Eco-Friendly and Large-Scale Fabrication of Microelectronic Silk-Based Enzymatic Biosensors

Silk fibroin (SF) from Bombyx mori is emerging as a sustainable biomaterial for enzyme-based biosensors due to its ability to stabilize enzyme activity and provide a robust matrix for biomolecule immobilization. Despite its integration into micro- and nanofabrication workflows remains underexplored. This study presents a novel, eco-friendly approach for integrating SF into cleanroom microelectronic workflows by depositing and processing SF at wafer level. Uniform SF films with tunable thicknesses ranging from $59\pm 5$ to $131 \pm 2$ nm were spin-coated onto silicon wafers and precisely patterned via electron beam lithography (EBL) using an optimized exposure dose of $1200\ \mu \mathrm{C}/\text{cm}^{2}$. Water was employed as the sole developer, avoiding the use of toxic organic solvents conventionally used in lithographic processes. SEM imaging confirmed high-resolution nano-patterning, with accurate and uniform hole sizes of $200 \pm 2$ nm. Enzymatic activity assays demonstrated that glucose oxidase (GOx) encapsulated in SF films retained over 90% activity after vacuum water annealing and 75% after chemically-mediated curing using green chemical compounds (GCC), representing a ~260-200% improvement over conventional methanol-based curing methods, which has previously limited the application of SF as a bio-modified photoresist in microelectronic biosensor fabrication. Notably, enzymatic activity measured after EBL exposure indicated that the lithographic process did not compromise the enzyme integrity. This protocol enables the scalable fabrication of a novel bio-functional photoresist based on SF combining precise patterning while ensuring enzyme stability, positioning SF as a sustainable material for next-generation biosensor and bioelectronic devices, and eliminating the need for hazardous chemicals in cleanroom fabrication.