Controlling Dispersion during Single-Cell Polyacrylamide-Gel Electrophoresis in Open Microfluidic Devices

Abstract

New tools for measuring protein expression in individual cells complement single-cell genomics and transcriptomics. To characterize a population of individual mammalian cells, hundreds to thousands of microwells are arrayed on a polyacrylamide-gel-coated glass microscope slide. In this “open” fluidic device format, we explore the feasibility of mitigating diffusional losses during lysis and polyacrylamide-gel electrophoresis (PAGE) through spatial control of the pore-size of the gel layer. To reduce in-plane diffusion-driven dilution of each single-cell lysate during in-microwell chemical lysis, we photo-pattern and characterize microwells with small-pore-size sidewalls ringing the microwell except at the injection region. To reduce out-of-plane-diffusion-driven-dilution-caused signal loss during both lysis and single-cell PAGE, we scrutinize a selectively permeable agarose lid layer. To reduce injection dispersion, we photopattern and study a stacking-gel feature at the head of each <1 mm separation axis. Lastly, we explore a semienclosed device design that reduces the cross-sectional area of the chip, thus reducing Joule-heating-induced dispersion during single-cell PAGE. As a result, we observed a 3-fold increase in separation resolution during a 30 s separation and a >2-fold enhancement of the signal-to-noise ratio. We present well-integrated strategies for enhancing overall single-cell-PAGE performance.

Hydrogels in Electrophoresis: Applications and Advances

“…47 Based on the on-chip Western blotting protocol and other thought-provoking reports from the same group, we would like to highlight two techniques that are related with the hydrogel. (1) The open microfluidic device [71][72][73][74] would be an alternative approach to avoid the difficulties brought by the gel volume change in a strictly confined environment. For example, when a gel swell-deswells upon solvent exchange or shrinks upon polymerization in a thin capillary, voids would appear that interfere with the analytical result.…”

<p>A brief review of cytotoxicity of nanoparticles on mesenchymal stem cells in regenerative medicine</p>

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<p>Multiplex loop-mediated isothermal amplification (multi-LAMP) assay for rapid detection of <em>mcr-1</em> to <em>mcr-5</em> in colistin-resistant bacteria</p>

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