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
Phenols, byproducts of large-scale production and use of man-made organics, will cause ecologically undesirable effects [
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, such as carbon nanotube [
In recent years, incorporation of the transition metal ions has caught more attention, which could enhance the optical, electrical, semiconducting, and surface properties of mesoporous materials [
Accordingly, in the present study, a facile method was firstly used to incorporate magnesium in the MCM-41 frameworks (Mg-MCM-41), and the catechol biosensor was fabricated by immobilizing laccase in Mg-MCM-41/PVA composite film. The Mg-MCM-41/PVA/Lac film modified Au electrode was expected to improve some disadvantages of amperometric laccase biosensor.
Laccase from
Nitrogen adsorption-desorption isotherm was measured at 77 K on a NOVA2000 Autosorb Sorption Analyzer (Quantachrome Corporation, USA). The specific surface area was calculated with the BET method. The pore size and pore volume were acquired from the adsorption branch of the isotherms by the BJH method. All the electrochemical measurements were carried out with a conventional three-electrode system using a PARSTAT 2263 electrochemical workstation (Princeton Corporation, USA). The MCM-41/PVA/Lac or Mg-MCM-41/PVA/Lac modified electrode was used as a working electrode with a saturated calomel electrode (SCE) as a reference electrode and a Pt wire as an auxiliary electrode. All experiments were carried out at room temperature.
The magnesium containing MCM-41, denoted hereafter as Mg-MCM-41, was synthesized as follows. The molar ratio of synthesis was 1 TEOS : 0.13 CTAB : 0.02 MgSO4 : 0.24 NaOH : 66.7 H2O, which was in accordance with the previous literatures [
Immobilization process was performed by the following method: 4 mg MCM-41 or 4 mg Mg-MCM-41 was dispersed into 5 mL 1 mg/mL laccase (in pH 4.8 acetate buffer) solution by ultrasonic for 30 min, respectively. The suspension was kept at 4°C for 24 h under stirring and then separated by centrifugation under the conditions of 8000 r/min for 15 min. Finally, the obtained MCM-41/Lac and Mg-MCM-41/Lac were mixed with 100
The Au electrode (4 mm diameter) was used as the substrate electrode. Before experiment, it was polished with 1.0, 0.3, and 0.05
Figure
Sample | BET surface area (m2/g) | Average pore diameter (nm) | Pore volume (cm3/g) |
---|---|---|---|
Mg-MCM-41 | 949.30 | 2.87 | 0.93 |
MCM-41 | 951.2 | 2.47 | 0.814 [ |
Nitrogen adsorption-desorption isotherm of Mg-MCM-41 and MCM-41.
EIS was used to investigate the electrochemical properties of Mg-MCM-41 modified electrode surfaces [
Nyquist plots of Mg-MCM-41/PVA/Lac/Au, MCM-41/PVA/Lac/Au, and bare Au electrode (inset) in 5 mM K3[Fe (CN)6]/K4[Fe (CN)6]/0.1 M KCl solution.
The respective semicircle diameters at the high frequency equalled the electron-transfer resistance (Rct) at the electrode surface. It was found that Rct of the Mg-MCM-41/PVA/Lac modified Au electrode was about 200 Ω, which was smaller than 300 Ω of MCM-41/PVA/Lac modified Au electrode and 4500 Ω of bare Au electrode. The results indicated that Mg-MCM-41/PVA could act as a superior electron-transfer interface between the EIS probe and electrode by accelerating the electron-transfer rate on the electrode surface effectively. In addition, the smaller Rct implied that the incorporation of Mg increased the electron-transfer rate on the electrodes surface effectively.
Cyclic voltammetries (CVs) were employed to characterize the electrochemical behavior of laccase immobilized on the Mg-MCM-41 and MCM-41. As shown in Figure
CVs of Mg-MCM-41/PVA/Lac/Au (A), MCM-41/PVA/Lac/Au (B), and Mg-MCM-41/PVA (C) electrodes in pH 4.8 acetate buffer solution at room temperature in presence of 0.05 mM catechol, respectively (50 mV*s−1).
