Lithographic patterning of immobilized enzymes in chitosan thin films for multi-layer, chemical/biological sensors

Patterning of immobilized enzyme in spin-cast chitosan thin films will enable the creation of multi-layer biologically-active devices. Bulk immobilized enzymes used in multi-layer devices, in contrast to surface functionalized devices; enable the implementation and characterization of multi-component assays. This paper demonstrates a procedure to immobilize enzymes into an aqueous chitosan solution, spin-cast that solution into a thin-film, and, finally, pattern that thin film using photolithography and oxygen plasma. Enzymes immobilized, deposited, and patterned using this process retain functionality, as shown with the example assay of szlig-D-galactosidase (szlig-Gal) and fluorescein di-szlig-D-galactopyranoside (FDG), a fluorogenic substrate for szlig-Gal. Hydrogel thicknesses of 200 nm to 1.5 mum (dry) were achieved and line widths down to 2 mum were observed. Finally, a multi-layer stack of chitosan hydrogel is demonstrated using a stopping layer of sputtered silicon dioxide (SiO₂).