Applications of capillary electrophoresis and laser-induced fluorescence detection to the analysis of trace species: from single cells to single molecules

摘要

Several separation and detection schemes for the analysis of small volume and amount of samples, such as intracellular components and single enzymes, were developed in this work. Laser-induced fluorescence (LIF) detection provides a very sensitive approach for both direct and indirect detection in capillary electrophoresis (CE);First, indirect LIF detection and capillary electrophoresis were used to quantify lactate and pyruvate in single red blood cells. By choosing a highly efficient fluorophore and adding 1% glucose to the running buffer to stabilize the system, a detection limit of around 20 attomoles was achieved for small anions, which resulted in the easy quantification of targeted anions in single erythrocytes;The measurement of the activity for sub-attomole enzymes inside single red blood cells presents a high challenge. The assay of specific enzyme activities was achieved by monitoring the highly fluorescent enzymatic reaction product, NADH. By adding proper non-fluorescent substrates into the running buffer, the enzymes will catalyze one specific reaction after they are separated into different zones and the CE flow is stopped. The fluorescent products were related to enzyme activity. Consequently, the enzyme activity can be quantified by monitoring the fluorescent product. At about biological pH 7.4, lactate dehydrogenase (LDH) isoenzyme activities were assayed for single red blood cells. A detection limit of 1.3 x 10[superscript]-21 moles for lactate dehydrogenase was achieved by the combination of on-capillary reaction and electrophoresis. The present approach is also applicable to the assay of multiple enzymes by introducing appropriate substrates. Since lactate dehydrogenase activity serves as a good marker for certain diseases, the ability to quantify individual isoenzymes at the single cell level is of clinical importance. Leukemia cells were analyzed to evaluate the value of LDH activity as a marker for the diagnosis of leukemia. From the single cell analysis, we found that LDH activity is not a unique marker for diagnosis of leukemia, although the LDH activity in leukemia cells is lower than that in normal white blood cells;Reactions of single LDH-1 molecules were investigated by monitoring the reaction product with LIF detection. By filling a narrow capillary tube with a very low concentration of LDH-1 and excess lactate and NAD[superscript]+, discrete product zones of NADH associated with individual LDH-1 molecules are formed. We can quantify molecular concentrations down to 10[superscript]-17 M, and can also measure their activities. From the products formed during two consecutive incubation periods, each LDH-1 molecule maintains the same distinct activity over a 2-hour period. We found that the same kind of enzyme molecules can have different activities, which vary in a factor of 4. The differences in activity might be caused by different stable conformation of LDH-1 enzymes.