## 3 Frustule Modification: Techniques and Applications
### Fustule Functionalization for Protein Immobilization  
Imobilization of proteins onto a solid support material allows improving their functionality by stabilizing their native conformation, which is of great benefit in several applications like catalysis, sensing, and drug delivery [84]. Diatom biosilica, in particular, offers several advantages if used as support material: its porosity, characterized by high values of specific surface area (up to 220 m\({}^{2}\) g\({}^{-1}\)), provides a large capacity for binding proteins and a very effective interaction with several adsorbates [53]; the size of frustule pores is large enough to allow for the incorporation of protein molecules, which have an equivalent hydrodinamic diameter of the order of nanometers [85]; its high chemical and mechanical stability and its hydrophilic properties allow for hydrogen bonds and electrostatic interactions with proteins; its transparency in a wide interval of the visible and infrared spectrum make it possible to analyze the immobilized proteins by optical spectroscopy; finally, its biocompatibility allows for in vivo applications. Among the possible in vitro techniques aimed at protein immobilization onto diatom biosilica, the most efficient are those based on the introduction of chemical functional groups able to irreversibly attach proteins to the hydrogenated silica frustule through covalent binding. Indeed silanol (\(\equiv\)Si-OH) and silanolate (\(\equiv\)Si-O\({}^{-}\)) groups on the frustule surface are not able to directly form stable covalent bonds with protein molecules and further chemically reactive functional species have to be introduced as cross-linkers. [PERSON] and co-workers [86] silanized _C. wailesii_ frustules using the alkoxysilane 3-aminopropyltrimethoxysilane (APS) followed by N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) as crosslinker in order to permit covalent bonding with anti-IgY (IgY: immunoglobulin Y) after exposure to UV light (see Figure 7). Conjugation with secondary antibody (horse radish peroxidase HRP) was verified by immunoblot chemiluminescence. Furthermore, in order to avoid multiple attachment sites via the amine groups with consequent distortion of the moiety, the authors tested a second method which exploits the carbohydrate side chains of the antibody through the oxidation of the hydroxil groups of the sugar residue on its Fc region to form aldehydes. In Ref. [87] [PERSON] et al. applied, for the same diatom species, a functionalization method usually implemented in chemical modification of oxidised porous silicon [88] and based on aminopropyltriethoxysilane (APTES) for silanization and glutaraldehyde (GA) as cross-linker. The obtained modified active surface is able to attach also DNA single strands other than proteins [89]. In the reported study, murine monoclonal antibody (MoAb) UN1, characterized by a specific reactivity with human thymocytes [90], has been used as molecular bioprobe to be attached to _C. availesii_ frustule. Futhermore, Protein A from _Staphilicoccus aureus_ has been used as a biospecific spacer arm in order to properly orient MoAb. Every single step of the functionalization process has been verified by means of FT-IR spectroscopy and rhodamine labelling of bioprobes has been used to test the quality of the functionalization process by means of fluorescence microscopy. The same protocol involving the use of APTES and GA has been used by [PERSON] et al. [91] for tyrosinase immobilization onto pennate diatom frustules. The obtained active biosilica was tested in degradation of phenolic compounds (phenol, para-cresol and phenyl acetate) in a batch system. The same authors reported lipase immobilization onto _Scenedesmus quadricaada_ frustules after bromoacetylation of the silanol groups and subsequent polymerization of 2-choloethyl acrylate (CEA). Finally, the terminal chloride groups were reacted with ethylene diamine to introduce amino groups that were cross-linked to lipase using GA.