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Chemical analysis of single cells requires methods for quickly and quantitatively detecting a diverse array of analytes from extremely small volumes (femtoliters to nanoliters) with very high sensitivity and selectivity. Microelectrophoretic separations, using both traditional capillary electrophoresis and emerging microfluidic methods, are well suited for handling the unique size of single cells and limited numbers of intracellular molecules. Numerous analytes, ranging from small molecules such as amino acids and neurotransmitters to large proteins and subcellular organelles, have been quantified in single cells using microelectrophoretic separation techniques. Microseparation techniques, coupled to varying detection schemes including absorbance and fluorescence detection, electrochemical detection, and mass spectrometry, have allowed researchers to examine a number of processes inside single cells. This review also touches on a promising direction in single cell cytometry: the development of microfluidics for integrated cellular manipulation, chemical processing, and separation of cellular contents.

Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sphingosine and sphingosine-1-phosphate. The assay was demonstrated to be capable of determining the in vitro activity of both kinase and phosphatase using purified enzymes. The K M of sphingosine kinase for its fluorescently labeled substrate was 38 ± 18 μM with a V max of 0.4 ± 0.2 μM/min and a k cat of 3900 s −1 . Pharmacologic inhibition of sphingosine kinase in a concentration-dependent manner was also demonstrated. Moreover, the fluorescent substrate was shown to be readily taken up by mammalian cells making it possible to study the endogenous activity of sphingosine kinase activity in living cells. The method was readily adaptable to the use of either bulk cell lysates or very small numbers of intact cells. This new methodology provides enhancements over standard methods in sensitivity, quantification, and manpower for both in vitro and cell-based assays.The sphingolipids sphingosine and sphingosine-1-phosphate (S1P) play crucial roles as signal transduction molecules involved in cell survival and migration. [1][2][3][4][5][6] These second messengers along with the sphingolipid metabolite ceramide are interconvertible, and their dynamic equilibrium is believed to be a determining factor in whether cells will live or die. 7 In addition to its role as an intracellular second messenger, S1P also acts as an extracellular ligand making it a pleiotropic signaling molecule with wide-ranging function from calcium homeostasis to chemotaxis. 4,[7][8][9] S1P is produced by phosphorylation of sphingosine by sphingosine kinase 1 and 2 (SK1 and SK2) and is reversibly dephosphorylated by two known mammalian phosphatases SPP1 and SPP2. 6,[9][10][11] In addition, S1P can be irreversibly degraded by a pyridoxylphosphate-dependent S1P lyase to hexadecenal and phosphoethanolamine. 6,12 The balance and interplay of these metabolic pathways remain to be fully elucidated. SK1 is thought to be oncogenic, and inhibitors of SK1 appear to act as effective chemotherapeutic agents in animal studies. 7,10,13,14 SK2 is involved in the immune response, and compounds directed at extracellular S1P signaling are showing great promise in clinical trials for autoimmune diseases. 10,[15][16][17][18] Thus, sphingosine signaling is proving to be extremely important in clinical medicine. [17][18][19][20][21] *Corresponding authors. Phone: 919-966-2291 (C.E.S. and N.L.A.). Fax: 919-962-2388 (C.E.S. and N.L.A.). cesims@unc.edu (C.E.S.); nlallbri@unc.edu (N.L.A. Although S1P plays a major role as an extracellular signaling molecule, it is predominately syn...

In vivo determinations of amino acids are important for improving our understanding of physiological states of biological tissue function and dysfunction. However, the chemically complex matrix of different biological fluids complicates the assay of this important class of molecules. We introduce a method for characterizing the amino acid composition of submicroliter volumes of vitreous humor perfusates. Low-flow push-pull perfusion sampling is compatible with collecting small volume samples in a complicated matrix that are potentially difficult to separate. An efficient, sensitive, and rapid analysis of amino acids from in vivo perfusates of the vitreous is presented with 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde (CBQCA) derivatitation and capillary electrophoresis (CE) separation with laser-induced fluorescence detection (LIF). Derivatization with CBQCA for up to 2 h provided high sensitivity and low detection limits at the nM level. Seventeen amino acids including D-serine (D-Ser) and D-aspartate (D-Asp) were resolved in less than 10 min. Importantly, D-Ser is separated from its enantiomeric pair. Characterization of vitreal amino acids with this assay technique will be useful for understanding ocular diseases and physiological mechanisms in vision.

A fast and simple method for the direct, simultaneous detection of nitrite (NO(2) (-)) and nitrate (NO(3) (-)) in rat striatum has been developed using a capillary electrophoresis separation of low-flow push-pull perfusion samples. The method was optimized primarily for nitrite because nitrite is more important physiologically and is found at lower levels than nitrate. We obtained a complete separation of NO(2) (-) and NO(3) (-) in rat striatum within 1.5 min. Optimal CE separations were achieved with 20 mM phosphate, 2 mM cetyltrimethylammonium chloride (CTAC) buffer at pH 3.5. The samples were injected electrokinetically for 2 s into a 40 cm x 75 microm ID fused-silica capillary. The separation voltage was 10 kV (negative polarity), and the injection voltage was 16 kV (negative polarity). UV detection was performed at 214 nm. The limits of detection obtained at a signal-to-noise ratio (S/N) of 3 for nitrite and nitrate were 0.96 and 2.86 microM. This is one of the fastest separations of nitrite and nitrate of a biological sample ever reported. Interference produced by the high physiological level of chloride is successfully minimized by use of CTAC in the run buffer.