To make effective biosensors, the detection molecules must be attached to a microelectronics-compatible surface in such a way that it is highly stable for long periods. Combinations of biomolecules and commonly used substrate materials such as glass, silicon or gold, however, are not known to be very stable over time. In the April 11 issue of Langmuir, researchers at Waseda University in Tokyo and at the University of Tokyo have described a photolithographic technique in which a highly stable diamond surface is coated with select amine groups. These groups then can be functionalized with various biomolecules, which stay attached to the diamond substrate even after multiple washings. The investigators chose diamond not only for its chemical stability, but also because it has high sensitivity in electrochemical reactions and can be deposited as a thin film on various substrates. They deposited an ∋8-μm-thick film of polycrystalline diamond onto a silicon substrate, then produced amine groups directly on the diamond’s surface by irradiating the material with 253.7-nm light from a halogen lamp in the presence of ammonia gas. They used a 100-nm-thick gold photomask and a coating of photoresist film to create an array of dots composed of the amine deposits. Where the gold and resist layers were removed during the photolithographic technique, they exposed the areas to C3F8, which created fluorine-terminated surfaces intended to prevent subsequent hybridization of the biomolecule. The researchers created arrays with dots that were 5, 10, 15 or 20 μm in diameter and used optical microscopy to confirm that the dots were regular in size and shape. They also used spatially resolved x-ray photoelectron spectroscopy to verify the differentiation of the surface areas with the amine groups and those terminated with fluorine. They functionalized the exposed amine groups to either complementary or noncomplementary sequences of DNA and hybridized them with DNA that was tagged with the fluorophore Cy5. Fluorescence imaging showed that the shape of the hybridized amine groups corresponded with those seen in the optical microscopy, although the noncomplementary DNA did not hybridize at all. Furthermore, fluorescence intensity stayed intact after up to 20 repeated cycles of hybridization and denaturization with sodium hydroxide.