# Diamonds for Life: Developments in Sensors for Biomolecules
## 3 Surface Modification of Diamond Thin Films
### Photochemical Reactions  
One of the first methods for the inclusion of different functional groups onto the surface of H-diamond resorted to processes initiated by photochemistry. In these, UV light is employed to attach simple NH\({}_{2}\) groups or bridging molecules, such as acid or amine-terminated alkenes, to the surface of the diamond film (Figure 2). The bridging molecules create a spacer arm that can subsequently form a covalent bond with a target-specific molecule, such as a DNA oligonucleotide chain or a protein. Moreover, surface modification by photochemical methods has advantages over wet-chemical modification because it allows a facile patterning of the substrate with functional groups. As a setback, the presence of two different functional groups in the amino alkenes, e.g., a double bond at one end and an amine at the other end, requires the use of protecting groups to ensure that the photochemical reaction occurs with the double bond site and to avoid reaction of the amine headgroups, which may cause etching of the substrate [24].  
The simplest form of functionalisation involves the generation of NH\({}_{2}\)-terminated surfaces in diamond. These can be obtained by the exposure of H-diamond to chlorine gas under irradiation with a 450 W Hg arc lamp (254 nm), with subsequent exposure of the chlorine-terminated to ammonia gas, also under irradiation. Both reactions are conducted at low pressure (_c.a._, 10\({}^{-7}\) atm) [25].  
Methods using spacer arms are preferred for a better spatial accommodation of large molecules such as DNA. The first report on the functionalisation of nanocrystalline diamond films with a DNA oligonucleotide sequence dates back to 2002 and employed 10-aminodec-1-ene (with the amine group being protected by trifluoroacetamide) to form a covalent bond with diamond carbons by irradiation with UV light (0.35 mW cm\({}^{-2}\), 254 nm) [26]. After deprotection, the primary amine was allowed to react with a heterobifunctional crosslinker, sulfosuccinidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SSMCC), to form a group that subsequently reacted with a thiol-modified DNA oligonucleotide chain.  
Alternatively, DNA oligonucleotide chains can be linked to H-diamond by an alkene bridge terminated with a carboxylic acid group at the other end. A commonly employed linker is 10-undecenoic acid, which reacts by activation with UV irradiation (20 h, 2.5 mW cm\({}^{-2}\), 254 nm) to form the bridging layer; subsequently, the free carboxylate groups are treated with 1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide (EDC), generating an active intermediate species that readily reacts with the amine groups of the desired DNA oligonucleotides (Figure 3A) [27; 28]. Activation of the carboxylate groups with EDC permits a much faster, high-yield reaction because the active intermediate is more reactive than the native COOH. Further development of this method compared the effect of reacting 10-undecenoic acid in the unprotected and protected forms (trifluoromethyl undecenoate), having shown that the use of protecting groups leads to a lower density of carboxylate groups on the surface of diamond [24]. This is likely due to the bulkier nature of the protected molecules that create a steric hindrance to the functionalisation reactions, reducing the density of the attached molecules.  
The inclusion of an antibody to the surface of H-diamond followed a process similar to that already described for DNA, in which trifluoroacetamide-protected 10-aminoodec-1-ene was employed in the first reaction step (Figure 3B). In a second step, the substrate was treated with a solution of glutaraldehyde, a protein cross-linking agent able to react with amines to form Schiff bases [29]. This method required, however, an extra treatment step (addition of 0.1 M solution of glycine in sodium cyanoborohydride coupling buffer) to remove unreacted aldehyde groups that could affect the stability of the surface of diamond [29].