Tyrosinase enzyme is a binuclear copper cluster containing monooxygenase contains a dicopper core in the active site [1]. This enzyme catalyzes the o-hydroxylation of monophenols and subsequent oxidation of the formed o-diphenols into the o-quinones. Depending on the electrochemical reduction of o-quinone, amperometric detection of phenol which is a high toxic compound can be achieved. For this aim spectrophotometric and chromatographic methods have been commonly used. Due to their lots of disadvantages, electrochemical enzyme sensors become promising alternative [2]. The performance of enzyme sensors strongly depends on the immobilization of the enzyme to the appropriate support. Many significant biological and chemical properties of chitosan attract attention as a potential support for enzyme immobilization [3]

In current study, tyrosinase enzyme was immobilized on chitosan support enhanced by magnetite nanoparticles. Electrochemical biosensor was constructed by coating this bionanocomposite film on 5mm diameter of working electrode. All electrochemical measurements were performed with a model PGSTAT 128N (AUTOLAB Instruments) having three electrode system (working electrode: tyrosinase–magnetite-chitosan modified glassy carbon, reference electrode: Ag/AgCl, auxiliary electrode: platinum wire). The interaction between the sensor component and their structure were characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. Electrochemical characterizations were also performed by cyclic voltammetry (CV) and impedance spectroscopy (EIS). The performance of electrochemical tyrosinase biosensor was tested by amperometric method in magnetically stirred PBS, followed by the addition of catechol solution as a phenolic compound.

The binding of tyrosinase enzyme to the bionanocomposite support was confirmed by FTIR and SEM analysis. According to the CV measurements, the reduction peak was observed attributed to the direct reduction of quinone on the electrode surface. EIS results showed decrease in mass transfer resistance indicate conducting properties of magnetite nanoparticles. Electrochemical sensing characteristics and analytical performance of developed sensor was also evaluated by amperometric detection of catechol. The designed sensor depicted wide linear range of catechol concentration with high correlation coefficient, high sensitivity, low detection limit and K_mvalue.

Acknowledgements

This study was supported financially by the Scientific and Technological Research Council of Turkey (TUBITAK Grant Number 114Z417).

 

References

[1] Abdullah J, Ahmad M, Karuppiah N, Henga LY, Sidek H, Sensors and Actuators B, 114 (2006) 604-609.

[2] Li GY, Jiang Y, Huang K, Ding P, Chen J, Journal of Alloys and Compounds, 466 (2008) 451–456

[3] Krajewska B, Enzyme and Microbial Technology, 35 (2004) 126–139