Adsorption of Laccase on the Surface of Nanoporous Gold and the Direct Electron Transfer between Them

Abstract

Nanoporous gold (NPG) with different pore sizes was obtained by simple dealloying and thermal annealing methods. The morphology of the NPG was characterized by scanning electron microscopy and nitrogen adsorption technique. Laccase was immobilized on the surface of the NPG by physical adsorption. Detailed studies were made on the effect of the pore size on laccase immobilization. NPG with pore size of 40−50 nm was demonstrated to be a suitable support for laccase immobilization. Compared with free enzyme, the optimum pH of immobilized laccase did not change; the optimum temperature, however, rose from 40 to 60 °C. Both thermal and storage stabilities of laccase improved markedly via the immobilization. Laccase immobilized on NPG (100 nm in thickness) was used for enzyme electrode construction. Direct electrochemistry of laccase on NPG supported by glassy carbon electrode (NPG/GC) was achieved with high efficiency due to the outstanding physicochemical characteristics of the NPG. The laccase-loaded NPG/GC electrode also exhibited a strong electrocatalytic activity toward O2 reduction. When stored at 4 °C for 1 month, the electrode showed no obvious changes in its response. All results presented in the paper indicated that NPG was an excellent carrier for laccase immobilization and would have potential applications in biofuel cell and/or biosensor areas.

An overview of dealloyed nanoporous gold in bioelectrochemistry

“…Firstly, enzymes confined within NPG display higher stability on exposure to high temperatures [49,50] and organic solvents [38] due to the nanopore providing a protective environment to the enzyme. After incubation at 50 °C for 2 h, only 6% of the initial activity of free laccase remained 35 in comparison to 60% for laccase immobilised on NPG [49].…”

“…After incubation at 50 °C for 2 h, only 6% of the initial activity of free laccase remained 35 in comparison to 60% for laccase immobilised on NPG [49]. The activity of xylanase immobilised on NPG was assayed at 50 °C and exhibited only a 25% loss after 10 cycles [39].…”

Nanoporous Gold Electrodes and Their Applications in Analytical Chemistry

“…Other favorable characteristics of nanoporous materials, in general, include the stabilization of the immobilized enzyme against denaturation, better thermal stability, and reduction in leaching [136,137]. Porous materials have been shown to be a good support for the immobilization of enzymes because the constrained environment can help stabilize the macromolecule under otherwise denaturing environments such as in an organic solvent or at high temperatures [138,139]. The research groups of Wang et al, for example, have recently studied the thermal and organic solvent stability of enzymes (lipase, 5 nm; catalase, 10 nm; and horseradish peroxidase, 4 nm) immobilized in nanoporous gold with an average pore size of 35 nm and concluded that they "demonstrated remarkable catalytic performance and stability" [139].…”

Characterization of Nanoporous Gold Electrodes for Bioelectrochemical Applications

“…Improved attachment of the np-Au film to GC has been reported by dropping a suspension of Nafion © on a dry np-Au / GC electrode. 9,26 This, however, would preclude the ability to modify the np-Au with thiols or any other potential linker molecules.…”