Fabrication of multiple-antibody arrays using a hydrogel microstamp

This work demonstrates a simple method for protein micropatterning. A new sugar poly(acrylate) hydrogel acts as a reusable `stamper' which deposits antibodies directly onto a silane modified capture surface. The antibodies are directly placed on the surface so a precise arrangement can be obtained with minimal cross-contamination among the antibody spots. Circular antibody spots have been created using this approach with diameters ranging from less than 20 to greater than 100 microns. Patterns were imaged using immunofluorescence microscopy wherein a fluorescently- labeled IgG antigen was selectively bound to the patterned (stamped) antibody. Signal to background ratios of greater than 25:1 have been routinely observed. Arrays with three antibodies were prepared and visualized by sequential exposure to fluorescently-labeled partner antigens. No cross-reactivity among the different antibody spots was observed. Engineered biosurfaces created using this new technology may be useful in the areas of multianalyte immunosensors, the assembly of protein ensembles, and cell patterning methods.