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Silver nanoparticles (AgNPs) have a large number of technological applications, such as photonic devices, molecular diagnostics, antimicrobial coating and biomedical devices. These nanomaterials can be synthesized using common fungi, which represents a green approach with the additional advantage of biocompatibility. Here we applied Tip-Enhanced Raman Spectroscopy (TERS) to obtain optical characterization with resolution beyond the diffraction limit of AgNPs produced by Rhodotorula glutinis and Rhodotorula mucilaginosa fungi. Those fungi are conventional found in ordinary soil of north part of Brazil, which biosynthesize core-shell AgNPs with protein-capping layer. With TERS we were able to investigate with lateral resolution of few nanometers the size and structure of the protein capping layer, how it interacts with the Ag core, and how sensitive the system (core + protein) is to visible light illumination. Despite the complex data generated which depends on the orientation of the protein, which is highly, it was possible to detect the stretching of the S - S sulfur bridge in amino acids. This stretch band is induced by a trans C α - S conformation acid on Rm-AgNPs, thus showing the potential of characterization of TERS in nanobiosystems. [ABSTRACT FROM AUTHOR]