Multiple sampling in single-cell enzyme assays using capillary electrophoresis with laser-induced fluorescence detection

Abstract

The single cell represents the basic unit of life and as such has become the focus of extensive research. Single-cell analysis is advantageous over conventional bulk cell methods as it allows complex and heterogeneous biological systems to be probed at their most basic level. The minimal sample size requirements also make it well suited to cell developmental studies, for confirming deleterious gene knockouts, or for any biomedical research where the amount of cells or tissue sample available for analysis is limited. This has led to a great deal of research devoted to chemical cytometry—the quantifying of analytes at the single-cell level [1, 2]. These analytes include drugs, neurotransmitters, amino acids, ions, carbohydrates, and peptides. There is also considerable interest in single-cell genomics and proteomics. Numerous techniques have been employed for single-cell analysis including flow cytometry, microfluidics, and capillary electrophoresis (CE) with electrochemical (ECD), mass spectrometry, or laser-induced fluorescence (LIF) detection. When studying enzymes, however, it is often more informative to determine enzyme activity rather than quantity by performing single-cell enzyme assays. Examining enzyme function at the single-cell level offers insights into complex and heterogeneous biochemical processes such as cell signaling pathways [3] and metabolism [4]. Single-cell enzyme assays typically use capillary electrophoresis with electrochemical or laser-induced fluorescence detection. CE-LIF and CE-ECD are well suited for single-cell analysis owing to their ultrasensitivity and small sample size requirements. Using CE-LIF, the conversion of a fluorescent substrate into fluorescent product is monitored by separating product from substrate molecules and then quantifying their relative abundances. There are many variations in single-cell enzyme assays, but in general two approaches are employed. In one approach individual cells are loaded on a CE capillary containing substrate for the enzyme of interest. The cell is lysed, releasing its enzymes, which are then free to interact with substrate. At any point after initiating the reaction, the CE voltage is applied and the amount of accumulated product is determined. This online approach is very sensitive since there is minimal dilution of cellular enzymes within the CE capillary. Using this approach the variation in lactate dehydrogenase activity in single erythrocytes has been determined [5]. A drawback to this method, however, is that the enzymatic reaction must occur in a CE-compatible buffer. The buffer requirements for CE are quite different to the native cellular environment and thus the measured activity may not reflect the enzyme’s true cellular activity. A second approach involves incubating or culturing cells in a medium that contains a fluorescent substrate. The substrate is taken into the cells and converted to product in a natural enzyme environment. Cells are then individually loaded onto a CE capillary. After cell lysis the relative amounts of substrate and product are determined by CE-LIF analysis. Experiments such as these also provide information regarding the cellular uptake of substrate, but are thus also limited to analyzing cell-permeable substrates or involve the use of techniques such as microinjection or electroporation to introduce substrate into the cell. This approach has been used to monitor the metabolism of a fluorescently labeled carbohydrate in individual cancer cells and correlate the observed metabolism with the cell cycle [4]. More recently, this method has been employed to examine the metabolism of a Ras-mimicking peptide by three different enzymes in single cells [6]. One limitation in both of these approaches is that the entire cell is consumed in the analysis. Thus only one measurement is possible, giving a snapshot of enzyme activity. Multiple sampling enhances the amount of information available form a single-cell study. For G. K. Shoemaker Department of Chemistry, University of Alberta, Edmonton, T6G 2G6, AB, Canada