)中的温度为20～40℃。Further preferably, the temperature in the step (2) is 20-40°C.
进一步优选的，所述步骤(2)中的搅拌速度为300～500rpm。Further preferably, the stirring speed in the step (2) is 300-500 rpm.
进一步优选的，所述步骤(2)中的反应时间为5～9h。Further preferably, the reaction time in the step (2) is 5-9 hours.
进一步优选的，所述步骤(2)中氯铂酸的浓度为0.08～0.32mM。Further preferably, the concentration of chloroplatinic acid in the step (2) is 0.08-0.32mM.
进一步优选的，所述步骤(2)中氯金酸的浓度为0.4～1.6mM。Further preferably, the concentration of chloroauric acid in the step (2) is 0.4-1.6 mM.
进一步优选的，所述步骤(2)中抗坏血酸的浓度为2～8mM。Further preferably, the concentration of ascorbic acid in the step (2) is 2-8 mM.
本发明的有益效果是：The beneficial effects of the present invention are:
1、本发明的制备方法选用铂这种过氧化氢酶活性很强的材料作为纳米颗粒催化活性中心，使合成的纳米颗粒具有很强的过氧化氢酶活性；1, the preparation method of the present invention selects the very strong material of this catalase activity of platinum as nanoparticle catalytic active center, makes the nanoparticle of synthesis have very strong catalase activity;
2、本发明的制备方法利用金和铂的协同作用，在金种子上包裹金和铂的双金属壳层，进一步增加纳米颗粒的过氧化氢酶活性；2. The preparation method of the present invention utilizes the synergistic effect of gold and platinum to wrap the double metal shell of gold and platinum on the gold seeds to further increase the catalase activity of the nanoparticles;
3、本发明的制备方法利用金与氨基、巯基和羧基等官能团相互作用强的特点，使合成的纳米颗粒易于修饰生物大分子。3. The preparation method of the present invention utilizes the characteristics of strong interaction between gold and functional groups such as amino, mercapto and carboxyl groups, so that the synthesized nanoparticles are easy to modify biomacromolecules.
4、本发明的制备方法与传统方法相比，此法简单、高效、通用性强，合成的纳米颗粒具有强且稳定的过氧化氢酶活性，为具有过氧化氢酶活性的纳米颗粒在生物分析和生物医学中的应用提供新的平台。4, the preparation method of the present invention compares with traditional method, and this method is simple, efficient, versatility is strong, the nanoparticle of synthesis has strong and stable catalase activity, is the nanoparticle with catalase activity in biological Analytical and biomedical applications provide new platforms.
附图说明Description of drawings
图1为本发明的强过氧化氢酶活性的纳米颗粒的合成原理图。Figure 1 is a schematic diagram of the synthesis of nanoparticles with strong catalase activity of the present invention.
图2中(A)为本发明实施例1中步骤(1)制备的金纳米颗粒和制得的Au@AuPtNPs的紫外-可见吸收光谱表征图；(B)为本发明实施例1制备的Au@AuPtNPs的X射线能谱表征图。(A) in Figure 2 is the ultraviolet-visible absorption spectrum characterization diagram of the gold nanoparticles prepared in step (1) in Example 1 of the present invention and the prepared Au@AuPtNPs; (B) is the Au prepared in Example 1 of the present invention X-ray energy spectrum characterization of @AuPtNPs.
图3中(A)为本发明实施例1的步骤(1)制备的13nm金纳米颗粒的透射电镜图；(B)为本发明实施例1的制备的Au@AuPtNPs的透射电镜图；(C)为本发明实施例1的制备的Au@AuPtNPs的高分辨透射电镜图；(D)为本发明实施例1的制备的Au@AuPtNPs的选区电子衍射图。Among Fig. 3 (A) is the transmission electron micrograph of the 13nm gold nanoparticles prepared in the step (1) of Example 1 of the present invention; (B) is the transmission electron micrograph of Au@AuPtNPs prepared in Example 1 of the present invention; (C ) is the high-resolution transmission electron microscope image of Au@AuPtNPs prepared in Example 1 of the present invention; (D) is the selected area electron diffraction image of Au@AuPtNPs prepared in Example 1 of the present invention.
图4为本发明的实施例制得的Au@AuPtNPs上的金元素(A)和铂元素(B)的X射线光电子能谱表征图。Fig. 4 is an X-ray photoelectron spectrum characterization diagram of gold element (A) and platinum element (B) on Au@AuPtNPs prepared in the embodiment of the present invention.
图5为本发明实施例1实验条件优化过程图，其中：(A)不同浓度氯金酸和氯铂酸；(B)不同浓度抗坏血酸；(C)不同反应温度；(D)不同反应时间。5 is a diagram of the optimization process of the experimental conditions of Example 1 of the present invention, wherein: (A) different concentrations of chloroauric acid and chloroplatinic acid; (B) different concentrations of ascorbic acid; (C) different reaction temperatures; (D) different reaction times.
