Surface-enhanced Raman spectroscopy (SERS) is a powerful technique for obtaining structural information of molecules in solution at low concentrations. While commercial SERS substrates are available, high costs prevent their wide-spread use in the medical field. One solution is to prepare requisite noble metal nanostructures exploiting natural nanostructures. As an example of biomimetic approaches, butterfly wing scales with their intricate nanostructures have been found to exhibit exquisite SERS activity when coated with silver. Selecting appropriate scales from particular butterfly species and depositing silver of certain thicknesses leads to significant SERS activity. For morphological observations we used scanning electron microscopes as well as a helium ion microscope, highly suitable for morphological characterization of poorly conducting samples. In this paper, we describe a protocol for carrying out SERS measurements based on butterfly wing scales and demonstrate its LOD with a common Raman reporter, rhodamine 6 G. We also emphasize what special care is necessary in such measurements. We also try to shed light on what makes scales work as SERS substrates by carefully modifying the original nanostructures. Such a study allows us to either use scales directly as a raw material for SERS substrate or provides an insight as to what nanostructures need to be recreated for synthetic SERS substrates.
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