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.

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“…These results clearly demonstrate that the application of the potential (+1.8 V) on the PGE surface exerts a positive effect on the adsorption of the oligonucleotides (Souza et al, 2011). The pretreatment of the carbon surface increases its roughness and hydrophilicity, thus facilitating the electrodes' adsorption to DNA (Pividori et al, 2000;Sabzi et al, 2008;Souza et al, 2011;Wang et al, 1996).…”

“…Then, the inosine-modified probe (guanine-free) was immobilized on the non-activated and activated PGE. The probe immobilization on the electrode surface occurred by adsorption, which is considered the simplest method to immobilize DNA because it does not require special reagents or nucleic acid modifications (Pividori et al, 2000;Souza et al, 2011).…”

“…Influence of the pretreatment on PGE surface The electrode surface was pretreated applying a potential of +1.8 V for 5 min in order to obtain a more sensitive and stable analytical signal Pournaghi-Azar et al, 2006;Souza et al, 2011). Then, the inosine-modified probe (guanine-free) was immobilized on the non-activated and activated PGE.…”

“…The electrochemical behavior of the PGE surface was studied using the differential pulse voltammetry (DPV) technique in 20 mM Tris-HCl buffer (pH 7.0) Pournaghi-Azar et al, 2006;Pournaghi-Azar et al, 2009;Souza et al, 2011). A scanning of the electrode potential was held between +0.3 V and +1.2 V, at a pulse amplitude of 50 mV and a scan rate of 20 mV s À1 .…”

“…The electrochemical activation of the surface polished PGEs was carried out applying a fixed potential of +1.8 V in 0.5 M acetate buffer solution (pH 5.0), no stirring, for 5 min Pournaghi-Azar et al, 2006;Souza et al, 2011).…”