Covalent immobilization of tyrosinase onto commercial multi-walled carbon nanotubes and its effect on enzymatic activity

Summary form only given. Multi-walled carbon nanotubes (MWCNTs) exhibit unique structural, electrical, mechanical, electrochemical, and chemical properties. Moreover, the possibility of modifying their surface properties through different methods has stimulated increasing interest in their application as components in biosensors. In this sense, it is possible to employ carbon nanotubes as support to immobilize enzymes. The purpose of this study was to immobilize the enzyme tyrosinase onto functionalized commercial MWCNTs via covalent bonding and study the enzyme activity of covalent bonded tyrosinase. Tyrosinase, an enzyme with a coupled binuclear copper complex in active site that catalyzes the hydroxylation of monophenols to o-diphenols (monophenolase activity) by oxygen, and also catalyzes the oxidation of o-diphenols to o-quninones (catecholase activity). Commercial MWCNTs underwent a two-stage treatment, namely acid treatment and two-steps diimide activated amidation before tyrosinase was immobilized onto functionalized MWCNTs. Commercial MWCNTs were first treated with sulphuric acid and nitric acid at a 1:3 ratio at 70°C to introduce carboxylated groups (-COOH) onto commercial MWCNTs. This was then followed with diimide-activated amidation by using a cross-linker, l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in the presence and absence of catalyst, N-hydroxysuccinimide (NHS). NHS acts as catalyst to stabilize amide bonding formed between diimide activated amidated MWCNTs and tyrosinase. The commercial MWCNTs and functionalized MWCNTs were characterized with FT-IR, FESEM, EDX, and TGA to examine the extent of the functionalization process. The characterization results showed that tyrosinase was successfully immobilized onto functionalized MWCNTs. The grey particles woven onto the functionalized MWCNTs thread as well as copper element in FESEM micrograph and EDX, respectively indicated the presence of tyrosinase onto functionalized MWCNTs. The pr- - esence of a peak (1382.74 cm^-1) pre-dominates in both tyrosinase immobilized diimide activated amidated MWCNTs in the presence and absence of catalyst, attributed to aliphatic C-N vibration. A peak (1639.16 cm^-1) assigned to the amide carbonyl also observed in FT-IR spectrum. A slight shift of decomposition temperature of commercial MWCNTs from 640°C to 520°C in acid treated MWCNTs indicated commercial MWCNTs had been successfully functionalized due to decomposition of structural of sample. The extent of enzyme immobilization onto functionalized MWCNTs was determined spectrophotometrically. 57% of tyrosinase was successfully immobilized onto functionalized MWCNT in the presence of NHS whilst that immobilized in the absence of NHS retained 65%. The voltammetry results using tyrosinase-NHS-graphite-MWCNTs modified working electrode revealed increasing limiting current values of reduction peak with increasing phenol concentrations when the applied potential versus Ag/AgCl was varied from +200 to -200 mV. This study has demonstrated the potential of using MWCNTs as support for enzyme immobilization and their application in biosensor technology.