Sensitive detection of dengue serotype-4 on lignin-derived graphene nanofibre and manganese oxide composite by DNA-sensing

Abstract

This research addresses the critical challenge of achieving precise DNA detection in complex biological samples by developing a novel, eco-friendly dengue virus serotype 4 biosensor. Utilizing laser-scribed graphene nanofibers derived from sustainable oil palm lignin, a cost-effective alternative to conventional graphene, the biosensor leverages the material's high surface area and conductivity for enhanced DNA immobilization and signal transduction. While lignin-derived graphene's heterogeneous structure poses challenges to electrochemical performance, this study overcomes these limitations by synthesizing laser scribed-graphene nanofibres (LSGNF) decorated with manganese oxide nanoparticles. Field-emission scanning electron microscopy images revealed a highly advantageous three-dimensional, interconnected network of thin, wrinkled sheets forming a porous scaffold, providing efficient electron transport and enhanced molecular interaction. Transmission electron microscopy further confirmed the successful integration of Mn₃O₄ nanoparticles onto the LSGNF surface. Fourier Transform Infrared Spectroscopy and Raman spectroscopy corroborated the formation of the LSGNF/Mn₃O₄ nanocomposite, demonstrating successful material synthesis. A dengue-specific DNA probe was then immobilized onto the nanocomposite surface, and its hybridization with complementary target DNA was evaluated. Successful DNA immobilization and hybridization were confirmed by the detection of phosphorus and nitrogen peaks using FTIR and X-ray photoelectron spectroscopy. Electrochemical impedance spectroscopy revealed the biosensor's high sensitivity, detecting dengue DNA at femtomolar concentrations (10⁻¹⁵ M) with a significant decrease in impedance upon hybridization and a high signal-to-noise ratio (SNR) of 3:1, surpassing typical DNA biosensors with SNRs of 1:1 to 2:1. The biosensor exhibited excellent selectivity, optimized between SMM, TMM and NC, stability over an 8-week study, and reproducibility across five repetitive IDEs. This promising advancement in dengue diagnostics offers a faster response, improved sensitivity, and cost-effectiveness for early disease detection and management.

Keywords: Dengue biosensor; Laser-scribed graphene; Manganese oxide nanoparticle; Nanomaterial.

Fig. 1

(a, b) FESEM images of Bare LSG. (c, d) FESEM images of LSGNF/Mn₃O₄. (e–k) Elemental mapping images of Bare LSG and LSGNF/Mn₃O₄.

Fig. 2

(a, b) Low magnification and (c) High magnification TEM images of Bare LSG; inset shows overlay of imaginary cubic shapes. (d, e) Low magnification and (f) High magnification TEM images of LSGNF/Mn₃O₄; inset shows overlay of imaginary cubic shapes.

Fig. 3

XRD spectra of Bare LSG, (i) before and (ii) after deposition of Mn₃O₄.

Fig. 4

Raman spectra of Bare LSG, (i) before and (ii) after deposition of Mn₃O₄.

Fig. 5

FTIR spectra of LSGNF/Mn₃O₄ bioelectrode upon (i) immobilization and (ii) hybridization of DNA sequences from Aedes Aegyptii serotype 4. Transmittance region: 700–1800 cm⁻¹.

Fig. 6

Survey scan of XPS core level spectra for (i) Bare LSG, (ii) immobilized (LSG/LSGNF/Mn₃O₄/ssDNA) and hybridized (LSG/LSGNF/Mn₃O₄/dsDNA).

Fig. 7

(a) Impedimetric curve of (i) Bare LSG, (ii) LSGNG/Mn₃O₄, (ii) immobilized LSGNF/Mn₃O₄ (probe), and (iii) hybridized LSGNF/Mn₃O₄ (target) bioelectrode; the inset shows the Randles equivalent circuit, where the parameters R_s, R_ct, Z_w, and CPE represent the bulk solution resistance, charge transfer resistance, Warburg impedance, and constant phase element respectively. (b) Impedance spectra of LSGNF/Mn₃O₄ hybridized with different concentrations of complementary target DNA (i–v) 1 fM to 1 nM, (c) illustrates the linear regression curve at different concentrations of target DNA with the linear equation: ΔR_ct = 1.964 × 10³x + 3.1837 × 10⁴, (R₂ = 0.96688), (d) bar chart showing specificity of the LSGNF/Mn₃O₄ bioelectrode against mismatching and non-complementary DNA, (e) reproducibility curve of 5 parallel bioelectrodes fabricated under similar processing conditions, (f) stability of LSGNF/Mn₃O₄ biosensors.