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A sensitive fluorescence detection system with an Hg-lamp as the excitation source and a photon counter as the detector for microchip CE (MCE) has been developed. O-Phthaldialdehyde (OPA, lambda(ex) = 340 nm) was employed to label the catecholamine neurotransmitters such as dopamine (DA), norepinephrine (NE), and amino acid neurotransmitters including alanine (Ala), taurine (Tau), glycine (Gly), glutamic acid (Glu), and aspartic acid (Asp). The separation of seven derivatized neurotransmitters was successfully performed in MCE and the detection limits (S/N = 3) for DA, NE, Ala, Tau, Gly, Glu, and Asp were 0.85, 0.49, 0.23, 0.15, 0.13, 0.18, and 0.29 fmol, respectively. The system was then successfully applied for separation and determination of neurotransmitters in rat pheochromocytoma (PC 12) cells, and the average amounts of analyte per cell from a cell population were 2.5 fmol for DA, 3.3 fmol for Ala, 8.2 fmol for Tau, 4.0 fmol for Gly, and 1.9 fmol for Glu, respectively. By single-cell injection mode, electrophoresis separation and quantitative measurement of Glu in individual PC 12 cells was obtained. The average value of Glu per cell from single PC 12 cells analysis was found to be 3.5 +/- 3.1 fmol.

A rapid and sensitive method was developed for the analysis of amino acids by microchip electrophoresis with Hg-lamp excitation fluorescence detection. Fluorescein-isothiocyanate (FITC) was chosen to estimate the sensitivity of this system, and the detection limit (S/N = 3) with FITC was 1.7 nM, which showed that the system was sensitive as well as simple. Two derivatizing agents, FITC and ortho-phthalaldehyde (OPA) were employed to label amino acids and were compared in the same fluorescence detection system with an Hg lamp as the excitation source. The separation parameters were optimized in detail. Optimum separation of OPA-labeled amino acids was obtained in less than 200 s with 20 mM borate buffer (pH 9.0) containing 20% acetonitrile and 10 mM beta-cyclodextrin. Detection limits for amino acids (alanine (Ala), taurine (Tau), glycine (Gly), glutamic acid (Glu), and aspartic acid (Asp)) of 0.38-1.0 muM were achieved. The method was successfully applied to analysis of amino acids in human vascular endothelial cells (ECV-304). The average amount of amino acids in single ECV-304 cells is estimated to be 5.84 fmol for Ala, 2.78 fmol for Tau, 1.15 fmol for Gly, 3.10 fmol for Glu, and 1.30 fmol for Asp.

A method for monitoring the biological exocytotic phenomena on a microfluidic system was proposed. A microfluidic device coupled with functionalities of fluorescence imaging and amperometric detection has been developed to enable the real-time monitoring of the exocytotic events. Exocytotic release of single SH-SY5Y neuroblastoma cells was studied. By staining the cells located on integrated microelectrodes with naphthalene-2,3-dicarboxaldehyde, punctuate fluorescence consistent with localization of neurotransmitters stored in vesicles was obtained. The stimulated exocytotic release was successfully observed at the surface of SH-SY5Y cells without refitting the commercial inverted fluorescence microscope. Spatially and temporally resolved exocytotic events from single cells on a microfluidic device were visualized in real time using fluorescence microscopy and were amperometrically recorded by the electrochemical system simultaneously. This coupled technique is simple and is hoped to provide new insights into the mechanisms responsible for the kinetics of exocytosis.