Supporting data: 3D projection electrophoresis for single-cell immunoblotting (Part 2)

Immunoassays and mass spectrometry are powerful single-cell protein analysis tools; however, interfacing and throughput bottlenecks remain. Here, we introduce three-dimensional single-cell immunoblots to detect both cytosolic and nuclear proteins. The 3D microfluidic device is a photoactive polyacrylamide gel with a microwell array-patterned face (x-y) for cell isolation and lysis. Single-cell lysate in each microwell is ‘electrophoretically projected’ into the 3rd dimension (z-axis), separated by size, and photo-captured in the gel for immunoprobing and confocal/light-sheet imaging. Design and analysis are informed by the physics of 3D diffusion. Electrophoresis throughput is >2.5 cells/s (70X faster than published serial sampling), with 25 immunoblots/mm2 device area (>10X increase over previous immunoblots). The 3D microdevice design synchronizes analyses of hundreds of cells, compared to status quo serial analyses that impart hours-long delay between the first and last cell. Here, we introduce projection electrophoresis to augment the heavily genomic and transcriptomic single-cell atlases with protein-level profiling.