PT  - JOURNAL ARTICLE
AU  - Samantha M. Grist
AU  - Andoni P. Mourdoukoutas
AU  - Amy E. Herr
TI  - 3D projection electrophoresis for single-cell immunoblotting
AID  - 10.1101/805770
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 805770
4099  - http://biorxiv.org/content/early/2019/10/16/805770.short
4100  - http://biorxiv.org/content/early/2019/10/16/805770.full
AB  - While single-cell resolution immunoassays and mass spectrometry are powerful protein analysis tools, key analytical bottlenecks remain in target specificity, analytical sensitivity, and measurement throughput. Advances are needed to complement single-cell genomics and transcriptomics tools. Here, we introduce highly parallel single-cell immunoblots designed to detect protein targets directly in unfixed mammalian cells. The 3D microfluidic device is comprised of a photoactive polyacrylamide gel, having one face (x-y) patterned with a high-density microwell array for cell isolation and lysis. From each microwell, single-cell lysate is ‘electrophoretically projected’ into the 3rd dimension (z-axis) for protein electrophoresis, photo-capture, and immunoprobing. Design guidelines for electrophoresis are informed by numerical simulation and empirical analyses of 3D diffusion of protein lysate. Unlike serial interrogation with capillary sampling interfaces, no automation or precision alignment is required for concurrent analysis of hundreds of cells. Importantly, cell separations are nearly synchronous (<10 s delay), whereas serial analyses impart hundreds of seconds of delay between analysis of the first and last cell in a population. We achieve an electrophoresis throughput of >2.5 cells/sec, which is 80X faster than serial sampling approaches, and perform 25 single-cell immunoblots per 1 mm2 of device area, a >10X increase over reported single-cell immunoblots. A straightforward device for parallel, synchronized single-cell immunoblotting, projection electrophoresis should advance integration of direct measurement of protein expression into the emerging single-cell atlas of genomic and transcriptomic profiles.