Surface-enhanced Raman spectroscopy based on enzyme-substrate specificity for structural analyses of α-amylase and...

Surface-enhanced Raman spectroscopy (SERS) shows unique advantages for the study of protein structure in water environment, because it can provide plenty of structural information of proteins in addition to very weak Raman scattering signal of water. To achieve the Raman-enhanced effect of proteins, the challenge is to prompt protein molecules to approach the surface of noble metal (such as Ag and Au). Based on the enzyme-substrate specificity, the Ag nanoparticles-encapsulated amylopectin (Amyp/AgNPs) colloid was prepared as an aqueous SERS substrate to obtain the Raman-enhanced effect of amylase. Dynamic light scattering analysis showed that the hydrodynamic diameter of the Amyp/AgNPs colloid was in the range of 40-700 nm. UV-vis spectroscopy and energy dispersive spectroscopy confirmed the formation of Ag nanoparticles in the Amyp/AgNPs colloid. The Ag nanoparticles in the Amyp/AgNPs colloid were observed as globular homogeneous particles in the sizes ranging from 10 to 20 nm using transmission electron microscopy (TEM). The crystalline Ag nanoparticles were further observed in the high-resolution TEM image. The obviously enhanced Raman bands assigned to the amino acid residues and the secondary structures of α-amylase and β-amylase appeared in their SERS spectra, which were further analyzed following the principal component analysis (PCA) process. The Raman-enhanced mechanism is considered as that the specificity of two amylases with amylopectin in the Amyp/AgNPs colloid may prompt them to approach the surface of Ag nanoparticles and then produce the significant SERS effect. The SERS strategy based on enzyme-substrate specificity is thus anticipated to apply for structural analysis of enzyme.