High‐sensitivity detection of biological amines using fast Hadamard transform CE coupled with photolytic optical gating

Abstract

Here, we report the first utilization of Hadamard transform CE (HTCE), a high-sensitivity, multiplexed CE technique, with photolytic optical gating sample injection of caged fluorescent labels for the detection of biologically important amines. Previous implementations of HTCE have relied upon photobleaching optical gating sample injection of fluorescent dyes. Photolysis of caged fluorescent labels reduces the fluorescence background, providing marked enhancements in sensitivity compared to photobleaching. Application of fast Hadamard transform CE (fHTCE) for fluorescein-based dyes yields a ten-fold higher sensitivity for photolytic injections compared to photobleaching injections, due primarily to the reduced fluorescent background provided by caged fluorescent dyes. Detection limits as low as 5 pM (ca. 18 molecules per injection event) were obtained with on-column LIF detection using fHTCE in less than 25 s, with the capacity for continuous, online separations. Detection limits for glutamate and aspartate below 150 pM (1-2 amol/injection event) were obtained using photolytic sample injection, with separation efficiencies exceeding 1 x 10(6) plates/m and total multiplexed separation times as low as 8 s. These results strongly support the feasibility of this approach for high-sensitivity dynamic chemical monitoring applications.

Review of recent advances in analytical techniques for the determination of neurotransmitters

“…[26, 27] The LODs reported by these researchers are comparable to those found with LIF. The purpose of many of these techniques was to develop new separations with improved LODs[15, 16, 23] or new, and more efficient, fluorophores. [11, 17, 19] These new fluorophores were 6-oxy-(N-succinimidyl acetate)9-( 2′-methoxycarbonyl) fluorescein,[11] o-phthaldialdehyde reacted with 5-((2-(and-3)-S-(acetylmercapto)succinoyl)amino)fluorescein,[17] and N-Hydroxysuccinimidyl fluorescein-O-acetate.…”

Hematopoietic stem cells, hematopoiesis and disease: lessons from the zebrafish model

“…Caged fluorescein dye, which changes color in response to a laser pulse, can be injected into embryos [61-63]. Laser activation of single cells, or groups of cells, allows the tracking of individual cell derivation over time.…”

Online photolytic optical gating of caged fluorophores in capillary zone electrophoresis utilizing an ultraviolet light‐emitting diode

“…Photolytic optical gating (POG) circumvents these issues by utilizing caged fluorophores that exhibit markedly decreased fluorescence quantum yields prior to photolysis. Samples are injected upon transient exposure to UV radiation, via photolytic uncaging of the fluorophore to generate highly fluorescent sample plugs . POG offers increased sensitivity compared to photobleaching by providing lower background signal and reduced noise .…”