Electrophoretic cytometry: High‐selectivity measurement of isoforms using microfluidics
Authors: Herr Amy E. 
Published: June 2021

From fundamental biosciences to applied biomedicine, high dimensionality data is increasingly important. In singe‐cell measurement tools, microfluidic design has underpinned the throughput, multiplexing and quantitation needed for this rich data. Genomics and transcriptomics are leading examples. Yet, measurement of proteins lags. While proteins and their dynamic forms are the downstream effectors of function, the immunoassay remains the de facto standard (flow cytometry, mass cytometry, immunofluorescence). We posit that to realize the full potential of high‐dimensionality cytometry, new approaches to protein measurement are needed.

In this talk, I will describe our ‘electrophoretic cytometry’ tools that increase target selectivity beyond simple immunoassays by introducing microfluidic single‐cell immunoblotting. Enhanced selectivity is essential for targets that lack high quality immunoreagents – as is the case for the vast majority of protein forms (proteoforms). I will share our results on highly multiplexed single‐cell western blotting and single‐cell isoelectric focusing that resolves single charge‐unit proteoform differences.

In fundamental engineering and design, I will discuss how the physics and chemistry accessible in microsystems allows both the “scale‐down” of electrophoresis to single cells and the “scale‐up” to concurrent analyses of large numbers of cells. Particular emphasis will be placed on precision control of fluids and materials transport in passive systems, with no pumps or valves. Precise reagent control allows for integration of cytometry with sophisticated sample preparation – the unsung hero of measurement science. I will discuss compartment‐specific, single‐cell western blotting for nucleo‐cytoplasmic profiling, which eliminates the need for complex image segmentation algorithms.

In applied biology, I will link our measurement science research to driving cytology needs, including understanding the role of protein signaling and truncated isoforms in development of breast cancer drug resistance and understanding protein signaling in individual circulating tumor cells.

Taken together, we view microfluidic design strategies as key to advancing protein measurement performance needed to address unmet gaps in quantitative biology and precision medicine.

Support or Funding Information

Research reported was supported in part by the National Cancer Institute of the National Institutes of Health (R21CA183679 to A.E.H.), a New Innovator Award from the National Institutes of Health (1DP2OD007294 to A.E.H.), an NSF CAREER award from the National Science Foundation (CBET‐1056035 to A.E.H.), a Diversity Supplement from the National Institutes of Health (to E.S.) and National Science Foundation Graduate Research Fellowships (DGE 1106400 to K.A.Y., J.V., and T.A.D.). We acknowledge the helpful feedback from students enrolled in the 2015 Single Cell Analysis course at Cold Spring Harbor Laboratory.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.