In recent years, capillary electrophoresis (CE) has been one of rapidly growing analytical techniques to study affinity interactions. Quick analysis, high efficiency, high resolving power, low sample consumption, and wide range of possible analytes make CE an indispensable tool for studies of biomolecules and, in particular, studies of their interactions. In the article, we discuss kinetic methods in CE. The spectrum of proven applications of kinetic CE methods includes: (i) measuring equilibrium and rate constants of protein-ligand interaction from a single experiment, (ii) quantitative affinity analyses of proteins, (iii) measuring temperature in CE, (iv) studying thermochemistry of affinity interactions, and (v) kinetic selection of ligands from combinatorial libraries. We demonstrate that new kinetic CE method can serve as a "Swiss army knife" in the development and utilization of oligonucleotide aptamers. Uniquely, they can facilitate selection of smart aptamers - aptamers with pre-defined binding parameters. We believe that further development of kinetic CE methods will provide a variety of methodological schemes for high-throughput screening of combinatorial libraries for affinity probes and drug candidates using CE as a universal instrumental platform.
Molecular cytometry refers to ultrasensitive analysis tools that are used to separate and identify entire classes of molecules in single cells. Recently, we described two molecular cytometry methods to analyze proteins at the single cell level. The first one was based on capillary gel electrophoresis with sheath-flow cuvette laser-induced fluorescence (LIF). A vacuum pulse was employed to introduce a single HT29 human colon cancer cell into the capillary. Once the cell was lysed, proteins were denatured with SDS, labeled with 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ), and then separated according to their size by using pullulan as the sieving matrix. The second one was based on submicellar capillary electrophoresis with sheath-flow cuvette LIF. Once a single cell was introduced and lysed, the cellular proteins were labeled with FQ and then separated in a submicellar buffer. This method has been applied to analysis of proteins in a single HT29 human cancer cell as well as single-cell stage Caenorhabditis elegans embryo.
Capillary electrophoresis with laser-induced fluorescence detection has the potential to become an important tool in analyzing the chemical contents of single cells. Singe-cell studies are important for understanding the molecular mechanisms of many biological processes, including serious disorders, such as concern and neurodegradative diseases. We developed a new technique, Metabolic Cytometry, that allows us to study complex metabolic pathways in single cells. The essence of the technique is the following. A fluorescently labeled metabolic probe is introduce by incubation into a cell where it is converted to a number of fluorescently labeled metabolites. After incubation the cell is injected into the capillary and lysed. The metabolites are then separated and detected using CE-LIF. If metabolic cytometry is combined with image cytometry, then metabolic activities can be correlated with cellular phenotype or genotype. We applied this powerful approach to correlate glycosylation with the cell's phase in the cell cycle.