Biosensors and Bioelectronics 21 (2006) 2320–2328 Amperometric glucose biosensor based on polymerized ionic liquid microparticles M. S´ anchez-Paniagua L´ opez b , D. Mecerreyes c , E. L ´ opez-Cabarcos b , B. L ´ opez-Ruiz a,∗ a Departamento de Qu´ımica Anal´ıtica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain b Departamento de F´ısico-Qu´ımica Farmac´ eutica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain c CIDETEC, Centro de Tecnolog´ıas Electroqu´ımicas, Parque Tecnol´ ogico de Miram´ on, Paseo Miram´ on 196, 20009 San Sebastian, Spain Received 13 September 2005 Available online 17 April 2006 Abstract A glucose amperometric biosensor based on the immobilization of glucose oxidase (GOx) in microparticles prepared by polymerization of the ionic liquid 1-vinyl-3-ethyl-imidazolium bromide (ViEtIm + Br − ) using the concentrated emulsion polymerization method has been developed. The polymerization of the emulsion dispersed phase, in which the enzyme was dissolved together with the ionic liquid monomer, provides poly(ViEtIm + Br − ) microparticles with entrapped GOx. An anion-exchange reaction was carried out for synthesizing new microparticles of poly(ViEtIm + (CF 3 SO 2 ) 2 N − ) and poly(ViEtIm + BF 4 − ). The enzyme immobilization method was optimized for biosensor applications and the following optimal values were determined: pH 4.0 for the synthesis medium, 1.23 M monomer concentration and 3.2% (w/w) cross-linking con- tent. The performance of the biosensor as a function of some analytical parameters such as pH and temperature of the measuring medium, and enzymatic load of the microparticles was also investigated. The effect of the substances which are present in serum samples such as uric and ascorbic acid was eliminated by using a thin Nafion layer covering the electrode surface. The biosensor thus prepared can be employed in aqueous and in non-aqueous media with satisfactory results for glucose determination in human serum samples. The useful lifetime of this biosensor was 150 days. © 2006 Elsevier B.V. All rights reserved. Keywords: Amperometric biosensor; Ionid liquid polymers; Glucose oxidase 1. Introduction Amperometric biosensors employing immobilized enzymes as biological component have been primarily used in aqueous media. However, due to the low solubility of many relevant compounds in aqueous solutions, the preparation of enzymatic sensors capable to operate in non-aqueous media acquires great analytical significance (Zaks and Klibanov, 1988; Wilkins et al., 2000; Campanella et al., 2003). In the present work we show that microparticles prepared by polymerization of ionic liquids could be used to fabricate biosensors that operate in non-aqueous media. Ionic liquids, also called molten salts, are organic salts with low melting point (<100 ◦ C), low flammability, high ionic con- ∗ Corresponding author at: Secci ´ on Departamental de Qu´ımica Anal´ıtica, Fac- ultad de Farmacia, Universidad Complutense, Ciudad Universitaria s/n, 28023 Madrid, Spain. Tel.: +34 91 394 1756; fax: +34 91 394 1754. E-mail address: bealopru@farm.ucm.es (B. L ´ opez-Ruiz). ductivity and a wide electrochemical window. In addition, they have minimal vapour pressure, high thermal stability and can be dissolved in a wide variety of organic and inorganic compounds including some polymers and minerals. The possibility to mod- ify the solubility of ionic liquid polymers by changing the anion makes them particularly attractive as enzymatic immobilization systems for biosensors. The synthesis of ionic liquid monomers (Bonhˆ ote et al., 1996) and polymers (Ohno et al., 2003) has been developed in the last decade. The direct polymerization of the ionic liquid monomer imidazolium, the subsequent modification of the polymer by an anion-exchange reaction, and the solubility of the resultant poly- mers in organic solvents has been recently reported (Marcilla et al., 2004). However, there are only a few examples of gels or microgels involving ionic liquids and polymers. Snedden et al. (2003) and Susan et al. (2005) prepared ionic gels by free radi- cal polymerization of vinyl monomers in ionic liquid. Muldoon and Gordon (2004) have reported the synthesis of polymer beads prepared by polymerization of imidazolium monomers and their 0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.02.019