ANALmcA CHIMICA zyxwvutsr ACTA zyxwvutsrqpon ELSEVIER Analytica Chimica Acta 313 (1995) 131-137 Sensitivity enhancement of fluorescent pH indicators using pH-dependent energy transfer Gabriella Gabor ‘, Suneet Chadha, David R. Walt * Max Tishler Laboratory for Organic Chemistry, Department of Chemishy, Tufts University, 62 Talbot Ave., Medford, u4 02155, USA Received 9 February 1995; revised 3 May 1995; accepted 3 May 1995 Abstract The concept of energy transfer is examined, in view of its application to enhance pH sensitivity of fluorescent dyes. Two fluorescent pH sensitive indicators are chosen such that they have nearly the same pK, values and have maximum spectral overlap between the emission spectrum of the donor and the excitation spectrum of the acceptor. In this work we studied the pH dependent energy transfer between 7-hydroxy-4-methylcoumarin-3-acetic acid (HCA) as the donor with fluorescein and 5- (and 6-j carboxy-4’,5’-dimethyl fluorescein (CDF) as the acceptors. The extent of energy transfer was measured experimentally by following the increase in acceptor emission at different pH values. The sensitivity of the acceptor dye is almost double when excited via energy transfer, provided the donor emission itself exhibits a similar dependence on pH. The pH sensitivity of fluorescein (AZ/ApH), was observed to increase from 0.28 when excited directly, to 0.71 when excited via energy transfer. CDF also shows a similar increase from 0.49 to 0.71. Keywords: Energy transfer; Fluorimetry; Dyes; Sensitivity enhancement 1. Introduction Fiber optic chemical sensors have led to renewed interest in indicator chemistry. The small size of optical fibers, insensitivity to electrical interference and lack of the need for a reference sensor make these devices potentially suitable for in situ applica- tions [l-6]. Indicators that are sensitive to analyte concentrations and exhibit changes in absorbance or luminescence form the basis of such sensors. Sensor fabrication involves immobilizing the active indica- tor on the tip of the optical fibers. * Corresponding author. ’ Present address: Israel Institute for Biological Research, Ness Ziona 70451, Israel. A number of optical sensors for pH have been described and are based on different chemical trans- duction mechanisms and optical principles [7]. Most pH indicators possess a limited dynamic range, therefore multiple indicators must be employed to obtain sensors that cover a wider pH range [S-lo]. Fluorescent indicators are typically weak acid dyes whose dissociated and undissociated forms have dif- ferent absorption or fluorescent properties in the pH range of interest. In many cases fluorescence occurs only from the excited state of the base form, e.g. fluorescein and certain coumarins [ll-131. Some indicators, such as hydroxypyrene trisulfonic acid (HITS), are fluorescent in both acid and base forms 1141 allowing pH changes to be followed by measur- ing the relative emission of both forms rather than 0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIOOO3-2670(95)00248-O