Amperometric catechol biosensor based on laccase immobilized on nitrogen-doped ordered mesoporous carbon (N-OMC)/PVA matrix

Abstract

A functionalized nitrogen-containing ordered mesoporous carbon (N-OMC), which shows good electrical properties, was synthesized by the carbonization of polyaniline inside a SBA-15 mesoporous silica template. Based on this, through entrapping laccase onto the N-OMC/polyvinyl alcohol (PVA) film a facilely fabricated amperometric biosensor was developed. Laccase from Trametes versicolor was assembled on a composite film of a N-OMC/PVA modified Au electrode and the electrochemical behavior was investigated. The results indicated that the N-OMC modified electrode exhibits electrical properties towards catechol. The optimum experimental conditions of a biosensor for the detection of catechol were studied in detail. Under the optimal conditions, the sensitivity of the biosensor was 0.29 A*M^-1 with a detection limit of 0.31 μM and a linear detection range from 0.39 μM to 8.98 μM for catechol. The calibration curve followed the Michaelis-Menten kinetics and the apparent Michaelis-Menten [Formula: see text] was 6.28 μM. This work demonstrated that the N-OMC/PVA composite provides a suitable support for laccase immobilization and the construction of a biosensor.

Keywords: N-OMC; PVA; biosensor; electrical properties; laccase.

Figure 1

TEM images of N-OMC taken (a) along and (b) perpendicular to the channel direction, (c) EDX graph of N-OMC, (d) XRD spectrum of N-OMC, and (e) nitrogen adsorption–desorption isotherm of N-OMC. Inset of figure 1(d): wide angle XRD patterns of OMC (A) and N-OMC (B).

Figure 2

XPS spectra of the N-OMC (a), N1s (b) and C1s (c) of N-OMC.

Figure 3

Nyquist plots of N-OMC/PVA/Lac/Au, OMC/PVA/Lac/Au and bare Au electrode (inset) in 5 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆]/0.1 M KCl solution.

Figure 4

(a) CV plots of N-OMC/PVA/Lac/Au (A), OMC/PVA/Lac/Au (B) and N-OMC/PVA/Au (C) electrodes in 0.1 M PBS (pH 5.0) containing 0.05 mM catechol. (b) Current versus the square root of scan rate plots of N-OMC/PVA/Lac/Au electrode in 0.1 M PBS (pH 5.0) containing 0.05 mM catechol. Inset of figure 4(b): CV plots of the N-OMC/PVA/Lac/Au electrode in 0.1 M PBS (pH 5.0) containing 0.05 mM catechol at different scan rates from inner to outer: 10, 30, 50, 80, and 100 mV*s⁻¹. (c) Calibration plot of pH vs potential. Inset of figure 4(c): CV plots of the N-OMC/PVA/Lac/Au electrode in 0.1 M PBS (pH 5.0) containing 0.05 mM catechol (pH: 4, 5, 6, 7 and 8).

Figure 5

(a) Effect of applied potential on current response of the N-OMC/PVA/Lac/Au electrode to 0.05 mM catechol in 5.0 PBS. (b) Effect of pH of PBS on current response of the N-OMC/PVA/Lac/Au electrode to 0.05 mM catechol at 0.45 V (vs SCE). pH of PBS: 4.0, 5.0, 6.0, 7.0, 8.0. (c) Effect of temperature on current response of the N-OMC/PVA/Lac/Au electrode to 0.05 mM catechol in 5.0 PBS at 0.45 V (vs SCE).

Figure 6

(a) The calibration curves of the N-OMC/PVA/Lac/Au electrode (A) and OMC/PVA/Lac/Au electrode (B) at 0.45 V (vs SCE). Inset: Amperometric responses of the N-OMC/PVA/Lac/Au electrode (A) and OMC/PVA/Lac/Au electrode (B) on successive additions obtained upon the successive addition of 0.5 mM catechol to stirred blank pH 5.0 PBS at 0.45 V (vs SCE). (b) The Lineweaver-Burk curves of the N-OMC/PVA/Lac/Au electrode (A) and OMC/PVA/Lac/Au electrode (B) at 0.45 V (vs SCE).