Chemical cytometry capillary electrophoresis

Chemical Cytometry Capillary Electrophoresis Analysis at the Level of the Single Cell... 

Chemical Cytometry oe Proteins, Biogenic Amines, and Metabolic Cascades by One-and Two-Dimensional Capillary Electrophoresis... 

We have developed two-dimensional CE systems for the characterization of proteins and biogenic amines. The use of this technology for chemical cytometry is similar to the use of onedimensional electrophoresis a cell is aspirated into the column, lysed, and its components labeled with FQ. For two-dimensional electrophoresis, components are separated based on CSE in the first-dimension capillary. Fractions are then transferred across an interface to a second capillary, where they undergo additional separation based on micellar electrokinetic chromatography (MECC) before detection by fluorescence. The voltage drop across the first capillary is set to zero during the second dimension separation, holding components stationary. In a typical experiment, 300 fractions are transferred between capillaries under computer control. 

The term of chemical cytometry was introduced by Dovichi and coworkers [ 1 ] in 2000. However the approach of micro-separation of single-cell contents has been known for half a century. Chemical cytometry, developed intensively along with fast development of capillary electrophoresis (CE) from late 1980s to early 1990s, is a term defined as the analysis of the chemical composition of individual cells. The general conceptual mode of chemical cytometiy includes the following steps [2] (i) cell sorting, capture, and injection into a capillary, (ii) ceU lysis inside the capillary, and (iii) separation of molecules of interest by CE and their detection by fluorescence or other means. 

In order to achieve a more comprehensive analysis of individual cells, methods that incorporate chemical separations have been developed to increase the number of parameters capable of being studied simultaneously. These techniques, termed chemical cytometry by Dovichi and coworkers [2], are used to detect and identify various components of single cells such as oligonucleotides, small molecules, and proteins, as well as to monitor enzyme activity. Capillary electrophoresis (CE), laser-induced fluorescence (LIF), and electrochemistry are the tools which are used in chemical cytometry this entry deals with capillary electrophoresis implemented in microfluidic devices. 

While an array of analytical techniques has been developed to study various aspects of cells (please see Cell Assays in Microfluidics), this entiy is focused on chemical cytometry. As mentioned above, chemical cytometry refers to methods in which intracellular constituents of a single cell are analyzed by means of a chemical separation. Such methods typically employ capillary electrophoresis for separations, combined with laser-induced fluorescence (LIF) or amperometry for detection. We refer readers to other entries for detailed descriptions of other methods used to evaluate the contents of cells ( Patch Clamp Measurements On-Chip, Mass Spectrometry on a Chip). 

In capillary-based chemical cytometry, a cell is injected into a capillary, where it is lysed, and then its contents are separated by electrophoresis. The separating contents are detected at or near the end of the capillary, as the analytes migrate past the detection zone. Microfluidic devices are well suited to replace capillaries for chemical cytometry. The channel dimensions (5-100 pm) and planar geometry allow for very efficient dissipation of Joule heat produced from large electric field gradients. The capacity to apply high fields (e.g., 500-1,000 V/cm) dramatically reduces separation time and minimizes band diffusion. Most importantly, the small channel dimensions can handle injection volumes ranging from nanoliters to himdreds of femtoliters [18] and thus result in minimal dilution of sample. 

Chemical cytometry is a highly specialized class of analytical techniques in which the contents of individual biological cells are evaluated by chemical separations (e.g., capillary electrophoresis). In contrast to the more conventional technique of flow cytometry, in which intact cells are evaluated for the presence of one or a few markers, in chemical c)flometry, each ceU to be analyzed is lysed, such that (theoretically) all of the chemical constituents can be detected, identified, and quantified. 

---