Determination of neurotransmitters in PC 12 cells by microchip electrophoresis with fluorescence detection.
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.
Shi
Baoxian
B
College of Chemistry and Molecular Science Wuhan University, Wuhan, PR China.
Huang
Weihua
W
Cheng
Jieke
J
D000818:Animals; D001923:Brain Chemistry; D002457:Cell Extracts; D004044:Dietary Proteins / Q000032:analysis; D004298:Dopamine / Q000032:analysis; D046209:Electrophoresis, Microchip / Q000295:instrumentation / Q000379:methods*; D018846:Excitatory Amino Acids / Q000032:analysis*; D005453:Fluorescence; D008856:Microscopy, Fluorescence; D018377:Neurotransmitter Agents / Q000032:analysis*; D009638:Norepinephrine / Q000032:analysis; D016716:PC12 Cells; D051381:Rats; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D002457:Cell Extracts; D004044:Dietary Proteins; D018846:Excitatory Amino Acids; D018377:Neurotransmitter Agents; C012551:single cell proteins; D004298:Dopamine; D009638:Norepinephrine