The Metallothionein Surface Enhanced Raman Spectroscopy Study

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 -NH₂ 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 -NH₂. 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.