Biosensors and Bioelectronics 24 (2008) 1053–1056 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Short communication Immobilization of a trienzymatic system in a sol–gel matrix: A new fluorescent biosensor for xanthine A. Salinas-Castillo, Isabel Pastor, Ricardo Mallavia, C. Reyes Mateo ∗ Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche E-03202, Alicante, Spain article info Article history: Received 7 May 2008 Received in revised form 18 July 2008 Accepted 25 July 2008 Available online 3 August 2008 Keywords: Fluorescent biosensor Sol–gel Multienzyme system Xanthine abstract In this work we report the development of a highly sensitive fluorescent multienzymatic biosensor for quantitative xanthine detection. This biosensor is built by the simultaneous encapsulation of three enzymes, xanthine oxidase, superoxide dismutase and peroxidase, in a single sol–gel matrix coupled to the Amplex Red probe. The sol–gel chemistry yields a porous, optically transparent matrix that retains the natural conformation and the reactivity of the three co-immobilized proteins. Xanthine determination is based on a sequence of reactions, namely catalytic oxidation of xanthine to uric acid and superoxide radical, and subsequent catalytic dismutation of the radical, resulting in the formation of hydrogen per- oxide, which reacts stoichiometrically with non-fluorescent Amplex Red to produce highly fluorescent resorufin. The optimal operational conditions for the biosensor were investigated. Linearity was observed for xanthine concentrations up to 3.5 M, with a detection limit of 20 nM, which largely improved the sensitivity of the current xanthine biosensors. The developed biosensor is reusable and remains stable for 2 weeks under adequate storage conditions. © 2008 Elsevier B.V. All rights reserved. 1. Introduction The development of new biosensor devices is currently the sub- ject of extensive research in areas such as clinical diagnostic, food technology, biomedical and environmental analysis (Borisov and Wolfbeis, 2008; Rich and Myszka, 2007; Xu et al., 2005; Velasco- García and Mottram, 2003; Wolfbeis et al., 2000). The importance of enzyme-based biosensors has increased considerably in the last years thanks to their exceptional properties which include high specificity, rapid response and low cost (Choi, 2004). The config- urations of enzymatic biosensors range from simple one-enzyme devices to multienzyme systems. Generally, additional enzymes are necessary because the products obtained in the first enzymatic reaction are not detected directly by a transducer and/or because the sensitivity and selectivity of the reaction of product recogni- tion by the transducer is improved. Consequently, the use of more than one enzyme species considerably extends the range of possi- ble applications of biosensors for detection of numerous biological substrates, but complicates the manufacture of these devices as well as their basic design (Wollenberger et al., 1993). One of the most important steps in the development of these kind of biosensors is the immobilization of the multienzymatic ∗ Corresponding author. Tel.: +34 966 658469; fax: +34 966 658758. E-mail address: rmateo@umh.es (C.R. Mateo). system in an appropriate solid support which integrates the biomolecules maintaining their functionality, allowing to per- form reactions in a sequential order while providing accessibility towards the target analyte and subsequent substrates (Xu et al., 2006). In recent years, the renaissance of sol–gel chemistry has pro- vided a versatile method for immobilizing and stabilizing a wide variety of enzymes and other biological molecules in transparent inorganic matrices. Compared to other immobilization methods, such as adsorption to solid supports, covalent attachment and poly- mer entrapment, the sol–gel glasses show numerous advantages, including entrapment of large amount of enzymes, thermal and chemical stability of the matrix, enhanced stability of the encap- sulated biomolecules, excellent optical transparency and flexibility in controlling pore size and geometry. Furthermore, thanks to the porous nature of the matrix, the immobilized proteins remain accessible to interact with external specific analytes with negligible protein leaching, making possible conduction of multienzymatic reactions (Pierre, 2004; Kandimalla et al., 2006; Jerónimo et al., 2007; Gupta and Chaudhury, 2007). Multienzymatic entrapment in the same sol–gel matrix allows sequential reactions to occur in a confined space, leading to high conversion efficiencies due to the proximity of analytes and inter- mediates to the enzymes active sites (Jin and Brennan, 2002). Several types of enzymatic biosensors have been developed based on two enzymes sol–gel immobilization (Martinez-Pérez et al., 2003; Pastor et al., 2004; Cui et al., 2007; Singh et al., 2007). 0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.07.052