Automated Capillary Electrophoresis System for Fast Single-Cell Analysis

Abstract

Capillary electrophoresis (CE) is a promising technique for single-cell analysis, but its use in biological studies has been limited by low throughput. This paper presents an automated platform employing microfabricated cell traps and a three-channel system for rapid buffer exchange for fast single-cell CE. Cells loaded with fluorescein and Oregon green were analyzed at a throughput of 3.5 cells/min with a resolution of 2.3 ± 0.6 for the fluorescein and Oregon green. Cellular protein kinase B (PKB) activity, as measured by immunofluorescence staining of phospho-PKB, was not altered, suggesting that this stress-activated kinase was not upregulated during the CE experiments and that basal cell physiology was not perturbed prior to cell lysis. The activity of sphingosine kinase (SK), which is often upregulated in cancer, was measured in leukemic cells by loading a sphingosine-fluorescein substrate into cells. Sphingosine fluorescein (SF), sphingosine-1-phosphate fluorescein (S1PF), and a third fluorescent species were identified in single cells. A single-cell throughput of 2.1 cells/min was achieved for 219 total cells. 88% of cells possessed upregulated SK activity, although subpopulations of cells with markedly different SK activity relative to that of the population average were readily identified. This system was capable of stable and reproducible separations of biological compounds in hundreds of adherent and nonadherent cells, enabling measurements of previously uncharacterized biological phenomena.

Single-cell sphingosine kinase activity measurements in primary leukemia

“…The three-channel system for physiologic buffer, air, and electrophoretic buffer was fabricated from PDMS (Figure S1 Electronic Supplementary Material) [33]. The channel for loading electrophoretic buffer was 3 cm in length, 0.5 cm in width, and 1.5 mm in depth.…”

“…Cells were lysed by adding 10 μL methanol and stored at −80°C until analysis. Lysate samples were diluted in electrophoretic buffer (27 mM monosodium phosphate and 10% 1-propanol at pH 7.3) and analyzed using a custom-built CE system, described previously [33]. The sample was separated in a 35 cm fused-silica capillary with an inner diameter of 30 μm.…”

Advances in Capillary Electrophoresis-Based Enzyme Assays

“…For example, a chip containing a channel system and a cell trap was fabricated that allowed transfer the content of a single trapped cell after lysis into a separation capillary [117][118][119]. The cells were lysed by a laser pulse and the content was injected electrokinetically into a 35 cm, 30 μm id, fusedsilica capillary.…”

Single cell kinase signaling assay using pinched flow coupled droplet microfluidics

“…This research encourages the use of different platforms that can isolate and assay the cells individually. Zarrine-Afsar and Krylov 6 and Dickinson et al 4 have previously reported capillary electrophoresis-based methods to monitor the activation of protein kinase A (PKA) and protein kinase B (PKB) in single cells. However, these methods involve either the cloning of cells to transiently express the GFP-tagged protein substrate or the use of immunofluorescence antibody staining to monitor kinase activity in trapped single cells.…”

“…The capillary electrophoresis system was automatized to analyze $3.5 cells/min. [4][5][6][7][8][9][10] Compared with these methods, the droplet based technology is attractive [11][12][13][14][15][16][17][18][19][20][21] because of its ultra high throughput cell encapsulations ($1000 droplets/s) [22][23][24] and accurate fluidic controls. Accordingly, a wide range of droplet base single cell polymerase chain reactions (PCR) were carried out in previous studies.…”