图6(A)为本发明不同温度合成Au@AuPtNPs的扫描电镜图；(B)为本发明不同温度合成Au@AuPtNPs的粒径统计分析。Fig. 6 (A) is a scanning electron microscope image of Au@AuPtNPs synthesized at different temperatures in the present invention; (B) is a statistical analysis of the particle size of Au@AuPtNPs synthesized at different temperatures in the present invention.
图7为本发明实施例1制得的Au@AuPtNPs过氧化氢酶活性随时间的稳定性变化图。Fig. 7 is a graph showing the stability of the catalase activity of Au@AuPtNPs prepared in Example 1 of the present invention over time.
图8为本发明实施例1制得的Au@AuPtNPs和过氧化氢酶的酶活性对比结果图，其中包括以过氧化氢为底物时米氏常数(Km)、最大反应速率(Vmax)和催化常数(Kcat)。Figure 8 is a comparison result of the enzymatic activity between Au@AuPtNPs and catalase prepared in Example 1 of the present invention, including the Michaelis constant (K m ) and the maximum reaction rate (V max ) when hydrogen peroxide is used as the substrate. ) and catalytic constant (K cat ).
具体实施方式detailed description
以下通过具体实施方式结合附图对本发明的技术方案进行进一步的说明和描述。The technical solutions of the present invention will be further illustrated and described below through specific embodiments in conjunction with the accompanying drawings.
实施例1Example 1
本实施例的技术路线如图1所示，具体实验条件摸索过程如图5和图6所示，具体步骤如下：The technical route of this embodiment is shown in Figure 1, and the process of exploring specific experimental conditions is shown in Figure 5 and Figure 6, and the specific steps are as follows:
(1)在250mL圆底烧瓶中加入100mL 0.01wt％氯金酸，连续搅拌下煮沸回流，匀速加入1mL 3wt％的柠檬酸钠还原氯金酸形成13nm金纳米颗粒(如图3)。实验过程中，溶液由淡黄色变为黑色，再变成红色。(1) Add 100mL 0.01wt% chloroauric acid to a 250mL round bottom flask, boil and reflux under continuous stirring, and add 1mL 3wt% sodium citrate at a uniform speed to reduce chloroauric acid to form 13nm gold nanoparticles (as shown in Figure 3). During the experiment, the solution changed from light yellow to black and then to red.
(2)在400rpm搅拌的条件下，向100mL 13nm AuNPs溶液中加入10mL含有1.6mM氯铂酸和8mM氯金酸的混合液。随后，缓慢加入5mL 80mM抗坏血酸，室温400rpm搅拌8h合成Au@AuPtNPs，即所述强过氧化氢酶活性的纳米颗粒。用紫外-可见分光光度计(UV-vis)、扫描电镜(SEM)、透射电镜(TEM)、高分辨透射电镜(HR-TEM)和X射线光电子能谱(XPS)分析合成好的Au@AuPtNPs，结果如图2、3、4和6所示。(2) Under the condition of stirring at 400 rpm, 10 mL of a mixture containing 1.6 mM chloroplatinic acid and 8 mM chloroauric acid was added to 100 mL of 13 nm AuNPs solution. Subsequently, 5 mL of 80 mM ascorbic acid was slowly added, and stirred at room temperature at 400 rpm for 8 h to synthesize Au@AuPtNPs, which are nanoparticles with strong catalase activity. The synthesized Au@AuPtNPs were analyzed by ultraviolet-visible spectrophotometer (UV-vis), scanning electron microscope (SEM), transmission electron microscope (TEM), high-resolution transmission electron microscope (HR-TEM) and X-ray photoelectron spectroscopy (XPS). , and the results are shown in Figures 2, 3, 4 and 6.
(3)测定Au@AuPtNPs过氧化氢酶活性：在200μL离心管中加入100μL 0.1M H2O2和2μL 2.5nM Au@AuPtNPs，混匀后在37℃下倒置放置反应10min，纳米颗粒催化H2O2分解产生的氧气集中于离心管顶端。将移液器取样体积调节至100μL，用移液器完全吸走离心管上端的氧气后再吸取密度为1g/mL的水，利用天平测定移液器吸取水的质量就能换算出水的体积。利用排水法计算Au@AuPtNPs纳米颗粒催化H2O2分解产生氧气的体积，结果如图7和8所示。(3) Determination of Au@AuPtNPs catalase activity: Add 100 μL 0.1M H 2 O 2 and 2 μL 2.5nM Au@AuPtNPs into a 200 μL centrifuge tube, mix well, place it upside down at 37°C for 10 min, and the nanoparticles catalyze H 2 The oxygen produced by the decomposition of O2 is concentrated at the top of the centrifuge tube. Adjust the sampling volume of the pipette to 100 μL, use the pipette to completely absorb the oxygen at the upper end of the centrifuge tube and then absorb water with a density of 1g/mL, and use a balance to measure the mass of the water absorbed by the pipette to convert the volume of the water. The volume of oxygen produced by the decomposition of H 2 O 2 catalyzed by Au@AuPtNPs nanoparticles was calculated by the drainage method, and the results are shown in Figures 7 and 8.
