金属硫蛋白的表面增强拉曼光谱研究
摘要
金属硫蛋白(MT)是一类低分子量、高半胱氨酸含量、能结合大量金属离子的蛋白质。近年来,MT在医学上的应用受到越来越多的重视。人们认识到MT可能与多种疾病密切相关,特别是Alzheimer's疾病等一些神经退化性疾病。因此,研究金属硫蛋白与不同金属结合的机理及其构型上的变化,对于研究某些疾病的病理和治疗方法有着重大的意义。
表面增强拉曼光谱(SERS)具有极高的检测表面物种的灵敏度,能从分子水平上获得物质详细的结构和化学组成信息。应用SERS技术,可望从分子水平上探究MT复杂的生理活性。但是,MT分子拉曼散射截面小,易受激光影响碳化分解,杂质影响严重。因此,选择合适的SERS基底和实验条件,得到重现性好、可信的SERS谱,进而从分子水平上研究MT生理特性引起空间结构的变化是一个极具挑战的课题。
本论文的工作和获得成果主要有:
1.探索了几种获取MT分子的SERS谱的方法。实验表明,自组装Au纳米粒子尽管可以制备出SERS活性高、重现性好的SERS基底,但是杂质会严重干扰MT的信号。粗糙金电极表面干净,可获得可信和有意义的SERS谱图。通过EDC/NHS交联反应将MT固定在修饰了MUA的粗糙金表面的方法,使MUA的信号得到了最大增强,但却得不到明显的MT的信号,不适合应用于MT的SERS研究。
2.研究了直接吸附在粗糙金表面的金属硫蛋白的SERS谱。结果表明,MT通过氨基与金作用;apo-MT与金的作用位点受制备方法影响:在溶液中预制的apo-MT通过半胱氨酸残基的巯基与金结合,在金表面制备的apo-MT通过氨基和巯基与金结合。从Cd_7-MT(Ⅱ),Cu_(12)-MT(Ⅱ),Cu_(20)-MT(Ⅱ)到Hg_7-MT(Ⅱ),随着金属-S键的逐渐增强,SERS基底上Au-S键的成分逐渐减少,基底对蛋白质二级结构的破坏减弱。
Metallothionein (MT) is a type of metalloproteins characterized by a large number of cysteine residues, low molecular weight and a remarkable ability to coordinate a large number of transition metal ions in the form of metal-thiolate clusters. In recent yeas, metallothionein draws more attention in medical application. It is recognized that MT may be closely related to many diseases, particularly Alzheimer's disease and other neurodegenerative diseases. Therefore, the studies of different metal-binding mechanisms and the structural changes of metallothionein are of great significances to the pathology of certain diseases.
SERS is an ultrasensitive analytical method to probe the surface species and can provide detailed information of the structure and chemical composition of the species on the molecular level. It is well-known that enhancement effect of SERS is closely connected with the suitable nanoscale roughness of the substrates or nanoparticles. And SERS can be used to understand the complicated physiological activities of protein on the molecular level. However, MT has small Raman scattering section, and easily become catbonaceous decomposition products by the laser. Impurities also confuse SERS spectra of MT. Therefore, it is a challenge to choose suitable SERS substrate and the experimental conditions to obtain credible SERS spectra with good reproducibility, and then to study the structural changes of MT caused by physiological characteristics on the molecular level.
The major works and results in the thesis are outlined as follows:
1. Several methods have been explored to obtain the SERS of metallothionein. Results showed that SERS substrates prepared by self-assembly nanoparticles usually had higher SERS-active and good reproducibility whereas the impurities seriously covered the signal of MT. Roughened Au substrate was clean to obtain reliable SERS signal. To obtain the enhanced SERS signal of MT, a direct immobilization of MT molecules onto roughened Au substrate was performed.
2. The SERS spectra of MT absorbed directly on roughened Au surfaces was studied. The results indicated that MT was adsorbed via -NH_2 group on roughened gold surfaces. The adsorption site of apo-MT on Au was determined by the preparation method of apo-MT. The apo-MT prepared in solution was adsorbed via the -SH group on Au whereas that prepared on the Au surface was adsorbed via -SH and -NH_2. Apo-MT immobilized on the SERS substrate could be easily decomposed into carbonaceous products under the laser beam whereas MT kept a stable composition. With the enhanced binding between the metal ions and the cysteine residues, the deformed secondary structure of MT was reduced.
