## 3 Frustule Modification: Techniques and Applications
### Frustule Metalization for Applications in Plasmonics  
Metallic-dielectric interfaces excited with optical radiation at specific wavelengths support the propagation of surface palsmon polaritons (SPPs), determined by collective oscillations of conducting electrons. In case of metallic nanoparticles or nanostructured metallic substrates the plasmonic field is strongly confined, giving rise to localized surface plasmons (LSPs) [61]. One of the most widespread applications of plasmonics is surface enhanced Raman spectroscopy (SERS), where LPs are exploited to enhance the Raman emission of molecules in proximity of the metallic nanostructures, compensating the low cross section characteristic of the process [62]. The enhancement factor (\(EF\)) is defined as:  
\[EF=\frac{I_{SERS}/N_{surf}}{I_{RS}/N_{vol}}, \tag{1}\]  
where \(I_{SERS}\) and \(I_{RS}\) are SERS and Raman intensities, \(N_{surf}\) is the average number of molecules adsorbed to the metallic substrate in the scattering area for the SERS measurements, and \(N_{vol}\) represents the number of molecules present in bulk in the scattering volume for spontaneous Raman scattering measurements. Two main approaches are usually followed in the realization of SERS substrates. On one hand, bottom-up chemical synthesis allows obtaining a plethora of metallic nanoparticles such as colloidal silver and gold nanoparticles [63], silica-coated metallic nanoparticles [64], monolayers of aligned metallic nanowires [65], metallic nanoprisms, nanocubes, nanostars, and nanosheets [66; 67; 68; 69], all ensuring high values of the enhancement factor but lacking in tunability, robustness and reproducibility. On the other hand, top-down nanolithographic techniques are used in the fabrication of plasmonic nano-antennas arrays [70; 71], subwavelength gold gratings [72], dielectric photonic crystals coupled with metallic nanoparticles [73], optical fibers provided with nanostructured, metallized facets [74], nanostructured metallic fishnets [75], and three-dimensional hollow nanostructures with tunable geometries [76], to name a few. Even though these solutions guarantee high reproducibility in the aquisition of the spectra, generally result expensive and prohibitive for large-scale production and routine practical applications. Being formed by a complex, intricate but still regular dielectric three-dimensional micro- and nano-structure, it is straightforward to hypothesize that diatom frustules, if properly metalized, are able to trigger plasmonic effects, presenting the additional but not negligible advantage to avoid both the loss of reproducibility proper of traditional bottom-up approaches and the prohibitive cost, complex fabrication and hard feasibility in mass production typically affecting top-down techniques. One of the the first attempts to use diatom frustules as SERS substrates consisted in the realization of silver replicas of _Synedra_ sp. and _Thalassiosira_ sp. valves by chemical removal of silica after frustule covering with evaporated silver [77]. The resulting metallic nanostructures, when used as SERS substrates, allowed obtaining enhancement factors up to \(\sim\)10\({}^{6}\) for rhodamine as analyte and assuming a 100% covering of the dye on the silver shell. Another approach used to metalize diatom frustules consists of the chemical attachment of metal nanoparticles to diatom biosilica, thus promoting the coupling of the guided-mode resonances supported by the valves to the LPs of the nanoparticles. The resulting hybrid photonic-plasmonic modes ensure high quality factors of the local electric field. [PERSON] et al. [78] obtained silver nanoparticles self-assembly to amine-functionalized _Pinularia_ sp. valves and tested the resulting hybrid nanostructures as SERS substrates.