Strategies in the optimization of DNA hybridization conditions and its role in electrochemical detection of dengue virus (DENV) using response surface methodology (RSM)

Abstract

In recent years, limited research has been conducted on enhancing DNA hybridization-based biosensor approaches using statistical models. This study explores the application of response surface methodology (RSM) to improve the performance of a DNA hybridization biosensor for dengue virus (DENV) detection. The biosensor is based on silicon nanowires decorated with gold nanoparticles (SiNWs/AuNPs) and utilizes methylene blue as a redox indicator. The DNA hybridization process between the immobilized DNA probe and the target DENV gene was monitored using differential pulse voltammetry (DPV) based on the reduction of methylene blue. Fourier-transform infrared spectroscopy (FTIR) and electrochemical impedance spectroscopy (EIS) were employed to confirm successful DNA hybridization events on the modified screen-printed gold electrode (SPGE) surface. Several parameters, including pH buffer, NaCl concentration, temperature, and hybridization time, were simultaneously optimized, with NaCl concentration having the most significant impact on DNA hybridization events. This study enhances the understanding of the role of each parameter in influencing DNA hybridization detection in electrochemical biosensors. The optimized biosensor demonstrated the ability to detect complementary oligonucleotide and amplified DENV gene concentrations as low as 0.0891 ng µL^-1 (10 pM) and 2.8 ng µL^-1, respectively. The developed biosensor shows promise for rapid clinical diagnosis of dengue virus infection.

Fig. 1. Systematic of the fabrication and mechanism detection of our developed biosensor.

Fig. 2. FTIR spectra of (a) modified electrode (b) before hybridization (c) after hybridization.

Fig. 3. Nyquist plots obtained for different modified SPGE (analysis of (a) bare SPGE, (b) SiNWs/SPGE (c) AuNPs-SiNWs/SPGE, (d) ssDNA probe/AuNPs/SiNWs-SPGE and (e) hybridized/AuNPs-SiNWs/SPGE in 1.0 mM [Fe(CN)6]^3−/4− containing 0f 0.1 M KCl at 0.20 V, frequency range 0.1 Hz to 100 KHz at amplitude 5 mV. Inset: equivalent circuit used to fit the EIS data; R_s, solution resistance; R_et, electron transfer resistance and C_dll, double layer capacitance.

Fig. 4. D response graph showing the effect of pH buffer, NaCl concentration, hybridization time and hybridization temperature on hybridization efficiency signal by our developed biosensor. Response surface curve of the influence of the interaction of various factors on the hybridization efficiency; (i) displays the influence of NaCl concentration and pH buffer on the hybridization efficiency; (ii) displays the influence of temperature and pH buffer on the hybridization efficiency; (iii) displays the influence of hybridization time and hybridization temperature on the hybridization efficiency; (iv) displays the influence temperature and NaCl concentration on the hybridization efficiency; (v) displays the influence temperature and NaCl concentration on the hybridization efficiency; (vi) displays the influence hybridization time and NaCl concentration on the hybridization efficiency; (vii) displays the influence hybridization time and temperature on the hybridization efficiency.

Fig. 5. The sensitivity and selectivity studies of developed DNA sensor; (a) DPV response of SiNWs/AuNPs-SPGE at different concentration of target DNA; (b) the comparison of calibration curves for the level of detection before and after optimization by the developed DNA sensors; (c) DPV response of the DNA biosensor for selectivity studies involving non-complementary, single-base mismatch, three-base mismatch, and complementary DNA sequences.

Fig. 6. (a) DPV response of SiNWs/AuNPs-modified electrode at different concentration of amplified ssDNA from blood spiked dengue virus; (b) calibration curve of the biosensor response at the different concentration ranging from 1.4 ng µL⁻¹ to 360 ng µL⁻¹ of genomic ssDNA concentration.