表面增强拉曼光谱(SERS)具有极高的检测表面物种的灵敏度,能从分子水平上获得物质详细的结构和化学组成信息。应用SERS技术,可望从分子水平上探究MT复杂的生理活性。但是,MT分子拉曼散射截面小,易受激光影响碳化分解,杂质影响严重。因此,选择合适的SERS基底和实验条件,得到重现性好、可信的SERS谱,进而从分子水平上研究MT生理特性引起空间结构的变化是一个极具挑战的课题。
本论文的工作和获得成果主要有:
1.探索了几种获取MT分子的SERS谱的方法。实验表明,自组装Au纳米粒子尽管可以制备出SERS活性高、重现性好的SERS基底,但是杂质会严重干扰MT的信号。粗糙金电极表面干净,可获得可信和有意义的SERS谱图。通过EDC/NHS交联反应将MT固定在修饰了MUA的粗糙金表面的方法,使MUA的信号得到了最大增强,但却得不到明显的MT的信号,不适合应用于MT的SERS研究。
2.研究了直接吸附在粗糙金表面的金属硫蛋白的SERS谱。结果表明,MT通过氨基与金作用;apo-MT与金的作用位点受制备方法影响:在溶液中预制的apo-MT通过半胱氨酸残基的巯基与金结合,在金表面制备的apo-MT通过氨基和巯基与金结合。从Cd_7-MT(Ⅱ),Cu_(12)-MT(Ⅱ),Cu_(20)-MT(Ⅱ)到Hg_7-MT(Ⅱ),随着金属-S键的逐渐增强,SERS基底上Au-S键的成分逐渐减少,基底对蛋白质二级结构的破坏减弱。
Metallothionein (MT) is a type of metalloproteins characterized by a large number of cysteine residues, low molecular weight and a remarkable ability to coordinate a large number of transition metal ions in the form of metal-thiolate clusters. In recent yeas, metallothionein draws more attention in medical application. It is recognized that MT may be closely related to many diseases, particularly Alzheimer's disease and other neurodegenerative diseases. Therefore, the studies of different metal-binding mechanisms and the structural changes of metallothionein are of great significances to the pathology of certain diseases.
SERS is an ultrasensitive analytical method to probe the surface species and can provide detailed information of the structure and chemical composition of the species on the molecular level. It is well-known that enhancement effect of SERS is closely connected with the suitable nanoscale roughness of the substrates or nanoparticles. And SERS can be used to understand the complicated physiological activities of protein on the molecular level. However, MT has small Raman scattering section, and easily become catbonaceous decomposition products by the laser. Impurities also confuse SERS spectra of MT. Therefore, it is a challenge to choose suitable SERS substrate and the experimental conditions to obtain credible SERS spectra with good reproducibility, and then to study the structural changes of MT caused by physiological characteristics on the molecular level.
The major works and results in the thesis are outlined as follows:
1. Several methods have been explored to obtain the SERS of metallothionein. Results showed that SERS substrates prepared by self-assembly nanoparticles usually had higher SERS-active and good reproducibility whereas the impurities seriously covered the signal of MT. Roughened Au substrate was clean to obtain reliable SERS signal. To obtain the enhanced SERS signal of MT, a direct immobilization of MT molecules onto roughened Au substrate was performed.
2. The SERS spectra of MT absorbed directly on roughened Au surfaces was studied. The results indicated that MT was adsorbed via -NH_2 group on roughened gold surfaces. The adsorption site of apo-MT on Au was determined by the preparation method of apo-MT. The apo-MT prepared in solution was adsorbed via the -SH group on Au whereas that prepared on the Au surface was adsorbed via -SH and -NH_2. Apo-MT immobilized on the SERS substrate could be easily decomposed into carbonaceous products under the laser beam whereas MT kept a stable composition. With the enhanced binding between the metal ions and the cysteine residues, the deformed secondary structure of MT was reduced.
引文
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