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PEG化纳米金制备方法及表征方法金纳米颗粒进行表面修饰在生物领域应用是非常重要的,不仅可以提高其稳定性,而且使其表面功能化,能更轻易的与抗体、酶等生物分子连接起来。常用含巯基配体对纳米金进行修饰,谷肌甘肤(GSH) ,巯基丙酸(MPA),半肌氨酸(cysteine),肌胺(cystamine),二巯基辛酸(DHLA)及聚乙二醇(PEG)等,其中最常见的是巯基聚乙二醇(PEG-SH),主要有以下几个优点: 1. PEG为高分子聚合物,将PEG修饰到纳米金颗粒上,纳米金胶体的稳定方式会由原来的静电稳定变为空间稳定,稳定状态不易受外界因素的影响; 2. PEG无抗原和致免疫性、无毒、生物相容性较好; 3. PEG在生理环境中对生物分子没有吸附或其他相互作用,有效减少了非特异性蛋白的吸附,延长了循环时间; 4. 采用端基不同的PEG,其一段连接在金纳米颗粒上,另一端的活性基团可以进行其他修饰,使纳米金颗粒表面性质多样化。 PEG化纳米金的制备方法: 金属纳米粒子的主要制备方法有物理法和化学法, 其中物理法主要有真空蒸镀法、软着陆法、电分散法和激光消融法等; 化学法主要有氧化还原法、电化学法、晶种法、微乳液法、相转移法、模板法。 氧化还原法: 该法通常是在含有高价金离子的溶液中加入还原剂,金离子被还原而聚集成纳米粒子。所用的还原剂包括经典的柠檬酸三钠、硼氢化钠、磷、十六烷基苯胺、聚乙二醇、聚苯胺等。其中以柠檬酸钠还原法zui为经典,就是以柠檬酸三钠为还原剂在热的水溶液中将[AuCl4]-还原,其制备方法简单,所得到的颗粒性质稳定。 微乳液法: 该法是将表面活性剂溶解在有机溶剂中,当表面活性剂的浓度超过临界胶束浓度(CMC)后形成亲水极性头向内、疏水有机链向外的液体颗粒结构,其内核可增溶水分子或亲水物质。微乳液一般由4种组分组成,即表面活性剂、助表面活性剂(一般为脂肪醇)、有机溶剂(一般为烷烃或环烷烃)和水。它是一种热力学稳定体系,可以合成大小均匀、粒径在10-20nm的液体颗粒。 晶种生长法: 晶种生长法是通过晶种合成反应制备金纳米颗粒,该法优点在于比一步法得到的颗粒粒径分布窄,可以通过控制反应条件zui终制备出粒径在50-110nm之间的金纳米球。同时,晶种生长法因其合成过程简便、高产量、长短径比可控及表面易于修饰等优点而成为合成胶体金纳米棒zui流行的方法。其基本原理是在反应溶液中加入一定量的金纳米颗粒晶种,在表面活性剂分子的作用下,晶种颗粒定向生长为一定长径比的金纳米棒。 光化学还原法: 光化学还原法是zui早用来制备金纳米棒的方法,在制备过程中,金酸与棒状的阳离子表面活性剂胶束结合在一起形成离子对,然后在紫外激发下电子从金属离子转移到配体中,从而使金离子还原成金。 纳米金的表面PEG化:用巯基聚乙二醇(PEG-SH)对金纳米球进行修饰,修饰过程为:将100μL浓度为5X 10-4mol/L的上述配体分别加入到1ml金纳米球溶液中,再将其搅拌30S以充分混合,然后将其放入4℃冰箱备用。 纳米金的表征方法: 为了全面而准确的表征纳米金的性质,通常使用多种手段同时对纳米金进行分析,常用的表征方法有:紫外可见光谱(Uv-vis)、红外光谱(FI-IR),扫描电子显微镜(SEM)或投射电子显微镜(TEM)、电动电位或电动电势(Zeta电位)、X射线衍射(XRD)、X射线光电子能谱、毛细管电泳(CE)等。 电子显微镜(SEM,TEM): SEM与TEM是观察纳米金粒子的直观方法,可以清楚的观察到粒子形貌,大小和分散度情况。 紫外可见(Uv-vis): 纳米金溶胶因其等离子共振作用,使得他在溶液中有特定的颜色。纳米金粒子表面包配体、历经大小和粒子形状不同时,粒子所表现出来的等离子共振吸收是不一样的。金纳米粒子的等离子体共振分为横向伸缩振动和纵向伸缩振动,在金粒子以球形存在于溶液中时,重要表现出横向伸缩振动,Uv-vis吸收在520nm左右,当纳米金溶胶中以棒状,线装,三角片状或其它非球形是,粒子主要表现出纵向伸缩振动,zui大吸收波长在600nm-1400nm处不等,zui大吸收波长越大,棒状与线装粒子的长径比越大,三角片的边厚比也越大,溶液表现出不同的颜色,如灰色、蓝色和深棕色等。 红外光谱(FI-IR): 每种物质的分子有其一定的分子结构,有一定的能级图,从而产生的红外吸收光谱,从而带来了分子能级图的信息。对红外光谱的分子,可以了解表面配体与纳米金之间的相互作用关系,对反应的机理及不同修饰剂对Au作用的方式与力的大小可以简单明了的分析。 Preparation method and characterization method of PEGylated gold nanoparticles Surface modification of gold nanoparticles is very important in biological applications, not only to improve their stability, but also to functionalize their surfaces, making it easier to connect with biomolecules such as antibodies and enzymes. Commonly used thiol-containing ligands to modify gold nanoparticles, glutathione (GSH), mercaptopropionic acid (MPA), cysteine (cysteine), sarcosine (cystamine), dimercaptooctanoic acid (DHLA) and polyethylene glycol Alcohol (PEG), etc., of which the most common is mercapto polyethylene glycol (PEG-SH), which has the following advantages: 1. PEG is a high molecular polymer. When PEG is modified on gold nanoparticles, the stability of gold nanoparticles will change from static stability to steric stability, and the stable state is not easily affected by external factors; 2. PEG has no antigen and immunogenicity, non-toxicity and good biocompatibility; 3. PEG has no adsorption or other interaction with biomolecules in the physiological environment, which effectively reduces the adsorption of non-specific proteins and prolongs the cycle time; 4. Using PEG with different end groups, one section of which is connected to gold nanoparticles, and the active group at the other end can be modified to diversify the surface properties of gold nanoparticles. The preparation method of PEGylated gold nanoparticles: The main preparation methods of metal nanoparticles are physical method and chemical method.Among them, the physical methods mainly include vacuum evaporation method, soft landing method, electric dispersion method and laser ablation method;Chemical methods mainly include redox method, electrochemical method, seed method, microemulsion method, phase transfer method and template method. Redox method: In this method, a reducing agent is usually added to a solution containing high-valent gold ions, and the gold ions are reduced and aggregated into nanoparticles. The reducing agents used include classic trisodium citrate, sodium borohydride, phosphorus, cetylaniline, polyethylene glycol, polyaniline, and the like. Among them, the sodium citrate reduction method is the most classic, that is, using trisodium citrate as a reducing agent to reduce [AuCl4]- in a hot aqueous solution, the preparation method is simple, and the obtained particles are stable in nature. Microemulsion method: The method is to dissolve the