Development of a hydroxyl radical ratiometric nanoprobe Matt King * , Raoul Kopelman Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA Accepted 6 December 2002 Abstract A hydroxyl radical nanoprobe has been developed by covalently attaching coumarin-3-carboxylic acid (CCA) to amine-functionalized polyacrylamide (AFPA) nanoparticles (40–100 nm in size). CCA is a non-fluorescent aromatic compound that reacts with hydroxyl radical (  OH) to produce a fluorescent product, 7-hydroxy-CCA (7-OH-CCA). Texas Red-Dextran (a reference dye) is encapsulated within the nanoprobe matrix to allow for ratiometric measurements to be made that correct for variations in source intensity, sample and instrument geometry and dilution. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Nanoprobe; PEBBLE; Hydroxyl radical 1. Introduction The hydroxyl radical (  OH) is one of the most reactive and shortest lived of the reactive oxygen species (ROS) [1]. The ROS include superoxide, singlet oxygen, nitric oxide, hydrogen peroxide and peroxynitrite. The lifetime of  OH in biological systems is believed to be about 1 ns [2]. Because of this, methods used to detect  OH have often been indirect, using markers such as lipid peroxidation, protein oxidation and the hydroxylation of DNA bases [1]. Other methods include ESR [5] (using a spin trap such as DMSO), HPLC [6] and fluorescence [3,4,7]. Two different methods can be used for the detection of  OH. One is the direct reaction of a probe molecule with  OH. The other method is to use a scavenger that creates a radical species with a longer life- time. The probe molecule then reacts with this radical species [7,8]. One of the main challenges of producing a functional OH probe is to have a reproducible method of generating  OH for the purposes of calibration and testing of the probe. One method is the photolysis of hydrogen peroxide by ultraviolet light [9]. This method is simple and clean but depends on the concentration of hydrogen peroxide and the intensity of light absorbed. In an another method, a redox active metal (typically an iron or copper chelate) catalyzes the dismutation of superoxide [8]. One example of this is the xanthine oxidase–[Fe(III)EDTA]  system shown in Eqs. (1)–(5). XanthineðXÞþ 2O 2 ! 2O 2  þ uric acid ðin the presence of xanthine oxidaseÞ (1) 2O 2  þ H þ ! H 2 O 2 þ O 2 (2) ½FeðIIIÞEDTA  þ O 2  $½FeðIIÞEDTA 2 þ O 2 (3) ½FeðIIÞEDTAþ O 2  þ 2H þ !½FeðIIIÞEDTA  þ H 2 O (4) ½FeðIIÞEDTA 2 þ H 2 O 2 !½FeðIIIÞEDTA  þ OH  þ  OH (5) The third method is the generation of  OH by the Fenton or Haber–Weiss system with a molecule such as ascorbic acid that can be oxidized to regenerate the metal catalyst [10]. Coumarin-3-carboxylic acid (CCA) reacts directly with  OH to produce the highly fluorescent compound, 7-hydroxy- coumarin-3-carboxylic acid (7-OH-CCA) (Fig. 1) [3], and this reaction has been shown to be specific for detection of  OH [3,4]. 7-OH-CCA has excitation maxima around 325 and 385 nm and emits at 450 nm. Sensors and Actuators B 90 (2003) 76–81 Fig. 1. Reaction of CCA with OH to produce 7-OH-CCA. * Corresponding author. E-mail address: kopelman@umich.edu (M. King). 0925-4005/03/$ – see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0925-4005(03)00100-X