Electrochemical Detection Platform Based on RGO Functionalized with Diazonium Salt for DNA Hybridization

Abstract

In this paper, we propose an improved electrochemical platform based on graphene for the detection of DNA hybridization. Commercial screen-printed carbon electrodes (SPCEs) were used for this purpose due to their ease of functionalization and miniaturization opportunities. SPCEs were modified with reduced graphene oxide (RGO), offering a suitable surface for further functionalization. Therefore, aryl-carboxyl groups were integrated onto RGO-modified electrodes by electrochemical reduction of the corresponding diazonium salt to provide enough reaction sites for the covalent immobilization of amino-modified DNA probes. Our final goal was to determine the optimum conditions needed to fabricate a simple, label-free RGO-based electrochemical platform to detect the hybridization between two complementary single-stranded DNA molecules. Each modification step in the fabrication process was monitored by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) using [Fe(CN)6]3−/4− as a redox reporter. Although, the diazonium electrografted layer displayed the expected blocking effect of the charge transfer, the next steps in the modification procedure resulted in enhanced electron transfer properties of the electrode interface. We suggest that the improvement in the charge transfer after the DNA hybridization process could be exploited as a prospective sensing feature. The morphological and structural characterization of the modified electrodes performed by scanning electron microscopy (SEM) and Raman spectroscopy, respectively, were used to validate different modification steps in the platform fabrication process.

A Novel Approach Using Reduced Graphene Oxide for the Detection of ALP and RUNX2 Osteogenic Biomarkers

“…However, the intensity ratio of the D and G bands (I D /I G ) increased from 0.77, measured for GO, to 1.22 in the case of RGO, clearly indicating an elevated level of defects in the structural framework of RGO caused by the reduction in graphitic domains when functional groups were removed from the GO surface. These results are not only confirmed by our previous studies [39,40] but also by other research groups like Das et al [42] and Bharath et al [43].…”

“…First, a bare SPCE was investigated (Figure 2A) to serve as a baseline for comparison with subsequent modifications. The image shows a clean, unmodified surface, typical for this substrate as observed in our previous studies [39,40]. Figure 2B presents the electrode modified with GO, showing thin (probably just few stratum of GO sheets), wrinkled, transparent layers covering the carbon electrode, a morphology that does not suffer drastic changes after GO electrochemical reduction, as observed in Figure 2C.…”

“…In this context, our paper presents a pilot study and focuses on the design phase and the identification of key parameters required to fabricate in the end a biosensor for ALP and RUNX2 detection. Our prior work paved the way for the current study, where we have specifically tailored the application towards detecting these specific osteogenic biomarkers, and this new direction represents a distinctive contribution, as our earlier efforts primarily centered on generic DNA sequences [39][40][41]. Therefore, we show that modifying screenprinted carbon electrodes (SPCEs) with RGO can lead to the design of a novel detection platform for specific biomarkers, i.e., ALP and RUNX2, with utmost importance for bone regeneration processes.…”

Critical Design Factors for Electrochemical Aptasensors Based on Target-Induced Conformational Changes: The Case of Small-Molecule Targets

“…In the electrochemical biosensors’ context, the electrochemically assisted covalent modification of the electrode surface is usually selected due to the possibility to easily control the surface reaction, and thus improve the overall reproducibility of the sensor fabrication process [ 1 ]. For example, the electrochemical reduction of aryl diazonium salts became a well-established tool for biosensor development [ 7 , 150 , 151 , 152 , 153 , 154 ]. Despite several challenges such as controlling the surface composition when preparing mixed layers, diazonium electrografting is simple, versatile, and, unlike the self-assembly method, involves short preparation times and long-term stability [ 155 ].…”

Solid Phase Oligo-DNA Extraction from Complex Medium Using an Aminated Graphene/Nitrocellulose Membrane Hybrid

“…Furthermore, the versatile nature of NH 2 -PEG-rGO extends beyond its role as a support material for ssDNA immobilization, encompassing the facile adsorption of ssDNA on the rGO lattice, which involves both covalent and non-covalent interactions, with a particular emphasis on non-covalent pi-stacking interactions as the primary pathway. Conversely, double-stranded (ds) DNA exhibits a weaker binding affinity to the rGO surface compared to ssDNA, primarily because of the electrostatic repulsion from the phosphate backbone [ 33 , 34 , 35 , 36 ].…”