Development of Amperometric Laccase Biosensor through Immobilizing Enzyme in Magnesium-Containing Mesoporous Silica Sieve (Mg-MCM-41)/Polyvinyl Alcohol Matrix
Magnesium-containing mesoporous silica sieve (Mg-MCM-41) provided a suitable immobilization of biomolecule matrix due to its uniform pore structure, high surface areas, fast electron-transfer rate, and good biocompatibility. Based on this, an amperometric biosensor was developed by entrapping laccase into the Mg-MCM-41/PVA composite matrix. Laccase from Trametes versicolor was assembled on a composite film of Mg-MCM-41/PVA modified Au electrode and the electrode was investigated by cyclic voltammetry, impedance spectroscopy, and chronoamperometry. The results indicated that the Mg-MCM-41/PVA/Lac modified electrode exhibited excellent catalytic activity towards catechol at room temperature in pH 4.8 acetate buffer solution. The optimum experimental conditions of biosensor for the detection of catechol were studied in detail. Under the optimal conditions, the linear range was from 0.94 to 10.23？μM with the sensitivity of 16.9227？A/M, the detection limit of 0.00531？μM, and the response time of less than 14？s. The Michaelis-Menten constant ( ) was estimated by Lineweaver-Burk equation and the value was about 1.01？μM. In addition, the biosensor exhibited high reproducibility and long-time stability. This work demonstrated that Mg-MCM-41/PVA composite provides a suitable support for laccase immobilization and construction of biosensor. 1. Introduction Phenols, byproducts of large-scale production and use of man-made organics, will cause ecologically undesirable effects [1]. Their determination is of great practical importance in evaluating the toxicity of an environmental sample. Several methods have been developed for this purpose, including spectrophotometry and high performance liquid chromatography [2]. These methods are expensive, time-consuming and sometimes require preconcentration. Many biosensors have been developed in the past using the catalytic activity of the redox enzymes such as tyrosinase, peroxidase, and laccase for phenol determination [3, 4]. Laccase (Lac) does not require H2O2 as cosubstrate and any cofactors for its catalysis. Hence, the constructions of the biosensor are much easier. Lac-based biosensors have been employed for the determination of a broad range of phenolic compounds [2]. The development of enzyme-based biosensor with excellent performance requires advances in the materials and method available for enzyme immobilization. So it is necessary to develop ideal immobilization materials and efficient immobilization method. A series of organic compounds and inorganic materials have been used as enzyme immobilization matrices,
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