## 3 Frustule Modification: Techniques and Applications
### Frustule Metalization for Applications in Plasmonics
Under non resonant condition and over a concentration interval of 10\({}^{-7}\)-10\({}^{-4}\) M, an increase in EF up to 12 times has been observed comparing the acquired spectra of rhodamine 6G dropped on valves and on a reference glass slide, respectively, both conjugated with silver nanoparticles. The same hybridization process has been exploited for the same diatom species in the development of a SERS-based sandwich immunoassaysystem aimed at the detection of antibody-antigen interaction [17]. In this case, the silver nanoparticles not only provide SERS enhancement, but are also used as substrates for antibody attachment. The experimental results proved that a detection limit down to 10 pg mL\({}^{-1}\) can be achieved, which is two orders of magnitude better than the one obtainable by the corresponding flat SERS substrates. A similar sandwich structure has been used for interleukin 8 (IL-8) detection in blood plasma, starting by the functionalization of _Pseudostaurosia trainori_ diatom frustules with gold nanoparticles conjugated with DTNB as Raman reporter and with specific antibodies as bioprobes (see Figure 5) [79]. Gold nanoparticles do not present significative oxidation, which usually affects silver. The experimental results showed an improved detection limit in comparison to the traditional enzyme-linked immunosorbent assay (ELISA). In particular, the detection limit for IL-8 in human blood plasma was found to be 6.2 pg mL\({}^{-1}\) versus 15.6 pg mL\({}^{-1}\) obtained by ELISA, while it equals 2.5 ng mL\({}^{-1}\) when using a glass-based SERS immune substrate. Certainly the most straightforward and simple way to obtain a diatom-based SERS substrate is by thermal evaporation of metal without any subsequent chemical removal of the biosilica. The choice of the species is also crucial since a peculiar frustule morphology and geometry can represent the optimal solution for a specific application. An example is described in Ref. [80], where uniform thermal deposition of gold on valves of _Pseudonitchia multistriata_ allowed the realization of SERS substrates for the efficient analysis of biochemical composition of cell membranes, in particular of red blood cells (RBCs) and B-leukemia REH cells. _P. multistriata_ valves are provided with a vertical, extruded side edge (see Figure 6b) which ensures an optimal interaction with cells avoiding steric hindrance. SERS signals from the membrane, with no interference from other cellular components (e.g., hemoglobin in the case of RBCs and nuclear components in the case of leukemia cells) are fundamental in the assessment of several pathologies. In particular, _P. multistriata_ frustules represent an example of natural _supergrating_, where different periodicities (see Figure 6a) interact optimizing the coupling with external optical radiation. When tested on a self-assembling biphenyl-4-thiol (BPT) monolayer (which uniformly covers the valve with a packing density of four molecules per nm\({}^{2}\)) and with a nominal thickness of 40 nm of gold deposited onto the valve, an EF of \((4.6\pm 0.9)\times 10^{6}\) is reached. A comparison between BPT spectra acquired onto the valve and outside the valve is reported  
Figure 5: Schematic representation of the SERS immunoassay described in Ref. [79], with indication of the single elements of the sandwich structure. Antibody immobilization, interleukin capture and a detail of the link between the frustule and the bioprobe through an amino group are also illustrated. Reproduced with permission. in Figure 6d. Furthermore, the authors envisage the use of free-standing, metalized _P. multistriata_ frustules within a microfluidic system in order to obtain mobile, optical driven SERS microsensors able to be dragged toward the target cell. A similar concept is at the basis of the work of [PERSON] et al. [81], where multiscale hierarchical mesocapsules have been obtained starting from in-situ growth of silver nanoparticles onto _Pinnularia_ sp. frustules.