Patterning of immobilized antibody layers via photolithography and oxygen plasma exposure

A novel technique for patterning immobilized antibody layers based upon photolithography and oxygen plasma exposure has been developed. Mouse monoclonal antibodies specific for thiabendazole (a post-harvest fungicide and veterinary anthelmintic) were covalently linked through free amine groups to aminosilanized silicon dioxide films using glutaraldehyde. Immobilized antibody layers were stabilized with sucrose, dehydrated, and stored refrigerated with desiccant. Photolithographic patterning was performed with a positive photoresist with modified bake temperatures and times, selective UV exposure with a contact mask, and aqueous alkaline solubilization of exposed resist. Exposed regions of immobilized antibody were then removed by exposure to a low power, radio frequency oxygen discharge. Residual resist was stripped with acetone. Successful patterning was demonstrated by challenging surfaces with goat anti-mouse antibody conjugated to tetramethylrhodamine isothiocyanate. Sucrose stabilization was necessary for antibody to undergo photoresist processing without loss of binding activity. Challenge with enzyme linked antigen of oxygen plasma exposed antibody layers demonstrated that plasma treatment completely neutralized antibody capture ability. Ellipsometry measurements of oxygen plasma exposed antibody layers indicated complete removal of immobilized antibodies. Fluorescent imaging demonstrated smallest line widths of 2-3 microns.