Measurements of Serotonin and Related Indoles Using Capillary Electrophoresis with Multiphoton-Induced Hyperluminescence

Gostkowski, Michael L.; Wei, Jing; Shear, Jason B.

doi:10.1006/abio.1998.2723

Cited by 26 publications

(31 citation statements)

References 22 publications

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“…Mirror-tilt adjustments are used to align and separate the focal spots. By using this optical system, multiphoton focal volumes Ͻ1 m 3 can be produced (10). Visible fluorescence from hydroxyindole photoproducts is epi-collected by using the ϫ100 objective, directed back along the laser-propagation path by the 90͞10 beamsplitter, and ref lected toward a bialkali photomultiplier tube (HC-125, Hamamatsu, Middlesex, NJ) by the dichroic mirror.…”

Section: Methodsmentioning

confidence: 99%

“…Molecules undergoing photoalteration were rendered undetectable, allowing fluorescent peaks to be detected on a low background. Studies on multiphoton-excited photoreactions of biological molecules (9)(10)(11), in which thermally labile fluorescent photoproducts are generated from nonfluorescent reactants, suggested that optical gating could be a useful means for producing plugs of evolving chemical mixtures for electrophoretic analysis. Note that in this case optical gating creates rather than destroys fluorescent molecules.…”

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confidence: 99%

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Microsecond electrophoresis

Plenert

Shear

2003

Proc. Natl. Acad. Sci. U.S.A.

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Although analysis strategies exist for probing a diverse array of molecular properties, most of these approaches are not amenable to the study of reaction intermediates and other transient species. Separations in particular can provide detailed information on attributes not readily measured by spectroscopy but typically are performed over time scales much longer than the life span of highly unstable compounds. Here we report the development of an electrophoretic strategy that dramatically extends the practical speed limit for fractionations and demonstrate its utility in examining transient hydroxyindole photoproducts. Fluorescent reaction intermediates are optically generated in femtoliter volumes within a flowing reagent stream and are differentially transported at velocities as large as 1.3 m⅐s ؊1 , thereby minimizing band variance and allowing multicomponent reaction mixtures to be resolved over separation paths as short as 9 m. Analyte migration times and band variances do not deviate significantly from basic theory for separations performed with fields that exceed 0.1 MV⅐cm ؊1 , indicating that effects from Joule heating are minor. We demonstrate the feasibility of achieving baseline resolution of a binary mixture in <10 s, nearly 100-fold faster than previously possible. Application of this approach to the study of a range of short-lived molecules should be feasible.A lthough of tremendous value, time-resolved spectroscopy ultimately is limited in its ability to probe transient solutionphase molecules. Accurate interpretation of spectroscopic data can be problematic when spectral features are broad or reaction environments contain multiple chemical species. Measurements of molecular transport behavior in defined fields or chromatographic flow environments can offer insights into properties not readily probed by spectroscopy alone and in many cases can be used to derive information on a large number of components within complex sample mixtures. Unfortunately, because such separation procedures typically require minutes or longer to complete, they have not been suitable for studies of highly unstable molecules.The speed of a separation is limited by the time required to transport a species of interest over a distance sufficiently long to isolate it from other detectable components. In chromatographic separations, the velocity of an analyte typically is restricted by its transfer rates between phases, with plate heights becoming prohibitively large under high flow-rate conditions. In contrast, capillary electrophoresis (CE) is not constrained by partitioning or adsorption-desorption kinetics and, consequently, has been adapted for analyses on low-and subsecond time scales (1-6). In CE, the migration time (t) of an analyte scales as the separation distance (L) and inversely with the applied field (E),where is the sum of the electrophoretic and electroosmotic mobilities. Under ideal circumstances in which diffusion is the sole determinant of spatial bandwidths, Jorgenson and Lukacs (7) showed that the number o...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Microsecond electrophoresis

Plenert

Shear

2003

Proc. Natl. Acad. Sci. U.S.A.

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“…Secondly, both 5HT and serotonin are induced to produce an unusual and characteristic green, ca. 500 nm, luminescence ('hyperluminescence') on multiphoton excitation using sub-picosecond near infrared laser pulses [11][12][13][14][15]. Shear et al [11] have shown that overall this is a 6-photon process, involving an initial 4-photon photochemical reaction leading to a transient product with a lifetime of hundreds of microseconds followed by two-photon excitation of the intermediate to produce the green fluorescence.…”

Section: Introductionmentioning

confidence: 99%

Identification and reactivity of the triplet excited state of 5-hydroxytryptophan

Dad¹,

Bisby²,

Clark³

et al. 2005

Journal of Photochemistry and Photobiology B: Biology

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Both the neurotransmitter serotonin and the unnatural amino acid 5-hydroxytryptophan (5HT), contain the 5-hydroxyindole chromophore. The photochemistry of 5HT is being investigated in relation to the multiphoton excitation of this chromophore to produce a characteristic photoproduct with green fluorescence ('hyperluminescence'). Laser flash photolysis (308 nm) of 5HT in aqueous solution at neutral pH produces both the neutral 5-indoloxyl radical (λ max 400-420 nm) and another transient absorption with λ max 480 nm and lifetime of 2 μs in deaerated solutions. Based on quenching by oxygen and β-carotene, the species at 480 nm is identified as the triplet excited state of 5HT. In acidic solution a new oxygen-insensitive intermediate with λ max 460 is assigned to the radical cation of 5HT. Time-resolved measurements of luminescence at 1270 nm have shown that the triplet state of 5HT is able to react with oxygen to form singlet excited oxygen ( 1 O 2 *) with a quantum yield of ∼0.1. However, 5HT has also been found to be an effective quencher of singlet oxygen with a second order rate constant of 1.3 × 10 8 dm 3 mol -1 s -1 . The results are discussed in the light of recent observations on the multiphoton-excited photochemistry of serotonin.

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“…After EOM analysis, the gel was silver stained to reveal the protein patterns of the cellular extracts (lanes [11][12][13][14][15][16][17]. Even with the high protein loads used in this study (ϳ100 g/sample), we observed sharp silverstained bands throughout the gel.…”

Section: Resultsmentioning

confidence: 99%