Geometry-induced injection dispersion in single-cell protein electrophoresis

Abstract

Arrays of microwells are widely used to isolate individual cells, facilitate high throughput cytometry assays, and ensure compatibility of those assays with whole-cell imaging. Microwell geometries have recently been utilized for handling and preparation of single-cell lysate, prior to single-cell protein electrophoresis. It is in the context of single-cell electrophoresis that we investigate the interplay of microwell geometry (circular, rectangular, triangular) and transport (diffusion, electromigration) on the subsequent performance of single-cell polyacrylamide gel electrophoresis (PAGE) for protein targets. We define and measure injector-induced dispersion during PAGE, and develop a numerical model of band broadening sources, experimentally validate the numerical model, and then identify operating conditions (characterized through the Peclet number, Pe) that lead to microwell-geometry induced losses in separation performance. With analysis of mammalian cells as a case study, we sought to understand at what Pe is the PAGE separation performance adversely sensitized to the microwell geometry. In developing design rules, we find that for the microwell geometries that are the most suitable for isolation of mammalian cells and moderate mass protein targets, the Pe is usually small enough (Pe < ∼20) to mitigate the effect of the microwell geometry on protein PAGE of single-cell lysate. In extreme cases where the largest mammalian cells are analyzed (Pe > ∼20), consideration of Pe suggests using a rectangular – and not the widely used circular – microwell geometry to maximize protein PAGE separation performance.