Single cell analysis by capillary electrophoresis with laser-induced native fluorescence detection

Abstract

<p>Individual mammalian cells were analyzed by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). This technique was chosen due to its high separation efficiencies, small sample volumes and sensitive detection. Native fluorescence was used, in which the analyte was not tagged with a fluorophore. 275-nm excitation gave attomole (amol = 10-18 mol) detection limits for the intracellular species of interest. Two projects are described in which hemoglobin (Hb) variants were determined in single red blood cells. In the third experiment, individual mast cells were degranulated on-column, and exocytosis and serotonin release were monitored temporally;First, single red blood cells, in which Hb molecules exist in their native, tetrameric states were analyzed. Upon injection and lysis of a cell, the tetramers were dissociated on-column into their respective polypeptide chains, separated and detected. Adult (normal and elevated A1) and fetal erythrocytes were analyzed. The amounts of glycated and total Hb were found to be uncorrelated;Second, an injection-based capillary isoelectric focusing technique was developed to separate Hb variants in single cells. Using dilute buffer conditions, the limit of detection (LOD) for Hb was 4 amol. In addition, a linear pH gradient was established along the capillary, which allowed variants differing by as little as 0.025 pI units to be resolved. The identification of variants with unknown pI values was also possible with this system;Third, the temporal evolution of on-column exocytotic release of serotonin from individual rat peritoneal mast cells (RPMCs) was monitored. The LOD for serotonin was 1.7 amol (S/N = 3; rms) with this system. The secretagogue was Polymyxin B sulfate, and was electromigrated into the capillary following injection of a single RPMC. Degranulation was induced and serotonin was released, the time courses of which were registered in the electropherograms. Following release, SDS was injected into the capillary to lyse the cell completely and to determine residual serotonin. With this procedure, events that are consistent with released serotonin from single sub-micron granules (250 aL each) were evident, which, to our knowledge, represent the smallest entities that have been analyzed with CE to date.</p>