The film growth of APTES is also dependent on temperature; with dendritic islands growing and fusing to cover the surface at 10°C, whereas at 40°C a homogeneous uniform film forms. Further elevated incubation temperatures (∼70°) leads to denser, stronger and better orientated films, without compromising the self-termination of the deposition. The position of the amine group has a strong effect on its chemistry in aqueous solution, compared to silanes that lack the amine or have the amine in a different position. It has a catalytic effect on the hydrolysation of the methoxy groups and can cause the formation of a six membered hydrogen bonded ring. Howarter and Youngblood's 71 work focused on optimising the reaction conditions -temperature, concentration and time -to establish a uniform thin film. They found that the concentration of APTES solution and the temperature of the incubation governed the surface roughness and receding contact angle, whereas they had minimal effect on the film thickness. It was the incubation time that controlled this characteristic. For low concentrations (1%) good films were produced when incubation time was limited to 1 hour and these did not appear to be temperature sensitive. To produce smooth thick films at higher concentrations PAs are not typically suited to SAM formation on silicon substrates as the Si-O-P bonds are easily cleaved by nucleophiles such as water or alcohols. The T-BAG method was established to allow the chemisorption of PAs onto a silicon surface. This method forms a densely packed layer of organic groups which prohibits the diffusion of water to the interface causing the SAM to be stable against hydrolysis. Whereas, silane groups may be more suited to silicon substrates, PAs appear to be superiorly anchored to metal oxide substrates, at pH 1-10. The pH and solvent of the phosphonate solution plays an important role in the formation of a stable layer. Varying the pH and using an organic solvent has been shown to stimulate the monolayer grafting and prevent a precipitation process. Another possible issue is that the solubility will the depend on the hydrophilic/hydrophobic nature of the organic backbone, though phosphonate ester analogues are often employed as they usually show no solubility issue. As they are not as well characterised as thiols and silanes, PAs are not as often adopted as a SAM for the immobilisation of antibodies. However, both (11-hydroxyundecyl)phosphonic acid and (12carboxydodecyl)phosphonic acid were assembled on a titanium substrate and cross-linked to a protein's amino group. The hydroxyl-terminated PA was determined to form a more ordered SAM than that of the carboxyl-terminated SAM. This hydroxyl-terminated layer could be utilised for the immobilisation of antibodies or antigens to an electrode surface.