Chemical mobilization following IEF enables single-point detection of an ideally stationary equilibrium electrophoresis mode. Despite prior studies exploring optimization of chemical mobilization conditions and recent insight from numerical simulations, understanding of both chemical mobilization mechanisms and the implications of mobilization on IEF analytical performance remains limited. In this study, we utilize full-field imaging of microchannel IEF to assess the performance of a range of canonical chemical mobilization schemes. We empirically demonstrate and characterize key areas where limited understanding of performance implications exists, including: the effects of mobilization solution pH and ion concentration, differences between ionic and zwitterionic mobilization, and diffusion as a source of zone broadening. We utilize Simul5 simulations to gain insight into the sources of the measured performance differences. Measurements of the location, linearity, and slope of the IEF pH gradient (via fluorescent pH markers imaged before and during mobilization) as well as mobilization-associated broadening of focused analytes were performed to quantify performance and determine the dominant sources of variability. Our results suggest that nonuniform broadening of the pH gradient and changes in the pH gradient linearity stem from conductivity nonuniformities in the separation channel and not diffusion-associated band broadening during mobilization.

To measure protein isoforms in individual mammalian cells, we report single-cell resolution isoelectric focusing (scIEF) and high-selectivity immunoprobing. Microfluidic design and photoactivatable materials establish the tunable pH gradients required by IEF and precisely control the transport and handling of each 17-pL cell lysate during analysis. The scIEF assay resolves protein isoforms with resolution down to a single-charge unit, including both endogenous cytoplasmic and nuclear proteins from individual mammalian cells.

The underlying physical properties of microfluidic tools have led to new biological insights through the development of microsystems that can manipulate, mimic and measure biology at a resolution that has not been possible with macroscale tools. Microsystems readily handle sub-microlitre volumes, precisely route predictable laminar fluid flows and match both perturbations and measurements to the length scales and timescales of biological systems. The advent of fabrication techniques that do not require highly specialized engineering facilities is fuelling the broad dissemination of microfluidic systems and their adaptation to specific biological questions. We describe how our understanding of molecular and cell biology is being and will continue to be advanced by precision microfluidic approaches and posit that microfluidic tools - in conjunction with advanced imaging, bioinformatics and molecular biology approaches - will transform biology into a precision science.