Immobilized, micro-patterned graphene nanoflake devices for high-throughput, uniform intracellular biomolecular delivery

Abstract

Developing a high-throughput, safe, and adaptable method for intracellular delivery remains a bottleneck hindering the translation of molecular therapeutics and genome engineering. Nano-sensitizing particle-activated photoporation results in highly efficient cell transfection, but the throughput is limited, and managing cytotoxicity associated with the internalization of nanoparticles remains a challenge. This study presents an immobilized, micropatterned reduced graphene oxide (rGO)-based scalable and biocompatible platform with an automated laser scanning interface for high-throughput photoporation-mediated intracellular delivery. The device comprised an ordered array of rGO islands of 10 µm diameter with an interisland spacing of 30 µm on a glass substrate of 2 cm × 2 cm dimensions. The device was aligned atop the seeded layer of cells, and a 1064-nm infrared nanosecond pulse laser (28 mJ cm⁻²) was scanned, inducing photothermal bubbles near the cell membrane. These bubbles rapidly expanded and collapsed, causing cell membrane deformation, thereby enabling uniform delivery to ∼5 × 10⁵ cells within 15 seconds. This platform successfully delivered various biomolecules including small-molecule dyes, siRNA, plasmids, and large-sized enzymes across different cancer cell lines and human mesenchymal stem cells (hMSCs). The best results were achieved with an enzyme transfection efficacy of 94% and a cell viability of 99% in hMSCs. This method provides a scalable and versatile bio-interface approach, paving the way for high-throughput intracellular delivery for therapeutic, diagnostic, and regenerative medicine applications and innovations.