Label-free electrochemical detection of the specific oligonucleotide sequence of dengue virus type 1 on pencil graphite electrodes

Abstract

A biosensor that relies on the adsorption immobilization of the 18-mer single-stranded nucleic acid related to dengue virus gene 1 on activated pencil graphite was developed. Hybridization between the probe and its complementary oligonucleotides (the target) was investigated by monitoring guanine oxidation by differential pulse voltammetry (DPV). The pencil graphite electrode was made of ordinary pencil lead (type 4B). The polished surface of the working electrode was activated by applying a potential of 1.8 V for 5 min. Afterward, the dengue oligonucleotides probe was immobilized on the activated electrode by applying 0.5 V to the electrode in 0.5 M acetate buffer (pH 5.0) for 5 min. The hybridization process was carried out by incubating at the annealing temperature of the oligonucleotides. A time of five minutes and concentration of 1 μM were found to be the optimal conditions for probe immobilization. The electrochemical detection of annealing between the DNA probe (TS-1P) immobilized on the modified electrode, and the target (TS-1T) was achieved. The target could be quantified in a range from 1 to 40 nM with good linearity and a detection limit of 0.92 nM. The specificity of the electrochemical biosensor was tested using non-complementary sequences of dengue virus 2 and 3.

Keywords: dengue virus; guanine oxidation; nucleic acid biosensor.

Figure 1.

The differential pulse voltammograms of guanine oxidation on (•) non-activated PGE, (○) activated PGE, (▾) TSF-1P (1 μM) immobilized on non-activated PGE and (Δ) TSF-1P (1 μM) immobilized on activated PGE. Voltammetric conditions: scanning potential steps, 20 mV/s; potential amplitude, 50 mV.

Figure 2.

The differential pulse voltammograms of guanine oxidation on (•) an activated PGE, (▾) TS-1T (1 μM) immobilized on an activated PGE and (○) TS-1P (1 μM) immobilized on an activated PGE. Voltammetric conditions: Scanning potential steps, 20 mV/s. Potential amplitude, 50 mV.

Figure 3.

The differential pulse voltammograms of guanine oxidation at TSF-1P (1 μM) immobilized activated for different times (•) 1 min, (○) 2 min, (▾) 5 min and (Δ) 10 min. Voltammetric conditions: scanning potential steps, 20 mV/s; potential amplitude, 50 mV.

Figure 4.

Current peaks of the guanine oxidation signal with different concentrations of the TS-1P modified activated PGE (0.1 μM, 1 μM, 5 μM and 10 μM). The results were plotted using the means of experiments performed in triplicate.

Figure 5.

The differential pulse voltammograms of guanine oxidation at (a) TS-1P (1 μM) immobilized on activated PGE before hybridization, (b) TS-1P (1 μM) immobilized on activated PGE after hybridization with complementary TS-1T (40 nM), (c) with PolyG-NC (40 nM), (d) with TS-2 NC (40 nM) (e) with TS-3 NC (40 nM) and (f) mixture of complementary and non-complementary (PolyG-NC, TS-2 NC and TS-3 NC) (40 nM each). Voltammetric conditions: Scanning potential steps, 20m V/s. Potential amplitude, 50 mV.

Figure 6.

Plot of ΔI (difference of guanine oxidation signal of the probe-modified PGE in the absence and presence of the target) vs. target concentration. Inset: related calibration graph at concentration range 1–40 nM for complementary target.