Determination of intracellular species at the level of a single erythrocyte via capillary electrophoresis with direct and indirect fluorescence detection

Abstract

Intracellular contents reflect the specific history of a cell including innate physiological heterogeneity as well as differing levels of exposure to environmental influences. A method capable of analyzing a variety of species from within a single human erythrocyte is demonstrated. Guided by a microscope, individual cells can be drawn into open capillaries of 10-microns i.d. On contact with a low ionic strength buffer solution, the cell lyses and releases its intracellular fluid. The ionic components are then separated by capillary electrophoresis. For glutathione, microderivatization with a fluorescent reagent can be accomplished in vitro with monobromobimane. The effects of extracellular oxidizing and reducing agents on the glutathione levels can thus be followed. For sodium and potassium, or any other ionic species, charge displacement of a fluorescent cation results in indirect fluorescence detection. The two detection modes are suitable for intracellular components present at low-attomole and sub-femtomole levels, respectively.