Ultrasensitive Peptide-Based Electrochemical Biosensor for Universal Diagnostic of Dengue

Abstract

Dengue is a neglected disease mainly affecting tropical and subtropical countries. The diagnosis of dengue fever is still a problem since most of it is made from whole or recombinant DENV proteins, which present cross-reactions with other members of the Flavivirus family. Therefore, there is still a huge demand for new diagnostic methods that provide rapid, low-cost, easy-to-use confirmation. Thus, in this study, we developed an affordable electrochemical biosensor for rapidly detecting immunoglobulin G (IgG) serological antibodies in the sera of DENV-infected patients. An identified linear B-cell epitope (DENV/18) specific for DENV 1-4 serotypes recognized by IgG in patient sera was selected as a target molecule after a microarray of peptides using the SPOT-synthesis methodology. After chemical synthesis, the DENV/18-peptide was immobilized on the surface of the working electrode of a commercially available screen-printed gold electrode (SPGE). The capture of DENV-specific IgG allowed for the formation of an immunocomplex that was measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) using a potassium ferrocyanide/ferricyanide ([Fe(CN)₆]^3-/4-) electrochemical probe. An evaluation of the biosensor's performance showed a detection limit of 100 µg mL^-1 for the synthetic peptides (DENV/18) and 1.21 ng mL^-1 in CV and 0.43 ng mL^-1 in DPV for human serum, with a sensitivity of 7.21 µA in CV and 8.79 µA in DPV. The differentiation of infected and uninfected individuals was possible even at a high dilution factor that reduced the required sample volumes to a few microliters. The final device proved suitable for diagnosing DENV by analyzing real serum samples, and the results showed good agreement with molecular biology diagnostics. The flexibility to conjugate other antigenic peptides to SPEs suggests that this technology could be rapidly adapted to diagnose other pathogens.

Keywords: IgG epitope; dengue; electrochemistry; serologic diagnostic; voltammetric biosensor.

Figure 1

The three-dimensional structure of the NS1-DENV-3 (AAT79552) protein, with the position of the DENV/3 epitope, was identified through SPOT synthesis. The epitope is pink within a model constructed using predicted protein structures I-TASSER. The image was created using PyMol.

Figure 2

Cyclic voltammograms (a,c) and differential pulse voltammetry (b,d) of the biosensor immobilized with the DENV/18 peptide: In (c,d) (Curve A) patient’s sera infected for DENV; (Curve B) non-infected human serum; (Curve C) buffer solution PBS.

Figure 3

Correlation between serum dilutions for DENV/18 peptide (904-SFIIDGPNTPEC-916) in the analyses between patient’s sera infected with DENV and non-infected human serum, (a) analysis by CV and (b) DPV.

Figure 4

Linearity of analysis of the biosensor for DENV/18 peptide, in the function of serum dilution with antibodies for DENV (a) analysis by CV and (b) DPV.

Figure 5

Surface scanning electron microscopy (SEM) analysis of the screen-printed gold electrode–SPGE; (a) bare, (b) L-Cys-coated gold electrode, (c) SPGE with L-Cys/GA, and (d) SPGE with L-Cys/GA/ DENV/18 peptide.

Figure 6

Atomic force microscopy (AFM) analysis and 3D images performed on the working electrode of the SPGE: (A) bare gold electrode, (B) L-Cys-coated gold electrode, (C) L-Cys/GA, (D) L-Cys/GA/DENV-18 peptide, (E) L-Cys/GA/DENV-18 peptide/human serum DENV-infected, (F) L-Cys/GA/DENV-18 peptide/human serum not infected with dengue.