Direct One-Step Immobilization of Glucose Oxidase in Well-Ordered Mesostructured Silica Using a Nonionic Fluorinated Surfactant J. L. Blin,* ,† C. Ge ´rardin, ‡ C. Carteret, § L. Rodehu ¨ser, ‡ C. Selve, ‡ and M. J. Ste ´be ´ † Equipe Physico-chimie des Colloı ¨des and Equipe Mate ´ riaux Tensioactifs, Polyme ` res et Colloı ¨daux, UMR 7565 UniVersite ´ Nancy 1/CNRS, Faculte ´ des Sciences, BP 239, F-54506 VandoeuVre-les-Nancy Cedex, France, and Laboratoire de Chimie Physique et Microbiologie pour l’EnVironnement, UMR7564 UniVersite ´ Nancy 1/CNRS rue de Vandoise, F-54600 Villers-le ` s-Nancy, France ReceiVed NoVember 9, 2004. ReVised Manuscript ReceiVed January 17, 2005 This work describes the immobilization of glucose oxidase (GOD) in mesostructured silica. The enzyme is incorporated into the silica framework via a direct one-step immobilization method. Results obtained by SAXS and nitrogen adsorption-desorption analysis clearly show that the channel arrangement of the recovered materials depends on the GOD loading. Indeed, when the hydrothermal treatment is performed at 60 °C for 2 days, well-ordered materials are obtained if the GOD concentration is lower than 3.2 mg per mL of micellar solution, and higher loading leads to the formation of wormhole-like structures. The efficiency of the immobilization was revealed by fluorescence and FTIR spectroscopy. It appears that there is a maximum loading of GOD, about 11 wt %, that can be incorporated into the matrix. Results also show that the surfactant plays the role of a pore-forming agent. Finally, we have shown that the entrapped enzyme maintains its activity. 1. Introduction In recent years, much interest has been attributed to the development of biosensors at the nanoscale level and promising results have first been obtained by Clark and Lyones. 1 These authors reported in 1962 an enzyme electrode for measuring glucose concentrations. Since this study, many papers dealing with enzyme-based biosensors have been published in the literature. 2-6 Because of its importance in the human metabolism, glucose is the most studied analyte. 7 Indeed, the development of a stable in vivo sensor could improve the regulation of glucose concentration and reduce complications related to diabetes. Therefore, many researches are focused on the immobilization of glucose oxidase (GOD). The making of biosensors requires the immobilization of a biomolecule on a solid surface. The immobilization should be irreversible and stable under potentially adverse reaction conditions. At the same time, high activity, good accessibility to analytes, and rapid response times should be kept, while leaching of the biomolecule has to be avoided. Conventional methods of enzyme immobilization include physical or chemical adsorption at a solid surface, 8 covalent binding or cross-linking to a matrix, 9 entrapment within a membrane, 10 and microencapsulation into polymer microspheres and hydrogels. 11,12 However, such techniques are not generic and can be used only for a limited range of biomolecules and applications. A promising method for the design of bio- sensors consists of the entrapment of the biomolecules in a silica matrix prepared via the sol-gel process. Indeed, these inorganic host supports exhibit several advantages such as physical rigidity, chemical inertness, simplicity of prepara- tion, tunable porosity, low-temperature encapsulation, optical transparency, negligible swelling, and mechanical stability. The sol-gel encapsulation of biomolecules was first reported in 1990 by Braun et al. 13 The authors showed that enzymes, trapped within a porous oxide matrix, retain their biological activity. It was also reported by Blyth et al. 14 and by Chung et al. 15 that metalloproteins, such as hemoglobin, encapsulated into porous sol-gel silica matrix kept their activity and could be used for the optical detection of small molecules. The * Author to whom correspondence should be addressed. Phone: +33-3-83- 68-43-70; fax: +33-3-83-68-43-22; e-mail: Jean-Luc.Blin@lesoc.uhp-nancy.fr. † Equipe Physico-chimie des Colloı ¨des. ‡ Equipe Mate ´riaux Tensioactifs. § Laboratoire de Chimie Physique et Microbiologie pour l’Environnement. (1) Clark, L. C.; Lyones, C. Ann. N.Y. Acad. Sci. 1962, 102, 29. (2) Bradley, D. Anal. Chem. 1997, 69, 454A. (3) Cammann, A. E. G.; Lemke, U.; Rohen, A.; Sander, J.; Wilken, H.; Winter, B. Angew. Chem. 1991, 103, 519. (4) Liu, B.; Hu, R.; Deny, J. Chem. 1997, 69, 2343. (5) Gough, D. A.; Lucisano, J. Y. Anal. Chem. 1995, 57, 2351. (6) Rhodes, R. K.; Shults, M. C.; Updike, S. J. Anal. Chem. 1994, 66, 1520. (7) Diabetes control and complications trial research group. N. Engl. J. Med. 1993, 329, 977. (8) Vandenberg, E. T.; Brown, R. S.; Krull, U. J. In Immobilized Biosystems in Theory and Practical Applications; Elsevier: Holland, 1983; p 129. (9) Weetall, H. H. Appl. Biochem. Biotechnol. 1993, 41, 157. (10) Doretti, L.; Ferrara, D.; Lora, S. Biosens. Bioelectron. 1993, 8, 443. (11) O’Driscoll, K. F. Methods Enzymol. 1976, 44, 169. (12) Scouten, W. H. Methods Enzymol. 1987, 135, 30. (13) Braun, S.; Rappoport, S.; Zusman, R.; Avnir, D.; Ottoglenghi, M. Mater. Lett. 1990, 10, 1. (14) Blyth, D. J.; Aylott, J. W.; Richardson, D. J.; Russel, D. A. Analyst 1995, 120, 2725. (15) Chung, K. E.; Lan, E. H.; Davidson, M. S.; Dunn, B. S.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1995, 67, 1505. 1479 Chem. Mater. 2005, 17, 1479-1486 10.1021/cm048033r CCC: $30.25 © 2005 American Chemical Society Published on Web 02/25/2005