Massively Parallel High-Throughput Single-Cell Patterning and Large Biomolecular Delivery in Mammalian Cells Using Light Pulses

The recent advancements of single-cell analysis have significantly enhanced the ability to understand cellular physiology when compared to bulk cellular analysis. Here a massively parallel single-cell patterning and very large biomolecular delivery is reported. Micro-pillar polydimethyl siloxane stamp with different diameters (40-100 & mu;m with 1 cm x 1 cm patterning area) is fabricated and then imprint distinct proteins and finally pattern single-cell to small clusters of cells depending on the micro-pillar diameters. The maximum patterning efficiency is achieved 99.7% for SiHa, 96.75% for L929, and 98.6% for MG63 cells, for the 100 & mu;m micro-pillar stamp. For intracellular delivery of biomolecules into the patterned cells, a titanium micro-dish device is aligned on top of the cells and exposed by infrared light pulses. The platform successfully delivers small to very large biomolecules such as PI dyes (668 Da), dextran 3000 Da, siRNA (20-24 bp), and large size enzymes (464 KDa) in SiHa, L929 and MG63 cells. The delivery efficiency for PI dye, Dextran 3000, siRNA, and enzyme for patterned cells are & AP;95 & PLUSMN; 3%, 97 & PLUSMN; 1%, 96 & PLUSMN; 1% and 94 & PLUSMN; 3%, with cell viability of 98 & PLUSMN; 1%. Thus, the platform is compact, robust, easy for printing, and potentially applicable for single-cell therapy and diagnostics.