Electrode Surface Confinement of Self-Assembled Enzyme Aggregates Using Magnetic Nanoparticles and Its Application in Bioelectrocatalysis Franc ¸ ois Mavre ´, † Me ´ lanie Bontemps, † Souad Ammar-Merah, ‡ Damien Marchal, † and Benoıˆt Limoges* ,† Laboratoire d’ Electrochimie Mole ´ culaire, UMR CNRS 7591, and Interfaces, Traitement, Organisation et Dynamiques des Syste ` mes (ITODYS), UMR CNRS 7086, Universite ´ de Paris 7, Denis Diderot, 2 place Jussieu, 75251 Paris Cedex 05, France Self-assembled enzyme aggregates, prepared from mag- netic iron oxide nanoparticles, avidin, and a biotinylated redox enzyme, were shown particularly useful for the simple, fast, and efficient construction of highly enzyme- loaded electrodes with the help of a magnet. The approach was illustrated in the case of the bioelectrocatalytic oxidation of NADH by a diaphorase oxidoreductase in the presence of a ferrocene mediator. Two different self- assembling procedures were tested, taking advantage of the spontaneous aggregation of the nanoparticles in the presence of avidin and also of the multivalency binding of biotinylated diaphorase toward avidin. Activities of the bound and unbound diaphorase were systematically controlled allowing determination of the number of active biotinylated diaphorase per nanoparticle incorporated within each magnetic enzyme aggregate. An active enzyme loading capacity of up to 2.35 nmol mg -1 was found for the best nanostructured enzyme assembly, which is 200 times better than for commercialized magnetic micrometer- sized beads coated with streptavidin and saturated with diaphorase. With the help of a permanent magnet, the magnetic enzyme aggregates were finally magnetically collected as a film on the surface of a small screen-printed carbon electrode and the catalytic currents recorded by cyclic voltammetry. From the analysis of the steady-state catalytic current responses and the kinetic rate constants of biotinylated diaphorase, it was possible to determine the enzyme concentration within the magnetic films. Owing to the high enzyme loading in the aggregates of nanoparticles (i.e., 130 μM), the catalytic current re- sponses were definitely higher than the ones measured at an electrode coated with a closed-packed monolayer of diaphorase or at an electrode covered with a film of magnetic micrometer-sized streptavidin beads saturated with diaphorase. Deposition of organized enzyme films on electrode surfaces, with thickness ranging from nano- to micrometer length, continue to attract increasing attention because of their broad biotechno- logical applications including biosensors 1-4 and bioprocesses. 5,6 With the aim to achieve nanostructured enzyme multilayer films with a high protein density, various immobilization strategies have been realized such as step-by-step attachment of enzyme layers through biospecific interactions, 7-11 covalent attachment, 12 or alternate layer assembly of oppositely charged macromolecules. 13-15 Although these strategies offer the possibility to build thin ordered enzymatic films of high catalytic activity, the step-by-step proce- dure is a slow and tedious process, and it cannot be easily transferred to industrial scale for the mass production of low-cost biosensors. To circumvent these drawbacks, a possible route consists in prearranging, in the bulk of a liquid phase, a three-dimensional nanostructured assembly of the enzyme and next to collect it as a regular film on the surface of an electrode. Such an idea can be put into practice with the help of magnetic nanoparticles that can be used as a unique building block in the design of an enzyme- based supramolecular network, assembled, for example, through multivalent and three-dimensional interactions, 16 to finally produce a magnetic solid phase that can be easily deposited as a film on the surface of an electrode by means of an external magnetic field. * To whom correspondence should be addressed. E-mail: limoges@ paris7.jussieu.fr. Telephone: (33) 1 44 27 28 01. Fax: (33) 1 44 27 76 25. † UMR CNRS 7591. ‡ UMR CNRS 7086. (1) Davis, F.; Higson, S. P. J. Biosens. Bioelectron. 2005, 21, 1. (2) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 1181. (3) Campas, M.; O’Sullivan, C. Anal. 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