## 3 Frustule Modification: Techniques and Applications
### Frustule Replicas  
One of the main advantages represented by the use of diatom cultures in nanotechnology is the near-zero cost availability of complex, three-dimensional micro- and nano-structures able to self-replicate at high rates with a remarkable reproducibility, giving rise to complex geometries and architectures hardly feasible even by means of the most advanced lithographic techniques [53; 80; 94]. Nevertheless, silica may be not the ideal choice for a specific application. In addition to metabolic insertion of ions in culture medium, which does not allow for the complete substitution of silica with other materials, other methods have been introduced through years aimed at the realization of accurate frustule replicas in different materials. High-refractive index frustule replicas can, for example, support wide and complete PBGs [53]. A case of metallic replica of a frustule has been already reported in Section 3.2, but other examples deserve to be mentioned. [PERSON] and coworkers [101] developed a low-cost, 2-step process based on a soft polymer mold in polydimethylsiloxane (PDMS) and subsequent transferring of the pattern from PDMS onto a hard photo-curable polymer to obtain a positive replica of the frustule (see Figure 9a). Actually the process can be applied to other kind of materials and the valve porous patterns can be tranferred to gels, precursors to ceramics and carbons, luminescent phosphors, salt and colloids. Depending on the starting diatom species, a huge variety of patterns can be transferred. The obtained replicas can find application as complex optical elements, masters for nanofabrication, biosensing devices and nanoreactors. Figure 8: Dose-response curve of the label-free photoluminescence-based biosensor described in reference [100]. Binding constant is also reported. Reproduced with permission. Silicon replicas of _Aulacoseira_ frustules have been synthesized by [PERSON] et al. [102] making use of a shape-preserving magnesiumic reduction. In the first stage of the process, MgO/Si replicas are obtained starting from a frustule-bearing steel boat and magnesium granules placed for 2.5 h in a furnace preheated at 650 \({}^{\circ}\)C, according to the following reaction:  
\[2\mathrm{Mg(g)}+\mathrm{SiO_{2}(s)}\to 2\mathrm{MgO(s)}+\mathrm{Si(s)} \tag{2}\]  
where (g) and (s) stand for gaseous and solid state, respectively. Subsequent treatments in HCl and HF solutions allowed the selective dissolution of magnesia. The chemical and structural composition of the obtained replicas have been scrutinized aby FT-IR spectroscopy, Energy Dispersive X-Ray Analysis (EDX) and X-ray diffraction (XRD). Silicon resulted as a crystalline phase characterized by an average crystallite size of 13\(\pm\)2 nm. Finally, since the obtained silicon frustules were charaterized by an impedence strongly dependent on the chemical composition of the environment, they have been succesfully tested as NO(g) sensors. [PERSON] et al. [103] obtained graphene replicas of _Aulacoseira_ sp. frustules by means of chemical vapor deposition (CVD) of methane followed by dissolution of the silica in hydrofluoric acid. During CVD, hydrocarbon species infiltrate into all the frustule surfaces, including the inner ones, and form a continuous graphene coating that retains the frustule shape in all its details. Since graphene is highly transparent to electron beams, this allows the visualization of the internal morphology of valves and girdles, revealing the complex, interconnected nanotubes linking their different layers. Mn-iron hydroxide hybrids have been obtained starting from diatom frustules by [PERSON] et al. [104]. The three-step process starts with a hydrothermal treatment in a KMnO\({}_{4}\) solution, which promotes the formation of a MnO\({}_{2}\) layer onto the diatom valve. Then, a FeSO\({}_{4}\cdot\)7H\({}_{2}\)O solution is used to promote MnO\({}_{2}\) tranfert into iron hydroxide through redox reaction between Fe\({}^{2+}\) and MnO\({}_{2}\). In the last step, silica is removed by a KOH solution etching.