Manipulating ionic strength to improve single cell electrophoretic separations

Abstract

A capillary electrophoresis system with ultrasensitive two-color laser-induced fluorescence detection was used to probe the effect of ionic strength on single cell separations of glycosphingolipids. Differentiated PC12 cells were incubated with two ganglioside substrates tagged with different fluorophores within the BODIPY family such that two distinct metabolic patterns could be simultaneously monitored. Aspiration of single differentiated PC12 cells suspended in a phosphate-buffered saline solution showed excessive peak dispersion, poor resolution, and peak efficiencies below 100,000 theoretical plates. Aspiration of single differentiated PC12 cells suspended in deionized water corrected peak dispersion. Average peak efficiencies ranged between 400,000 and 600,000 theoretical plates. Improved performance was due to the dilution of the high salt concentrations inside of single neuronal-like cells to produce field amplified sample stacking. Single cell separations showed the highest resolution when aspiration of single differentiated PC12 cells suspended in deionized water were separated using a running buffer of high ionic strength. The improvement in resolution allowed for the identification of analytes not previously detected in single cell metabolism studies.

Design and Application of Sensors for Chemical Cytometry

“…However, this method of detection requires the design of a fluorescent reporter. 29–31 Electrochemical detection also achieves limits approaching single molecule detection. 32 However, this technique can only be used for electroactive species, and is prone to interference from electrode fouling by sample components.…”

“…These microseparation methods have been used to quantify organelle pH, proteins, peptides, lipids, gasotransmitters, small molecules and metal ions in single cells. 24–27, 29, 37–48 The method employs electrokinetic separation within a small-diameter, fused-silica capillary taking advantage of the differential mobility of analyte molecules under an applied electric field.. 5, 21 An asset of this method is that any modification to an analyte or reporter almost always results in a new mobility under appropriate separation conditions. Fluorescence detection is typically performed on-column by interrogating the capillary with a focused light source, generally a laser, and detecting emitted light with a photomultiplier tube (PMT) or photodiode.…”

“…84,85 These aspects represent unique opportunities for chemical cytometry, and present challenges that capillary electrophoresis with fluorescence detection (CE-F) is uniquely suited to tackle since CE-F offers exquisite separation capabilities, ultra-high sensitivity and dynamic range, and compatibility with both aqueous and organic buffer systems. 10, 29, 30, 43, 47, 85–90…”

Contemporary sample stacking in analytical electrophoresis

“…A FASS‐CE system with ultrasensitive two‐color LIF detection was used by Keithley et al. to probe the effect of ionic strength on single‐cell separations of glycosphingolipids. The BGE consisted of 100 mM Tris and 100 mM CHES with 20 mM SDS and 5 mM α‐CD.…”

Single Cell Manipulation Technology

“…Cell manipulation technology, which is applied to sort cells, separate cells, trap cells, isolate cells, move cells, recover cells and so on [9][10][11][12][13][14], combined with microfluidic technique make the ways of manipulation cells at single-cell-level more precision and convenient than ever before. According to different cell manipulation forces generation principles, cell manipulation technology can be divided into optical manipulation technique, magnetic manipulation technique, electrical manipulation technique, acoustic manipulation technique and mechanical manipulation technique.…”