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

Abstract

We report the use of multiphoton-excited photochemistry to generate highly fluorescent products from hydroxyindoles fractionated in submicron capillary electrophoresis channels. In this approach, the near-infrared (750 nm) output from a modelocked titanium:sapphire laser is focused at the outlet of a 0.6-μm i.d. capillary, producing pulse intensities of ∼1012W cm−2within a femtoliter focal volume. Hydroxyindole molecules migrating through the outlet aperture of the capillary intersect the beam focus, where absorption of three to four photons (∼1.65 eV photon−1) initiates a photobleaching reaction. The resultant hydroxyindole photoproducts produce broadband visible emission (λmax≈ 500 nm) when excited with two additional near-IR photons and appear substantially more resistant to photobleaching than the parent hydroxyindoles. This multiphoton-induced conversion of analytes to hyperluminescent derivatives thus offers a more sensitive approach than UV fluorescence for detecting extremely small quantities of material. Mixtures of the hydroxyindoles serotonin (5-hydroxytryptamine), 5-hydroxytryptophan, and 5-hydroxyindole acetic acid are reliably characterized (relative error ≈10%) in 100 s, with detection limits as low as ∼70 zmol (∼42,000 molecules). The sensitivity of this measurement strategy improves on the best previously reported results for capillary separations of indoles by more than one order of magnitude.