Fiber-Optic Time-Resolved Fluorescence Sensor for in Vitro Serotonin Determination STEPHANE MOTrlN,* CANH TRAN-MINH, PIERRE LAPORTE, RAYMOND CESPUGLIO, and MICHEL JOUVET Laboratoire de Biotechnologie, Ecole Nationale Superi6ure des Mines de St-Etienne, F-42023 St-Etienne Cedex 2, France (S.M., C.T.); Laboratoire T.S.I., URA CNRS 842, Universitd Jean Monnet, F-42023 St-Etienne Cedex 2, France (P.L.); and D6partement de Mgdecine Expgrimentale, INSERM U 52, Universit6 Claude Bernard, F-69373 Lyon Cedex 08, France (R.C., M.J.). At pH 7 and with the excitation at wavelengths above 315 nm, previously unreported fluorescence of 5-HT (5-hydroxytryptamine) is observed. Two fluorescence bands were observed for 5-HT; the first emits at around 390 nm with an associated lifetime near 1 ns, and the other (well known) emits at 340 nm with an associated lifetime of 2.7 ns. With both static and time-resolved fluorescences, the spectral and temporal effects of the excitation wavelength were studied between 285 and 340 nm. With these basic spectroscopic properties as a starting point, a fiber-optic chemical sensor (FOCS) was developed in order to measure 5-HT with a single- fiber configuration, nitrogen laser excitation, and fast digitizing tech- niques. Temporal effects including fluorescence of the optical fiber were studied and compared with measurements both directly in cuvette and through the fiber-optic sensor. Less than thirty seconds are required for each measurement. A detection limit of 5-HT is reached in the range of 5 ~M. Our system, with an improved sensitivity, could therefore be a possible and convenient "tool" for in vivo determination of 5-HT. Index Headings: Spectroscopic techniques; Fluorescence; Time-resolved spectroscopy; Serotonin; Optical fluorometric sensors. INTRODUCTION Historically different approaches (anatomical, neuro- physiological, and pharmacological) have helped to es- tablish a relationship between the level of brain serotonin (5-hydroxytryptamine or 5-HT) and the alternation of the different states of vigilance. New techniques have offered the possibility of studying the activity of sero- toninergic neurons throughout the sleep-wake cycle. 1 Methods of measuring 5-HT or 5-HIAA (5-hydroxyin- doleacetic acid) levels in vivo, which allow the investi- gation of its release and metabolism, are divided into two categories, voltammetry and dialysis (push-pull perfu- sion). 2-4 In vivo voltammetry is associated with good an- atomical resolution because of the small electrode sizes. Compound specificity, however, is the major limitation of the voltammetric technique) Dialysis is also associ- ated with advantages and limitations. Dialysis allows a greater specificity of compound detection, but the thick- ness Of the dialysis probe limits anatomical resolution. In addition, the dialysis probe has a relatively short "functional" life in vivo, and the necessity for long time intervals between successive measurements limits the association of compound release with specific behavioral events. 6 Received 14 October 1992; revision receiVed 17 December 1992. * Author to whom correspondence should be sent. These limitations lead us to search for an alternative in vivo monitoring technique which would involve a min- iature sensor characterized by rapidity, specificity, and sensitivity of measurements. In this paper, we present the feasibility of in vitro rapid determination of 5-HT (less than 30 seconds) by a fiber-optic chemical sensor (FOCS). The single-fiber configuration is preferred, rath- er than the double-fiber or multiple-fiber configuration, despite an increase in white noise associated with single fibersY In addition, the injection and collection of light complicate the setup. For the possible extension to an in vivo sensor, the interactions between the sensor and media must be the simplest in order to miniaturize, ster- ilize, and minimize problems of incompatibility of ma- terials Such as the resins used for the attachment of two fibers. Detection limit sensitivity depends on experi- mental parameters. These parameters are critical for the magnitude of the fluorescence noise and the efficiency of the single-fiber optical coupler configuration. Specificity was investigated, and the signal produced by 5-HIAA was also characterized. Relationships between results and choice of instrumentation are discussed. One such choice was the use of the time-resolved flu- orescence technique. Such a method has been widely used in fields as diverse as molecular biology, polymer science, and solid-state physics, s In most cases, the aim is to provide only qualitative measurements. Neverthe- less, quantitative methods appear necessary, especially when the steady-state fluorescence intensity is not suf- ficient. 9-11 Two approaches for quantitative applications have emerged. The first approach uses a fiber optic with the construction of an extrinsic time-resolved fluores- cence optrode for remote sensing 12 and a semi-intrinsic optrode for detection of chemical compounds which have no fluorescence. 13,14 The second approach involves the field of time-resolved fluorescence instrumentation in or- der to obtain a rapid and simplified technique. 1~,~6 Photophysical properties of 5-HT are mainly due to the 5-hydroxyindole chromophore. In order to selectively measure 5-HT by a remote sensor using optical fibers, we examined specific photophysical properties for this group with regard to indole compounds (e.g., tryptophan) and especially between 5-HT and 5-HIAA within the group of 5-hydroxyindolic compounds. Some differences in UV absorbance are found in polar solvents between simple indoles and indoles which are substituted in the 590 Volume 47, Number 5, 1993 0003-7028/93/4705-059052.00/0 APPLIED SPECTROSCOPY © 1993 Society for Applied Spectroscopy