Immobilization of horseradish peroxidase on nanoporous copper and its potential applications

Nanoporous copper (NPC) with a pore size of 100-200nm was prepared by simply dealloying Al₆₀Cu₄₀ alloy in a 5wt.% HCl solution. The NPC was characterized by scanning electron microscopy and nitrogen adsorption techniques. Horseradish peroxidase (HRP) was immobilized on NPC by adsorption. Compared with free enzyme, the thermal stability of the immobilized enzyme was greatly improved due to the multiple attachments between the enzyme molecule and the NPC surface. After 2h incubation at 50°C, the immobilized HRP retained ca. 90% of the initial activity while only ca. 10% initial activity remained for the free enzyme. The interaction between HRP and the porous surface also made the K_m and K_cat values of the immobilized enzyme increase (from 0.43 to 0.80mM) and decrease (from 8.1×10³ to 2.2×10³min^-1), respectively. Based on the good electric conductivity and electrocatalytic activity of the NPC electrode, an electrochemical biosensor for O-phenylenediamine (OPD) was made. The calibration curve of the biosensor was linear from 0.5μM to 14.5μM OPD with a sensitivity of 0.37μAμM^-1. The stability and reproducibility of the biosensor were also demonstrated to be good. When positioned at -0.45V for 200s, its current response toward 10μM OPD remained ca. 80% of its initial value. For five HRP-loaded NPC electrodes, the relative standard deviation (RSD) of the current response toward 10μM OPD was ca. 4.5%. All these results indicated that NPC was a good support for the HRP immobilization and its low price would facilitate its large-scale application.