Oligonucleotide immobilization on micropatterned streptavidin surfaces

Abstract

We describe a simple procedure for photolithographic patterning of streptavidin on silicon substrates. Long wavelength UV (365 nm) light was used to direct the covalent attachment of photoactivatable biotin onto silylated silicon wafers. Fluorescently labeled streptavidin was found to bind only in areas exposed to the light. We used this procedure to selectively pattern streptavidin inside microwells etched in silicon, and we investigated the binding characteristics of biotinylated oligonucleotides of lengths, n = 16, 54 and 99 bases. The binding curves were found to fit the functional form of the Langmuir isotherm, with binding saturation proportional to n(-3/4).

Figure 1

(A) Side view of scheme used for photochemical attachment of photoactivatable biotin on MPTS-functionalized SiO₂. A photomask is used to direct UV light to specific areas of the substrate. (B) Proposed orientation of immobilized streptavidin.

Figure 2

Micropatterned streptavidin surfaces. (A) Fluorescence micrograph of Cy2-labeled streptavidin patterned on a flat MPTS-functionalized SiO₂ surface. Fluorescent streptavidin is shown in white. (B) Fluorescent scan of Cy2-streptavidin-coated microwells. Each microwell is 2 × 2 mm² × 200 µm deep, and spaced 2 mm apart.

Figure 3

(A) Binding capacity of oligonucleotides versus amounts added for 99 base (circles), 54 base (triangles) and 16 base (squares) lengths. (B) Expanded view of dotted section of (A). The solid curves are Langmuir isotherm functions fitted to the experimental data (Discussion).