Publication
612
Anal. Chem. , 79, 187 - 184, 2007.
DOI:
10.1021/ac061367a
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Electrode surface confinement of self-assembled enzyme aggregates using magnetic nanoparticles and its application in bioelectrocatalysis |
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François Mavré, Mélanie Bontemps, Souad Ammar-Merah, Damien Marchal and Benoît Limoges
Laboratoire d' Electrochimie Moléculaire, UMR CNRS 7591, and Interfaces, Traitement, Organisation et Dynamiques des Systèmes (ITODYS), UMR CNRS 7086, Université de Paris 7, Denis Diderot, 2 place Jussieu, 75251 Paris Cedex 05, France
Self-assembled enzyme aggregates, prepared from magnetic 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 M),
the catalytic current responses 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. |