Immobilization of horseradish peroxidase on nanoporous copper and its potential applications

Nanoporous copper (NPC) with a pore size of 100–200 nm was prepared by simply dealloying Al60Cu40 alloy in a 5 wt.% 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 2 h 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 Km and Kcat values of the immobilized enzyme increase (from 0.43 to 0.80 mM) and decrease (from 8.1 × 103 to 2.2 × 103 min−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 0.5 μM to 14.5 μM OPD with a sensitivity of 0.37 μA μM−1. The stability and reproducibility of the biosensor were also demonstrated to be good. When positioned at −0.45 V for 200 s, its current response toward 10 μM OPD remained ca. 80% of its initial value. For five HRP-loaded NPC electrodes, the relative standard deviation (RSD) of the current response toward 10 μM 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.