In the past decade, capillary electrophoresis has demonstrated increasing utility for the quantitative analysis of single cells. New applications for the analysis of dynamic cellular properties demand sampling methods with sufficient temporal resolution to accurately measure these processes. In particular, intracellular signaling pathways involving many enzymes can be modulated on subsecond time scales. We have developed a technique to rapidly lyse an adherent mammalian cell using a single electrical pulse followed by efficient loading of the cellular contents into a capillary. Microfabricated electrodes were designed to create a maximum voltage drop across the flattened cell's plasma membrane at a minimum interelectrode voltage. The influence of the interelectrode distance, pulse duration, and pulse strength on the rate of cell lysis was determined. The ability to rapidly lyse a cell and collect and separate the cellular contents was demonstrated by loading cells with Oregon Green and two isomers of carboxyfluorescein. All three fluorophores were detected with a separation efficiency comparable to that of standards. Parallel comparison of electrical lysis to that produced by a laser-based lysis system revealed that the sampling efficiencies of the two techniques were comparable. Rapid cell lysis by an electrical pulse may increase the application of capillary electrophoresis to the study of cellular dynamics requiring fast sampling times.

Pteridines are a class of compounds excreted in urine, the levels of which are found to elevate significantly in tumor-related diseases. For the first time, we have developed a method, based on high-performance capillary electrophoresis (HPCE) and laser-induced fluorescence (LIF) detection, to monitor the pteridine levels in urine. HPCE provides better separation than high-performance liquid chromatography and the LIF detector enables us to detect minute amounts of pteridines in body fluid. Eight different pteridine derivatives were well separated in 0.1 M Tris-0.1 M borate-2 mM EDTA buffer (pH 8.75) using a 60-cm fused-silica capillary (50-micron i.d., 35-cm effective length), six of which were detected and characterized in urine samples from normal persons and different cancer patients. The detection limits of these pteridines are under 1 x 10(-10) M. The levels of neopterin, pterine, xanthopterin, and pterin-6-carboxylic acid were found to be significantly elevated in urine excreted by cancer patents, while the level of isoxanthopterin dropped in these patients. No significant change of biopterin level was found between healthy individuals and cancer patients. This method can be used in clinical laboratories either for cancer monitoring or for precancer screening.

The viscosities of some polymer solutions for DNA separation in capillary electrophoresis are generally very high, which makes them hard to pump into the capillaries. We have developed a novel sieving buffer, based on low-molecular-weight hydroxypropylmethylcellulose, to separate DNA fragments. The viscosity of this sieving matrix was at least 1 order of magnitude lower than that of traditional buffers with similar sieving effect. The influence of additives such as urea and mannitol was investigated. It was found that the double-stranded DNA (ds DNA) fragments began to denature in 3.5 M urea, and 7 M urea can denature the ds DNA completely. The presence of mannitol will decrease the overlap threshold of the polymer solution (the concentration at which the polymer molecules begin to entangle with each other), which makes it possible to separate DNA fragments in a polymer solution of relatively low concentration. The influence of the electrical field was also investigated, and it was found that the mobility of DNA fragments up to 2000 bp in length did not change greatly with different electric fields. This phenomenon implies that the DNA fragments at this range do not change their conformation with the increase of electric field as was previously believed. The possible mechanism for the separation of DNA fragments is also discussed.