Novel DNA Biosensor for Direct Determination of Carrageenan

Abstract

A novel disposable electrochemical biosensor based on immobilized calf thymus double-stranded DNA (dsDNA) on the carbon-based screen-printed electrode (SPE) is developed for rapid biorecognition of carrageenan by using methylene blue (MB) redox indicator. The biosensor protocol for the detection of carrageenan is based on the concept of competitive binding of positively charged MB to the negatively charged dsDNA and carrageenan. The decrement in the MB cathodic peak current (i_pc) signal as a result of the released MB from the immobilized dsDNA, and attracted to the carrageenan can be monitored via differential pulse voltammetry (DPV). The biosensor showed high sensitivity and selectivity to carrageenan at low concentration without interference from other polyanions such as alginate, gum arabic and starch. Calibration of the biosensor with carrageenan exhibited an excellent linear dependence from 1-10 mg L^-1 (R² = 0.98) with a detection limit of 0.08 mg L^-1. The DNA-based carrageenan biosensor showed satisfactory reproducibility with 5.6-6.9% (n = 3) relative standard deviations (RSD), and possessing several advantages such as simplicity, fast and direct application to real sample analysis without any prior extensive sample treatments, particularly for seaweeds and food analyses.

Figure 1

The chemical structures for three basic types of carrageenans.

Figure 2

Schematic competitive electrostatic interaction of dsDNA and carrageenan towards binding with MB and electrical setup of the DNA device. (a) High DPV signal obtained after accumulation of MB in the dsDNA. (b) Lower DPV signal generated after immersion of the dsDNA electrode in the carrageenan solution. (c) The three-electrode system consists of a carbon SPE working electrode, Ag/AgCl reference electrode and a glassy carbon counter electrode.

Figure 3

(a) Differential pulse voltammograms of (i) SPE and (ii) dsDNA-SPE in 20 mM Tris-HCl buffer containing 10 mM NaCl at pH 7.0 after accumulation with MB indicator for 300 s. (b) Effect of dsDNA loading on the MB-dsDNA-SPE response in a mixed 20 mM Tris-HCl/10 mM NaCl buffer at pH 7.0.

Figure 4

(a) Effect of MB concentration on the bare SPE and dsDNA-SPE response in 20 mM Tris-HCl buffer containing 10 mM NaCl (pH 7.0) with 7 min MB accumulation period. (b) DPV peak current signal of MB at dsDNS-SPE from 3-11 min MB accumulation periods. DPV response was measured in 20 mM Tris-HCl buffer in the presence of 10 mM NaCl at pH 7.0. (c) Cyclic voltammograms of dsDNA-SPE interacted with 100 µM MB at different time durations at a scan rate of 50 mV s⁻¹.

Figure 5

Effect of Na⁺ ion concentration on the MB i_pc response. DPV experiment was conducted on the MB-dsDNA-SPE in a mixed 20 mM Tris-HCl/10 mM NaCl electrolyte at pH 7.0.

Figure 6

The DNA-based biosensor response towards various polyanions between concentration range of 1 mg L⁻¹ and 10 mg L⁻¹.