Immobilization of horseradish peroxidase on nanoporous copper and its potential applications

• Published in: Bioresource technology 2010-12, Vol.101 (24), p.9415-9420
• Main Authors: Qiu, Huajun, Lu, Lu, Huang, Xirong, Zhang, Zhonghua, Qu, Yinbo
• Format: Article
• Language: English
• Subjects: APPLICATIONS; Biological and medical sciences; Biosensors; Biotechnology; Copper; Copper - chemistry; COPPER ALLOYS (40 TO 99.3 CU); COSTS; Electrochemical biosensor; Electrochemical Techniques; Electrodes; Enzyme immobilization; Enzymes; Enzymes, Immobilized - metabolism; Fundamental and applied biological sciences. Psychology; Horseradish peroxidase; Horseradish Peroxidase - metabolism; Hydrogen-Ion Concentration; Immobilization; Immobilization of enzymes and other molecules; Immobilization techniques; Kinetics; Methods. Procedures. Technologies; MICROSTRUCTURES; Nanomaterials; Nanoparticles - chemistry; Nanostructure; Peroxidase; Phenylenediamines - metabolism; PORE SIZE AND SHAPE; POROSITY; Porous copper; Surface chemistry; Temperature; THERMAL STABILITY; Time Factors; Various methods and equipments
• ISSN: 0960-8524; 1873-2976
• DOI: 10.1016/j.biortech.2010.07.097

Nanoporous copper (NPC) with a pore size of 100–200 nm was prepared by simply dealloying Al 60Cu 40 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 K m and K cat values of the immobilized enzyme increase (from 0.43 to 0.80 mM) and decrease (from 8.1 × 10 3 to 2.2 × 10 3 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.