Electrode Surface Confinement of Self-Assembled Enzyme Aggregates Using Magnetic Nanoparticles and Its Application in Bioelectrocatalysis

Abstract

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.

Chemical Profiling of Re-Du-Ning Injection by Ultra-Performance Liquid Chromatography Coupled with Electrospray Ionization Tandem Quadrupole Time-of-Flight Mass Spectrometry through the Screening of Diagnostic Ions in MSE Mode

“…This method has become one of the most frequently applied approaches in the area of fast chromatographic separations [ 17 – 19 ]. High-resolution tandem mass spectrometry can provide a more specific and accurate mass as long as the co-eluting compounds possess different m/z values [ 20 ]. And the isotopic abundances and the elemental composition of fragment ions are greatly conducive to the structural elucidation of unknown compounds.…”

Magnet-assisted assembly of 1-dimensional hollow PtCo nanomaterials on an electrode surface

“…24,25 Attachment of the hollow PtCo nanomaterials on an electrode surface with the aid of a magnet Growing interest in the construction of functional thin films has motivated considerable research activity in an assembly technique based on magnetic interaction, which represents a unique technique to present a rapid and controllable approach for the formation of functional group modified magnetic material films on solid surfaces. [16][17][18][19] The principle for the assembly of hollow PtCo nanomaterials on a magnetic electrode surface is illustrated in Fig. 3.…”

“…This property has found some applications. [16][17][18][19] For example, highly enzyme loaded electrodes have been prepared by deposition of magnetic enzyme aggregates on an electrode surface with the aid of a magnet. 16 Similarly, a sensitive and stable Ru(bpy) 3…”

New Micro‐ and Nanotechnologies for Electrochemical Biosensor Development

“…The biosensor exhibited a linear range from 8.3 × 10 − 8 to 7.0 × 10 − 5 mol l − 1 , and a detection limit of 2.5 × 10 − 8 mol l − 1 for catechol detection. Mavr é et al [140] prepared self -assembled aggregates of iron oxide nanoparticles, avidin, and a biotinylated diaphorase oxidoreductase for bioelectrocatalytic oxidation of NADH. This method represented a very simple, fast, and effi cient route for the construction of highly loaded enzyme electrodes.…”