# Nanostructured Biosilica of Diatoms: From Water World to Biomedical Applications
## 2 Chemical Modification of the Frustule Surface  
Chemical modification of porous drug carriers is known to be an essential factor controlling diffusion and drug delivery rate [30]. Many other properties, such as biocompatibility and cellular uptake, can be improved by binding proteins or bio-materials on the surface of drug carriers [31]. Surface modifications not only affect molecular interactions but also control the identification of biomolecules offering interesting perspectives for targeted-drug delivery. The ability to be easily modified with different molecules makes diatom frustules useful in biomedicine. Biomedical applications necessitate intense purification treatments of diatom frustules to obtain a safe and biocompatible material. Before purification, the features of diatom frustules are completely masked by the presence of many impurities on their surface, such as organic and inorganic compounds from the environment. [PERSON] et al. [32] described an accurate purification procedure for diatomite, a sedimentary rock white to yellow, composed of fossilized skeletons of diatoms. The siliceous skeletons contain silicon dioxide (SiO\({}_{2}\)) and small amounts of associated compounds, such as inorganic components and traces of iron, alkali metals, and other constituents [33]. Diatomite powder was suspended in a Piranha solution (2 M H\({}_{2}\)SO\({}_{4}\), 10% H\({}_{2}\)O\({}_{2}\)) and intensively washed to remove traces of sulfuric acid. Subsequently, a solution 5.0 M of hydrochloric acid (HCl) was added to diatomic dispersion to remove the inorganic compounds (Al\({}_{2}\)O\({}_{3}\), FeO\({}_{3}\), CaCO\({}_{3}\), CaO, K\({}_{2}\)O, Na\({}_{2}\)O, MgO) without altering the siliceous structure. The removal of impurities from the frustules modified their composition and exposed the reactive hydroxyl groups (Si-OH) on the surface, which facilitates the binding of biomolecules on diatom biosilica walls [34; 35; 36]. As a result of this attractive property, the number of reports including diatom functionalization strategies has enormously increased. A significant drawback of immunocomplex-based biosensors is that chemical labeling techniques are generally required for analyte sensing. Current label-free biosensors for immune-complex detection depend on surface plasmon resonance (SPR), electrochemical or impedance measurements, all of which involve technical and costly instruments [37; 38]. To face this problem, [PERSON] et al. [39] modified photoluminescence (PL)-active frustules from _Pinnularia_ sp. with an anti-trinitrotoluene (TNT) single-chain variable fragment (scFv) to make a TNT-biosensor based on PL-detection. For this purpose, surface silanol groups on diatom frustules were aminated by reaction with 3-aminopropyl-trimethoxysilane (APTMS), and the scFv was covalently attached to aminated biosilica through the crosslinking-agent disuccinimidyl suberate (DSS). The authors assumed that the estimated scFv coverage on diatom frustules was approximately \(2.5\times 10^{4}\) molecules per \(\upmu\)m\({}^{2}\), or 40% of the total surface area, which is more than enough for the fabrication of a selective antibody-based biosensor for TNT label-free detection. Silane-based chemistry is one of the most exploited modifications of the diatom surface since it offers a facile way to attach a biological or chemical molecule to the surface. [PERSON] et al. [40] exploited the advantages of silanized diatom frustules using 3-aminopropylriethoxysilane (APTES) to bind biomolecules on the diatom frustule likewise. They proposed a chitosan (Chi) grafting on _Amphora_ frustules (Chi@AF) to encapsulate the chemotherapeutic anthracycline antibiotic Doxorubicin (DOX) in the frustule ultrastructure. The Chi-grafting on the AF surface was obtained by linking the amino groups of the polysaccharide to APTES-functionalized diatom frustules in the presence of glutaraldehyde as a cross-linker.