Mg-MCM-41/PVA modified Au electrodes exhibited one pair of redox peaks ascribed to reduction of catechol, which proved the electrocatalytic properties of Mg-MCM-41. Mg-MCM-41/PVA/Lac also exhibited one well-defined redox peak, which means that catechol could carry through the direct electrochemical reaction on the surface of Mg-MCM-41/PVA/Lac electrodes. The peak current of Mg-MCM-41/PVA/Lac modified Au electrodes was 2.59 times that of Mg-MCM-41/PVA modified Au electrodes, which may be attributed to coreduction of catechol by the laccase immobilized in Mg-MCM-41 and Mg-MCM-41 or the activating effect of laccase by Mg incorporated in Mg-MCM-41.
Figure
Current versus the square root of scan rate plots of Mg-MCM-41/PVA/Lac/Au electrode in pH 4.8 acetate buffer solution with 0.05 mM catechol. Inset: CVs of the PVA/Lac/Au electrode in pH 4.8 acetate buffer solution with 0.05 mM catechol at different scan rates from inner to outer: 10, 30, 50, 70, and 90 mV*s−1.
To improve the performance of the biosensor, the effect of applied potential on the response of proposed biosensor to catechol was investigated. The effect of applied potential on the response current was shown in Figure
(a) Effect of applied potential on the current response of the Mg-MCM-41/PVA/Lac/Au electrode to 0.05 mM catechol in pH 4.8 acetate buffer solution. (b) Effect of pH of acetate buffer solution on the current response of the Mg-MCM-41/PVA/Lac/Au electrode to 0.05 mM catechol at 0.45 V (versus SCE). (c) Effect of temperature on the current response of the Mg-MCM-41/PVA/Lac/Au electrode to 0.05 mM catechol in pH 4.8 acetate buffer solution at 0.45 V (versus SCE).
The effect of solution pH on response current was studied between pH 4.0 and 6.0. As shown in Figure
The effect of temperature on the current response of Mg-MCM-41/PVA/Lac modified Au electrodes was also studied in Figure
Figure
(a) The steady-state current response of Mg-MCM-41/PVA/Lac (A) and MCM-41/PVA/Lac (B) modified Au electrodes at 0.45 V to 0.05 mM catechol supported by acetate buffer solution (pH 4.8) at room temperature, respectively. (b) The calibration curves of the Mg-MCM-41/PVA/Lac/Au electrode (A) and MCM-41/PVA/Lac/Au (B) at 0.45 V (versus SCE). Inset: amperometric responses of the Mg-MCM-41/PVA/Lac/Au electrode (A) and MCM-41/PVA/Lac/Au (B) on successive additions of obtained upon the successive addition 0.5 mM catechol to stirred blank pH 4.8 acetate buffer solution at 0.45 V (versus SCE). (c) The Lineweaver-Burk curves of the Mg-MCM-41/PVA/Lac/Au electrode (A) and MCM-41/PVA/Lac/Au (B) (versus SCE).
Figure
Kinetic studies of the immobilized laccase were performed at various concentrations of catechol. The apparent Michaelis-Menten constant (
The reproducibility of the Mg-MCM-41/PVA/Lac/Au electrode was evaluated by comparing the response currents of 10 enzyme electrodes prepared. The relative standard deviation (RSD) was 5.2% when 0.05 mM catechol was determined. A reproducible current response with a RSD of 4.6% was observed for 30 successive assays of 0.05 mM catechol. The long-term stability was investigated by measuring a catechol solution intermittently, and the electrode was stored at 4°C by immersing in 0.1 M acetate buffer solution when it was not in use. The results showed that the response current maintained more than 91% of its initial value after 30 days, indicating the good stability.
In the present study, a functionalized Mg-MCM-41 with good electrocatalytic properties has been synthesized. With the prominent advantages of large surface area, uniform mesopores, and remarkable electrocatalytic properties, an amperometric biosensor was developed by immobilizing laccase into the pores of Mg-MCM-41. The enzyme molecules assembled on Mg-MCM-41/PVA exhibited a high bioactivity and stability, and Mg-MCM-41 was demonstrated as suitable candidate to immobilize enzyme. The assembled laccase biosensor exhibited high sensitivity, low detection limit, good stability, and acceptable reproducibility for the determination of catechol. This work demonstrated that the Mg-MCM-41/PVA composite provides a support for laccase immobilization and construction of biosensors.
The authors declared that there is no conflict of interests regarding the publication of this paper.
The authors gratefully acknowledge the financial support from the Natural Science Foundation of China (Grant no. 51301117) and the Natural Science Foundation for Young Scientists of Shanxi Province, China (Grant nos. 2013021003–1 and 2013021013–5).