Amperometric biosensor for sulfite determination in beverages using glassy carbon modified with hybrid nano-material electrode in simple flow injection system

This work presented the development of sulfite biosensor for determination of sulfite in a simple flow injection system. The biosensor was developed based on the hybrid materials, composed of carboxylic functionalized carbon nanotubes, poly (diallyldimethy-Iammoniachloride) and gold nanoparticle (CNTs-PDDA-AuNPs) coated on a glassy carbon (GC) electrode, which constructed an effective immobilization matrix and made the immobilized components hold high stability and bioactivity. Sulfite oxidase (SOx) was immobilized to CNTs-PDDA-AuNPs and cytochrome C composites film by using glutaraldehyde (Glu). This developed biosensor (GC/CNTs-PDDA-AuNPs/SOx) was applied in flow injection analysis (FIA). Performances for determination of sulfite were investigated using two approaches including direct method or detection the oxidation of sulfite and indirect method or detection the reduction of hydrogen peroxide. Flow through electrochemical cell using GC/CNTs-PDDA-AuNPs/SOx as a working electrode, Ag/AgCl as a reference electrode and Pt wire as a counter electrode was applied for the detection.
In the direct method, oxidation of sulfite was studied at the GC/CNTs-PDDA-AuNPs/Sox using a solution of 0.1 M phosphate buffer (pH 8.0) as a carrier and applying a potential of 0.3 V. The proposed method exhibits linear calibration over the range of 2-200 mg L-1 of sulfite with slope of 204.66 nA mg-1 L and correlation coefficient of 0.9991. The detection limit (3) was 1.3 mg L-1. The developed biosensor also provided good precision (RSD=3.8%) for sulfite signal (5 mg L-1, n=20) with a rapid sample throughput (57 samples h-1).
In the indirect method, response current is based on the reduction of hydrogen peroxide which produced during the enzyme-catalyzed reaction. Reduction of hydrogen peroxide was studied at the developed biosensor (GC/CNTs-PDDA-AuNPs/SOx) using cyclic voltammetry (CV) and amperometry. The optimization potential for the amperometric detection was -0.4 V. The a amperometric detection of sulfite was performed in flow injection system using solution of 0.1 M phosphate buffer (pH 8.0) as a carrier and applying a potential of -0.4 V. Linear concentration dependence is observed in the range between 500 to 1500 mg L-1. The regression equation is given by y=7.61x-1792.3 (r2=0.9697), when y and x are the area of peak current (nA) and sulfite concentration (mg L-1). The detection limit (3) was 44.25 mg L-1, %RSD-2.93. Sample throughput was 65 samples h-1. From experimental results, direct method offers higher sensitively and wider dynamic range than indirect method.