The covalent attachment of thiol-modified DNA oligomers; to self-assembled monolayer silane films on fused silica and oxidized silicon substrates is described. A heterobifunctional crosslinking molecule bearing both thiol- and amino-reactive moieties was used to tether a DNA oligomer (modified at its terminus with a thiol group) to an aminosilane film formed on silica surfaces. A variety of aminosilanes, crosslinkers and treatment conditions have been tested to identify optimal conditions for DNA immobilization using this approach. The DNA films which result have been characterized using UV spectroscopy, water contact angle measurement, radiolabeling and hybridization methods.

The deep ultraviolet (λ < ∼250 nm) photochemistry of chemisorbed organosilane self-assembled films of the type R(CH2) n SiO−surface where n = 0, 1, 2 and R = phenyl, naphthyl, or anthracenyl is explored. Photochemistry is examined using 193 and 248 nm laser irradiation as well as deep ultraviolet lamp sources. It is demonstrated for a variety of systems, including single and multiple rings as well as heterocycles, that the primary photochemical mechanism is cleavage of the Si−C bond. Photocleavage of the organic group generates a polar, wettable silanol surface that is amenable to subsequent remodification by organosilane chemisorption, allowing the fabrication of high-resolution patterns of chemical functional groups in a single molecular plane. The use of patterned monolayers as templates of reactivity for subsequent selective chemical reactions is demonstrated.

Two photolithographic methods are described for the formation of patterned single or multiple DNA species on SiO2 substrates. In the first approach, substrates are treated with a photochemically labile organosilane monolayer film. Irradiation of these surfaces with patterned deep UV (193 nm) light results in patterned chemically reactive groups which are then reacted with heterobifunctional crosslinking molecules. Covalent attachment of modified synthetic DNA oligomers to the crosslinker results in stable DNA patterns. Alternatively, a photoresist is spin-coated over a silane film which had been previously modified with the heterobifunctional crosslinker. Upon patterned irradiation and subsequent development, the underlying crosslinker-modified layer is revealed, and is then reacted with a chemically modified DNA. Feature dimensions to 1 micron are observed when a single fluorescent DNA is attached to the surface. By performing sequential exposures, we have successfully immobilized two distinguishable DNA oligomers on a single surface. Synthetic DNA immobilized in this manner retains the ability to hybridize to its complementary strand, suggesting that these approaches may find utility in the development of miniaturized DNA-based biosensors.

The atomic force microscope (AFM) measures force and displacement with high sensitivity and submillisecond temporal resolution. By functionalizing the AFM probe with specific chemical groups or macromolecules it is possible to characterize the chemical and physical properties of single molecules on the nanometer scale. In this paper we discuss the key issues that must be addressed when designing and characterizing a successful immobilization chemistry, and describe the chemistry we developed to covalently immobilize oligonucleotides in a specific orientation.

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