A Sensitive and Selective Label-Free Electrochemical DNA Biosensor for the Detection of Specific Dengue Virus Serotype 3 Sequences

Abstract

Dengue fever is the most prevalent vector-borne disease in the world, with nearly 100 million people infected every year. Early diagnosis and identification of the pathogen are crucial steps for the treatment and for prevention of the disease, mainly in areas where the co-circulation of different serotypes is common, increasing the outcome of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Due to the lack of fast and inexpensive methods available for the identification of dengue serotypes, herein we report the development of an electrochemical DNA biosensor for the detection of sequences of dengue virus serotype 3 (DENV-3). DENV-3 probe was designed using bioinformatics software and differential pulse voltammetry (DPV) was used for electrochemical analysis. The results showed that a 22-m sequence was the best DNA probe for the identification of DENV-3. The optimum concentration of the DNA probe immobilized onto the electrode surface is 500 nM and a low detection limit of the system (3.09 nM). Moreover, this system allows selective detection of DENV-3 sequences in buffer and human serum solutions. Therefore, the application of DNA biosensors for diagnostics at the molecular level may contribute to future advances in the implementation of specific, effective and rapid detection methods for the diagnosis dengue viruses.

Keywords: DNA biosensors; dengue fever; differential pulse voltammetry; electrochemical detection; guanine oxidation.

Figure 1

Flowchart of the selection criteria used to design the DENV-3 probe.

Figure 2

Electrochemical signals of different concentrations of DENV-3 probe onto pencil graphite electrodes (PGE). Differential pulse voltammetry (DPV) was used for electrochemical analysis based on guanine oxidation. Experimental conditions: Scanning potential range between +0.5 V and +1.2 V and scan rate of 0.05 V/s. The results represent the average of triplicates carried out at each DENV-3 probe concentration.

Figure 3

Current signals obtained for different DENV-3 target sequence concentrations after hybridization with probe-modified PGEs. Inset: Related calibration graph at a concentration range of 10–100 nM for the target sequence. Experimental conditions: Scanning potential range between +0.5 V and +1.2 V and scan rate of 0.05 V/s.

Figure 4

Differential pulse voltammograms corresponding to bare PGE (a), probe-modified PGE before (b) and after hybridization with 250 nM of target sequence (c) in 20mM Tris-HCl buffer solution (pH 7.0). Experimental conditions: Scanning potential range between +0.5 V and +1.2 V and scan rate of 0.05 V/s.

Figure 5

Differential pulse voltammograms for guanine oxidation of (a) bare PGE; (b) probe-modified PGE; (c) probe-modified PGE after hybridization with DENV-3 sequence; (d) non-complementary sequence and (e) a mixed solution of DENV-3 sequence and non-complementary sequence. Experimental conditions: Scanning potential range between +0.5 V and +1.2 V and scan rate of 0.05 V/s.

Figure 6

Current peaks related to guanine oxidation of the probe-modified-PGE after (a) and before hybridization with DENV-3 (b); in the presence of non-complementary sequences (c) and in a solution mixed with DENV-3 and non-complementary sequences (d), all diluted in human serum. Experimental conditions: Scanning potential range between +0.5 V and +1.2 V and scan rate of 0.05 V/s.