High-selectivity cytology via lab-on-a-disc western blotting of individual cells

Abstract

Cytology of sparingly available cell samples from both clinical and experimental settings would benefit from high-selectivity protein tools. To minimize cell handling losses in sparse samples, we design a multi-stage assay using a lab-on-a-disc that integrates cell handling and subsequent single-cell western blotting (scWestern). As the two-layer microfluidic device rotates, the induced centrifugal force directs dissociated cells to dams, which in turn localize the cells over microwells. Gravity-based sedimentation then settles the cells down into the microwells, where the cells are lysed and subjected to scWestern. Taking into account cell losses from capillary loading, centrifugation, and lysis-buffer exchange, our lab-on-a-disc device handles cell samples with as few as 200 cells with 75% cell settling efficiencies. Over 70% of microwells contain single cells after the centrifugation. In addition to cell settling efficiency, cell-size filtration from a mixed population of two cell lines is also realized by tuning the cell flight-of-time during centrifugation (58.4% settling efficiency with 6.4% impurity). Following the upstream cell handling, subsequent scWestern is demonstrated by detection of four proteins (GFP, β-TUB, GAPDH, and STAT3) in a glioblastoma cell line. By integrating the lab-on-a-disc cell preparation and scWestern analysis, our platform measures proteins from sparse cell samples at a single-cell resolution.

Single-Cell Omics Analyses Enabled by Microchip Technologies

“…This method enabled Hughes et al to not only detect proteins but also distinguish isoforms. It was applied to the analysis of rat neural stem cells, glioblastoma cells, CTCs, and different subcellular compartments (157,159,160).…”

Recent advances in protein analysis by capillary and microchip electrophoresis

“…125 Use of centrifugation for cell preparation allows for a more robust integration with single-cell Westerns for cell samples with as a few as 200 cells/low density cell suspensions with minimal cell loss. An array of microwells is molded to a polyacrylamide gel electrophoresis layer.…”

Single-cell technologies for profiling T cells to enable monitoring of immunotherapies

“…By repetition of antibody-stripping and re-probing, it could detect up to eleven different proteins across thousands of single cells in the same experiment [41–43]. By utilizing a combination of lab-on-a-disc cell device and the scWB analysis, it was possible to quantify protein from less than one hundred cells [44]. The same group further developed an approach termed single cell isoelectric focusing (scIEF) using isoelectric point (pI) difference to separate protein isoforms [45].…”