Mixed urease/amphiphile LB films and their application for biosensor development Aidong Zhang a,b , Yanxia Hou a,b , Nicole Jaffrezic-Renault a, * , Jialiang Wan b , Alexey Soldatkin a,c , Jean-Marc Chovelon d a IFOS-LPC1, UMR CNRS 5621, Ecole Centrale de Lyon, 36 Ave Guy de Collongue, BP 163, F69131 Ecully Cedex, France b Department of Chemistry, Central China Normal University, Wuhan 430079, PR China c IMBG, National Academy of Sciences of Ukraine, Kyiv 252143, Ukraine d LACE, UMR CNRS 5634, Claude Bernard University-Lyon1, 69622 Villeurbanne Cedex, France Received 1 June 2001; received in revised form 27 November 2001; accepted 30 November 2001 Abstract The optimal conditions for the formation of a stable mixed urease/octadecylamine (ODA) film at the air – water interface are determined. This film is efficiently transferred onto the hydrophobized surface of a pH-ISFET, and the features of the urea enzymatic field-effect transistor (ENFET) are determined: detection limit 0.2 mM, response time 15 s, and dynamic range 0 – 20 mM. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Mixed LB films; Urea ENFET; Urease; Octadecylamine; Behenic acid 1. Introduction Langmuir–Blodgett technology has been considered as a convenient tool for designing artificial systems (LB films) with biological functions such as biosensors [1]. The ultra- thin, structure-ordered films with the incorporation of bio- molecules much resemble the biological membrane environment [2] and, thus, the preservation of a high enzymatic activity is expected. Until now, many kinds of biosensors have been designed with the LB films as the biosensitive part; however, few papers are concerned with urease mixed LB films [3]. The aim of this work is focused on the formation of stable mixed urease/amphiphile Lang- muir monolayers at the air – water interface, the monolayer transfer and the biosensor characteristics of the enzymatic field-effect transistor (ENFET) obtained by the deposition of the LB film on a pH-ISFET. 2. Experimental Urease (EC 3.5.1.5) from Jack Beans with a specific activity of 800 U mg À1 was used. Langmuir experiments were performed on a Langmuir trough from NIMA (model 611). The chloroform solution of octadecylamine (ODA) or behenic acid (BA) with a concentration of 1 mg/ml was spread (22 Al used for ODA and 25 Al for BA each time) onto the subphase. Infrared spectroscopy was performed on Perkin-Elmer 2000 (GB) with a standard ATR accessory. The response of the ISFET modified by the mixed LB film to the addition of urea solutions was conducted with a laboratory ISFET meter (IMT, Neuchatel, Switzerland) in a phosphate buffer solu- tion with a pH of 5.5. 3. Results and discussions The adsorption of urease in the subphase onto the ODA or BA monolayer can reach equilibrium after about 2–3 h, and the associating process is much similar to that of the reported BuChE/ODA monolayer [4]. The adsorption proc- ess is driven by the electrostatic interaction between urease and ODA or BA. In the case of ODA, at a pH value of the subphase of 5.5, the positively charged ODA head can interact with the negatively charged portion of the urease surface and induce the formation of a stable mixed Lang- muir monolayer at the air–water interface. A similar ad- sorption process can occur, but with a reverse electrostatic 1567-5394/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S1567-5394(02)00045-2 * Corresponding author. Tel.: +33-472-186-420; fax: +33-478-331-140. E-mail address: Nicole.Jaffrezic@ec-lyon.fr (N. Jaffrezic-Renault). www.elsevier.com/locate/bioelechem Bioelectrochemistry 56 (2002) 157 – 158