Electrode surface confinement of self-assembled enzyme aggregates using magnetic nanoparticles and its application in bioelectrocatalysis.

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 microM), 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.