Label-Free DNA Biosensor Based on Reduced Graphene Oxide and Gold Nanoparticles

Abstract

Currently available DNA detection techniques frequently require compromises between simplicity, speed, accuracy, and cost. Here, we propose a simple, label-free, and cost-effective DNA detection platform developed at screen-printed carbon electrodes (SPCEs) modified with reduced graphene oxide (RGO) and gold nanoparticles (AuNPs). The preparation of the detection platform involved a two-step electrochemical procedure based on GO reduction onto SPCEs followed by the electrochemical reduction of HAuCl₄ to facilitate the post-grafting reaction with AuNPs. The final sensor was fabricated by the simple physical adsorption of a single-stranded DNA (ssDNA) probe onto a AuNPs-RGO/SPCE electrode. Each preparation step was confirmed by morphological and structural characterization using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy, respectively. Furthermore, the electrochemical properties of the modified electrodes have been investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The results demonstrated that the introduction of AuNPs onto RGO/SPCEs led to an enhancement in surface conductivity, a characteristic that favored an increased sensitivity in detection. The detection process relied on the change in the electrochemical signal induced by the binding of target DNA to the bioreceptor and was particularly monitored by the change in the charge transfer resistance of a [Fe(CN)₆]^4-/3- redox couple added in the test solution.

Keywords: DNA detection; biosensor; electrochemistry; gold nanoparticles; graphene.

Figure 1

SEM images recorded at 2000× magnification (30 µm scale) for RGO/SPCEs functionalized with AuNPs from (A) 1 mM, (B) 5 mM, (C) 10 mM, and (D) 15 mM HAuCl₄ solutions in 0.5 mM H₂SO₄.

Figure 2

SEM images recorded at 100,000× magnification (500 nm scale) for RGO/SPCEs functionalized with AuNPs from (A) 1 mM, (B) 5 mM, (C) 10 mM, and (D) 15 mM HAuCl₄ solutions in 0.5 mM H₂SO₄.

Figure 3

High-resolution C1 XPS spectra of bare SPCE modified with (A) GO, (B) RGO, and (C) AuNPs. The orange curve represents the C1s high-resolution spectrum of GO, RGO, and AuNPs-RGO thin layer, respectively, on the surface of the electrode.

Figure 4

High-resolution XPS Au 4f spectra of SPCE modified with RGO and AuNPS.

Figure 5

(A) CV and (B) EIS Nyquist plot recorded in 1 mM [Fe(CN)₆]^3–/4–, 0.1M KCl, for RGO/SPCE functionalized with AuNPs from 1 mM, 5 mM, 10 mM, and 15 mM HAuCl₄ solutions in 0.5 mM H₂SO₄.

Figure 6

(A) CV and (B) EIS Nyquist plot recorded in 1 mM [Fe(CN)₆]^3–/4–, 0.1 M KCl, for bare SPCE, GO/SPCE, RGO/SPCE, and AuNPs-RGO/SPCE.

Figure 7

(A) CV and (B) EIS Nyquist plot recorded in 1 mM [Fe(CN)₆]^3–/4–, 0.1 M KCl, for bare SPCE, GO/SPCE, and AuNPs-GO/SPCE.

Figure 8

(A) CV curves of AuNPs-RGO/SPCE at different scan rates in 1 mM [Fe(CN)₆]^3–/4– solution with 0.1M KCl. (B) Plot of anodic and cathodic peaks current vs. square root of scan rates.

Figure 9

(A) Anodic peaks in CV (after baseline correction) recorded in 1 mM [Fe(CN)₆]^3–/4–, 0.1 M KCl, for (a) AuNPs-RGO/SPCE modified with (b) 500 nM DNA probe and hybridized with (c) 1 nM, (d) 10 nM, (e) 50, and (f) 100 nM DNA target. (B) Charge transfer resistance recorded by EIS in 1 mM [Fe(CN)₆]^3–/4–, 0.1 M KCl, for AuNPs-RGO/SPCE modified with 500 nM DNA probe and hybridized with 1 nM, 10 nM, 50, and 100 nM DNA target.

Figure 10

(A) Typical chronocoulometric curves illustrated for dsDNA/AuNPs-RGO/SPCE electrodes using 100 μM Ru(NH₃)³⁺ as a redox indicator. (B) The calculated oligonucleotides surface density before (ssDNA) and after hybridization with 100 nM DNAt (dsDNA) suggests the formation of dsDNA after DNA target addition, with lower affinity for the modified SPCE surface.