surfactant in an organic solvent. When the concentration of the surfactant exceeds the critical micelle concentration (CMC), a liquid particle structure with hydrophilic polar heads inwards and hydrophobic organic chains outwards is formed. Solubilizes water molecules or hydrophilic substances. Microemulsions generally consist of four components, namely surfactant, co-surfactant (usually aliphatic alcohol), organic solvent (usually alkane or cycloalkane) and water. It is a thermodynamically stable system that can synthesize liquid particles of uniform size and particle size of 10-20nm. Seed Growth Method: The seed crystal growth method is to prepare gold nanoparticles through the seed crystal synthesis reaction. The advantage of this method is that the particle size distribution is narrower than that obtained by the one-step method, and gold nanoparticles with a particle size between 50-110 nm can be finally prepared by controlling the reaction conditions. ball. At the same time, the seed growth method has become the most popular method for the synthesis of colloidal gold nanorods due to its advantages of simple synthesis process, high yield, controllable aspect ratio and easy surface modification. The basic principle is that a certain amount of gold nanoparticle seeds is added to the reaction solution, and under the action of surfactant molecules, the seed particles grow directionally into gold nanorods with a certain aspect ratio. Photochemical reduction method: Photochemical reduction is the earliest method used to prepare gold nanorods. During the preparation process, gold acid is combined with rod-like cationic surfactant micelles to form ion pairs, and then electrons are transferred from metal ions to ligands under UV excitation. in the body, thereby reducing the gold ions to gold. Surface PEGylation of gold nanoparticles: The gold nanospheres were modified with thiol polyethylene glycol (PEG-SH). The modification process was as follows: 100μL of the above ligands at a concentration of 5X 10-4mol/L were added to 1ml of gold nanospheres. In the solution, stir it for 30S to mix well, and then put it in a 4°C refrigerator for later use. Characterization of gold nanoparticles: In order to fully and accurately characterize the properties of gold nanoparticles, multiple methods are usually used to analyze gold nanoparticles at the same time. The commonly used characterization methods are: ultraviolet-visible spectroscopy (Uv-vis), infrared spectroscopy (FI-IR), scanning electron microscopy ( SEM) or transmission electron microscopy (TEM), zeta potential or zeta potential (Zeta potential), X-ray diffraction (XRD), X-ray photoelectron spectroscopy, capillary electrophoresis (CE), etc. Electron Microscopy (SEM, TEM): SEM and TEM are intuitive methods to observe gold nanoparticles, which can clearly observe the particle morphology, size and dispersion. Ultraviolet Visible (Uv-vis): The nano-gold sol has a specific color in solution due to its plasmon resonance. The plasmon resonance absorption exhibited by the gold nanoparticles is different when the surface of the gold nanoparticles is different in size and shape. The plasmonic resonance of gold nanoparticles is divided into lateral stretching vibration and longitudinal stretching vibration. When the gold particles exist in the solution in a spherical shape, the lateral stretching vibration is mainly exhibited, and the Uv-vis absorption is around 520 nm. , linear, triangular flake or other non-spherical particles, the particles mainly exhibit longitudinal stretching vibration, the maximum absorption wavelength ranges from 600nm to 1400nm, the larger the maximum absorption wavelength, the greater the aspect ratio of rod and wire particles, The larger the edge-to-thickness ratio of the triangular piece, the solution showed different colors, such as gray, blue and dark brown. Infrared Spectroscopy (FI-IR): The molecules of each substance have a certain molecular structure and a certain energy level map, resulting in an infrared absorption spectrum, which brings the information of the molecular energy level map. For the molecules of the infrared spectrum, the interaction between the surface ligands and the gold nanoparticles can be understood, and the reaction mechanism and the mode of the effect of different modifiers on Au and the magnitude of the force can be analyzed simply and clearly. |