Determination of fluorogen-labeled neurotransmitters at the zeptomole level using two photon excited fluorescence with capillary electrophoresis

Two-photon excited (2PE) fluorescence detection is demonstrated to be a highly sensitive means for analyzing fluorogen-labeled neurotransmitters fractionated in submicrometer capillary electrophoresis channels. In this approach, fluorescamine-labeled neurotransmitters that have been electrophoretically separated in 620-nm-i.d. capillaries intersect the focused output from a near-infrared mode-locked titanium-sapphire laser positioned at the capillary outlet. Extremely high peak, laser intensities (similar to 10(11)-10(12) W cm(-2)) facilitate the nearly simultaneous absorption of two near-IR photons (lambda(ex) approximate to 780 nm) to excite fluorescamine derivatives ordinarily excited with a single, near-ultraviolet photon (lambda(ex) approximate to 390 nm). Rapid cycling of analytes through the fluorescent excited state and low background from scatter and out-of-sample luminescence combine to make 2PE fluorescence a highly sensitive approach for detecting minute quantities of neurotransmitters. In these studies, mixtures of the fluorescamine derivatives of dopamine, glycine, and glutamate are fractionated reproducibly in several minutes, with instrumental mass detection limits as low as 13000 molecules (similar to 20 zmol). These detection levels are similar to 100 fold lower than have been achieved previously for fluorescamine-based assays. Analytes can be derivatized at concentrations equal to the limit of quantitation with no loss in sensitivity; hence, characterization of neuronal samples at the zeptomole level appears feasible, provided that efficient on-column labeling procedures can be implemented.