Covalent Immobilization of Tyrosinase onto Multi-walled Carbon Nanotubes and Its Potential Use In Phenol Biosensing


  • Shafinaz Shahir
  • Boon Kai Tai
  • Zaiton Abdul Majid
  • Nor Aziah Buang



Tyrosinase, Mul ti-walled carbon nnanotubes, Biosen sor, 1 –ethyl -3-(33-dimethylaminop ropyl) carbodiimidde, Phenol,


The possibility of modifying the surface properties of multi-walled carbon nanotubes (MWCNTs) has stimulated increasing interest in their application as components in biosensors. In this sense, it is possible to employ surface modified MWCNTs as support to immobilize biomaterials such as enzymes. In this study the enzyme tyrosinase was immobilized onto functionalized MWCNTs (fMWCNTs) via covalent bonding and activity of immobilized tyrosinase was measured via electrochemical detection of phenol. MWCNTs were first treated with sulphuric acid and nitric acid with ratio 1 : 3 at 70ºC to introduce carboxylated groups (-COOH). The carboxyl moieties were then activated by treatment with a cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to enable tyrosinase immobilization via amide bonding. FTIR spectra of tyrosinase immobilized fMWCNTs showed the presence of peaks attributing to aliphatic C-N (1382 cm-1) and amide carbonyl (1639 cm-1) vibrations which confirmed successful covalent immobilization of tyrosinase onto fMWCNTs. Electrochemical measurements using tyrosinase-fMWCNTS-CPE revealed increasing limiting current values of reduction peak with increasing phenol concentrations at -200mV. The appearance of the reduction current indicates that the immobilization process retained the biological activity of the covalently bonded tyrosinase on fMWCNTs surface. This study has demonstrated the potential of using MWCNTs as support for enzyme immobilization and their application in biosensor technology.


H. Dai, Carbon nanotubes: Opportunities and challenges, Surface Science, 500, (2002), 218-241.

D.H.Lin ,and B.S.Xing, Tannic acid adsorption and its role for stabilizing carbon nanotubes suspensions, Environ. Sci. Technol, 42 (2008), 5917–5923.

W.Yang, P.Thordarson, J.J.Gooding, S.P.Ringer, and F.Breet, Carbon nanotubes for biological and biomedical applications, Nanotechnology, 18 (2007).

K. Balasubramanian, and M.Burghard, Biosensors based on carbon nanotubes, Anal Bioanal Chem, 385, (2006), 452-468.

N.Duran, M.A.Rosa, A.D’Annibale, and L.Gianfreda, Application of laccases and tyrosinase (phenoloxidases) immobilized in different supports: A Review, Enzyme and Microbial Technology, 31, (2002), 907-931.

P.Liu, Modification of carbon nanotubes with polymers, European Polymer Journal, 41, (2005), 2693-2703.

G.A.Rivas, M.D.Rubianes, M.C. Rodriguez, N.F.Ferreyra, G.L.Luque, M.L.Pedano, S.A. Miscoria, and C.Parrado, Carbon nanotubes for electrochemical biosensing, Talanta, 74 (2007), 291-307.

H.Dai, Y.Zhang, D.Wang, and R.J.Chen, Noncovalent Sidewall Functionalization of Single-Walled Carbon Nanotubes for Protein Immobilization, J. Am. Chem. Soc., 123(2001), 3838-3839.

J.J.Gooding, R.Wibowo, J.Liu, W.Yang, D.Losic, S. Orbons, F.J.Mearns, J.G.Shapter, and D.B.Hibbert, Protein Electrochemistry Using Aligned Carbon Nanotube Arrays, J. Am. Chem. Soc., ( 2003), 9006-9007.

L.Wang, Q.Ran, Y.Tian, S.Ye, J.Xu, Y.Xian, R.Peng and L.Jin, Covalent grafting tyrosinase and its application in phenolic compounds detection, Microchim Acta, (2010), 217-223.

C.Vedrine, S.Fabiano and C.Tran-Minh, Amperometric tyrosinase based biosensor using an electrogenerated polythiophene film as an entrapment support, Talanta, 59 (2003), 535-544.

S.Wada, H.Ichikawa, and K.Tatsumi, Removal of phenols from wastewater by soluble and immobilized Tyrosinase, Biotechnology and Bioengineering, 42, (1993), 854-858.

H.Notsu, T.Tatsuma and A.Fujishima, Tyrosinase-modified boron-doped diamond electrodes for the determination of phenol derivatives, Journal of Electrochemical Chemistry 532, (2002), 86-92.

G.Seetharam and B.A.Saville, Degradation of phenol using tyrosinase immobilized on siliceous supports, Water Research, 37, (2003),436-440.

K.Jiang, L.S. Schadler, R.W. Siegel, X. Zhang, H. Zhang and M. Terrones, Protein immobilization on carbon nanotube via a two-step process of diimide-activated amidation, Journal of Material Chemistry, 14(2003), 37-39.

J. Kathi and K.Rhee, Surface modification of multi-walled carbon nanotubes using 3-aminopropyltriethoxysilane, Journal of Materials Science, 43(2008), 33-37.

C.Cai, J.Chen, and T. Lu, Direct electron transfer of glucose oxidase on the carbon nanotube electrode, Science in China Series B: Chemistry, 47(2004), 113-119.

K,Kalantar-zadeh and B. Fry, Nanotechnology-Enabled Sensors. USA: Springer Science+Business Media. (2008), 374-380. [19] F.H.Gonjoy, J.Nastalczyk, Z.Roslaniec, and K.Schulte, Surface modified multi-walled carbon nanotubes in CNT/epoxy-composites, Chemical Physical Letter, 370 (2003), 820-824.