本领域普通技术人员可知，本发明的成分和参数在下述范围内变化时，仍能够得到与上述实施例相同或相近的技术效果，皆属于本发明的保护范围：Those of ordinary skill in the art will know that when the components and parameters of the present invention change within the following ranges, the same or similar technical effects as those of the above-mentioned embodiments can still be obtained, and all belong to the protection scope of the present invention:
一种强过氧化氢酶活性的纳米颗粒的制备方法，包括如下步骤：A method for preparing nanoparticles with strong catalase activity, comprising the steps of:
(1)将氯金酸溶液在连续搅拌下煮沸回流，同时匀速加入柠檬酸钠溶液以还原氯金酸形成粒径12～14nm金纳米颗粒，其中氯金酸溶液与柠檬酸钠溶液的体积比为100:1～1.5，且氯金酸溶液中含有0.01～0.02wt％的氯金酸，柠檬酸钠溶液中含有2.8～3.5wt％的柠檬酸钠；(1) The chloroauric acid solution is boiled and refluxed under continuous stirring, and at the same time, the sodium citrate solution is added at a constant speed to form 12-14nm gold nanoparticles with a particle size of chloroauric acid, wherein the volume ratio of the chloroauric acid solution to the sodium citrate solution is It is 100:1～1.5, and the chloroauric acid solution contains 0.01～0.02wt% chloroauric acid, and the sodium citrate solution contains 2.8～3.5wt% sodium citrate;
(2)向步骤(1)制得的物料中加入氯金酸和氯铂酸的混合溶液，然后缓慢加入抗坏血酸溶液，10～50℃的温度下以100～800rpm的速度搅拌反应2～12h，以使上述金纳米颗粒上包裹金铂双金属壳层，即得所述强过氧化氢酶活性的纳米颗粒，其中在该步骤的反应体系中，氯铂酸的浓度为0.04～0.064mM，氯金酸的浓度为0.2～3.2mM，抗坏血酸的浓度为1～16mM，且上述氯铂酸和氯金酸的质量比为10～40:100，氯铂酸和氯金酸的总质量与抗坏血酸的质量比为13.4～53.5:100，金纳米颗粒与氯铂酸和氯金酸总质量的质量比为2.7～11.2:100。(2) Add a mixed solution of chloroauric acid and chloroplatinic acid to the material obtained in step (1), then slowly add ascorbic acid solution, stir and react at a speed of 100-800 rpm at a temperature of 10-50°C for 2-12 hours, In order to wrap the gold-platinum bimetallic shell on the above-mentioned gold nanoparticles, the nanoparticles with strong catalase activity are obtained, wherein in the reaction system of this step, the concentration of chloroplatinic acid is 0.04-0.064mM, and the chloride The concentration of gold acid is 0.2～3.2mM, the concentration of ascorbic acid is 1～16mM, and the mass ratio of above-mentioned chloroplatinic acid and chloroauric acid is 10～40:100, the total mass of chloroplatinic acid and chloroauric acid and ascorbic acid The mass ratio is 13.4-53.5:100, and the mass ratio of the gold nanoparticles to the total mass of chloroplatinic acid and chloroauric acid is 2.7-11.2:100.
优选的，所述步骤(2)中，温度为20～40℃，搅拌速度为300～500rpm，反应时间为5～9h，反应体系中：氯铂酸的浓度为0.08～0.32mM，氯金酸的浓度为0.4～1.6mM，抗坏血酸的浓度为2～8mM。Preferably, in the step (2), the temperature is 20-40°C, the stirring speed is 300-500rpm, and the reaction time is 5-9h. In the reaction system: the concentration of chloroplatinic acid is 0.08-0.32mM, and the concentration of chloroauric acid is 0.08-0.32mM. The concentration of ascorbic acid is 0.4-1.6mM, and the concentration of ascorbic acid is 2-8mM.
以上所述，仅为本发明的较佳实施例而已，故不能依此限定本发明实施的范围，即依本发明专利范围及说明书内容所作的等效变化与修饰，皆应仍属本发明涵盖的范围内。The above is only a preferred embodiment of the present invention, so the scope of the present invention cannot be limited accordingly, that is, equivalent changes and modifications made according to the patent scope of the present invention and the content of the specification should still be covered by the present invention In the range.
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