Sandwich-type graphene electrochemical sensor for nucleic acid detection of SARS-CoV-2

Abstract

Graphene and its derivatives exhibit excellent electrical and mechanical properties, including a high specific surface area, excellent electron mobility, and good biocompatibility, which make them ideal materials for fabricating biosensor devices. Nevertheless, sensors based on pure graphene sensors still have certain limitations. For example, the number of dangling chemical bonds on the graphene surface is insufficient, which restricts the chemisorption of target molecules on the graphene surface. Additionally, graphene tends to stack and self-polymerize due to the presence of strong π-π interactions, van der Waals forces, and high surface energy, which leads to limitations in its semiconductor applications. The incorporation of other nanomaterials (e.g., metals, metal oxides, and conductive polymers) into graphene sheets has been demonstrated to prevent graphene agglomeration and improve the nanostructure. Conductive polymers have been the subject of considerable interest within the context of electronic device manufacturing and the development of electrochemical sensors. This is due to a number of factors, including their low cost, simple preparation, high electrical conductivity, and high compatibility with modern electronic devices. Polypyrrole (PPY), a widely used conductive polymer, has attracted attention, particularly in electrode modification. PPY exhibits excellent electrical conductivity, redox reversibility, biocompatibility, and environmental stability while also offering low production costs, making it an attractive option for use as a conductive polymer. Concurrently, the distinctive structural characteristics of graphene and its oxides render them prospective conductive fillers for conductive polymers. Consequently, the incorporation of graphene into polymers can compensate for their inherent limitations and enhance the long-term stability of sensing materials. The combination of graphene and conducting polymers represents a powerful means of preparing modified electrodes with good electrochemical properties, which have been successfully applied to the electrochemical detection of various biomolecules. For example, Oliveira et al. developed an electrochemical gene sensor based on PPY and graphene quantum dots for the detection of the PML/RARα fusion gene in childhood acute promyelocytic leukemia. As a graphene derivative, reduced graphene oxide (rGO) is similar to graphene in numerous aspects, including favourable electrical conductivity, flexibility, low cytotoxicity, hydrophilicity, a substantial surface-area-to-volume ratio, and elevated chemical resistance. These attributes render rGO an exemplary matrix for nanocomposites. Owing to the presence of hydrophilic and reactive functional groups, rGO is ideal for use in biosensors. The hydrophilic nature of rGO is instrumental in the assembly of biosensors, enabling the fabrication of sensing platforms through techniques such as drop-casting, spin-coating, ink-jet printing, and processing of electrode materials. In the present study, a “sandwich” DNA hybridisation strategy was employed to construct an electrochemical DNA sensor based on PPY-rGO composite nanomaterials. PPY-rGO nanocomplexes were initially prepared by electrochemical deposition and subsequently modified on the surface of a screen-printed carbon electrode (SPCE) to increase the conductivity of the electrode, with SARS-CoV-2 serving as the target. The PPY-rGO nanocomplexes possess a substantial specific surface area and excellent conductivity, in addition to providing many attachment sites for the subsequent electrodeposition of AuNPs by cyclic voltammetry (CV). This enables the immobilization of a greater number of single-stranded DNA (ssDNA) probes, thereby enhancing the sensitivity and specificity of the sensor for the detection of target molecules. To further improve the specificity of detection, two DNA probes were designed on the basis of a sandwich hybridization strategy. One is a specific capture DNA (CDNA) with a sulfhydryl tag, and the other is a signal DNA (SDNA) with a biotin moiety, which can bind to horseradish peroxidase-streptavidin biofunctionalized gold nanoparticles (SA-HRP-AuNPs). Hybridization of the CDNA, target DNA (tDNA), and SDNA on the electrode surface formed a sandwich structure, whereby the SA-HRP-AuNPs bound to the biotin moiety. The detection of tDNA sequences was achieved via differential pulse voltammetry (DPV), which measures the current change of the sensor in hydrogen peroxide (H2O2) and hydroquinone (HQ) as the solvent electrochemical test solution. Electrochemical characterization and sensor performance testing were performed via a convenient electrochemical workstation and PSTrace software from PalmSens (Houten, Netherlands). SPCE electrodes were

1. Introduction

Graphene and its derivatives exhibit excellent electrical and mechanical properties, including a high specific surface area, excellent electron mobility, and good biocompatibility, which make them ideal materials for fabricating biosensor devices [1]. Nevertheless, sensors based on pure graphene sensors still have certain limitations. For example, the number of dangling chemical bonds on the graphene surface is insufficient, which restricts the chemisorption of target molecules on the graphene surface. Additionally, graphene tends to stack and self-polymerize due to the presence of strong π-π interactions, van der Waals forces, and high surface energy, which leads to limitations in its semiconductor applications [2].

The incorporation of other nanomaterials (e.g., metals, metal oxides, and conductive polymers) into graphene sheets has been demonstrated to prevent graphene agglomeration and improve the nanostructure [3]. Conductive polymers have been the subject of considerable interest within the context of electronic device manufacturing and the development of electrochemical sensors. This is due to a number of factors, including their low cost, simple preparation, high electrical conductivity, and high compatibility with modern electronic devices [4]. Polypyrrole (PPY), a widely used conductive polymer, has attracted attention, particularly in electrode modification. PPY exhibits excellent electrical conductivity, redox reversibility, biocompatibility, and environmental stability while also offering low production costs, making it an attractive option for use as a conductive polymer [5]. Concurrently, the distinctive structural characteristics of graphene and its oxides render them prospective conductive fillers for conductive polymers [6]. Consequently, the incorporation of graphene into polymers can compensate for their inherent limitations and enhance the long-term stability of sensing materials. The combination of graphene and conducting polymers represents a powerful means of preparing modified electrodes with good electrochemical properties, which have been successfully applied to the electrochemical detection of various biomolecules [7]. For example, Oliveira et al. [8] developed an electrochemical gene sensor based on PPY and graphene quantum dots for the detection of the PML/RARα fusion gene in childhood acute promyelocytic leukemia.