Electrochemical label-free and sensitive nanobiosensing of DNA hybridization by graphene oxide modified pencil graphite electrode

Abstract

Based on the strong interaction between single-stranded DNA (ss-DNA) and graphene material, we have constructed a novel label-free electrochemical biosensor for rapid and facile detection of short sequences ss-DNA molecules related to hepatitis C virus 1a using graphene oxide modified pencil graphite electrode. The sensing mechanism is based on the superior adsorption of single-stranded DNA to GO over double stranded DNA (ds-DNA). The intrinsic guanine oxidation signal measured by differential pulse voltammetry (DPV) has been used for duplex DNA formation detection. The probe ss-DNA adsorbs onto the surface of GO via the π– π* stacking interactions leading to a strong background guanine oxidation signal. In the presence of complementary target, formation of helix which has weak binding ability to GO induced ds-DNA to release from the electrode surface and significant variation in differential pulse voltammetric response of guanine bases. The results indicated that the oxidation peak current was proportional to the concentration of complementary strand in the range of 0.1 nM–0.5 μM with a detection limit of 4.3 × 10⁻¹¹ M. The simple fabricated electrochemical biosensor has high sensitivity, good selectivity, and could be applied as a new platform for a range of target molecules in future.

Introduction

In recent years, sensitive, effective and rapid detection of specific biomolecules have attracted much attention due to their potential roles in medical diagnosis, genetic screening, biological engineering, food quality analysis and environmental protection. DNA sequence detections have various applications such as detection of target genes, discrimination and classification of various organisms and also detection of genetic based disorders. Various DNA biosensors have been developed including fluorescence techniques [1], [2], surface plasma resonance spectroscopy [3], [4], [5], [6], quartz-crystal microbalance [7], [8], electrochemiluminescence [9], electrochemical [10], [11], [12], [13], [14], [15] and so on. Electrochemical detection has attracted a great deal of attention in the development of biosensors because of low background, simplicity operation, fast response time, high sensitivity, miniaturization, cost effectiveness, and etc. New kind of carbon materials carbon nanotube (CNT), graphene (GN) and graphene oxide GO based DNA sensors have attracted considerable attention in recent years due to a number of the outstanding electronic, thermal and mechanical properties and good chemical stability [16], [17], [18], [19], [20], [21], [22].

Various electrochemical DNA biosensors based on graphene or its derivatives have been developed [19], [23], [24], [25], [26], [27]. Although these approaches have high sensitivity, hybridization indicators or labeled DNA probes are usually needed. Most convenient electrical readout technique is electrochemical impedance spectroscopy (EIS) by using electrochemical redox indicator like [Ru(phen)₃]²⁺, [Fe(CN)₆]^3-/4-, which has been shown to be well suited for hybridization detection [28], [29], [30], [31]. To overcome probe labeling or indicator usage, increasing researches have been made to develop label-free electrochemical DNA biosensor. Signal transduction induced directly from oxidation of guanine or adenine moieties in DNA strands (label-free detection) makes the principle of DNA hybridization detection in direct strategy and seems to be a simple, less time consuming and more applicable strategy in comparison with the others.

The GN modified carbon electrodes were used for the sensitive determination of hybridization based on guanine oxidation signal [32]. Their strategy is similar to unmodified electrodes and both ss-DNA and ds-DNA adsorb on the GN modified electrode surface. Researches showed that GN and GO has superior binding to ss-DNA over rigid ds-DNA and has been used for the fabrication of biosensors for detecting nucleic acids [30], [33], [34], proteins [35], [36], and small molecules [37], [38], [39]. Recently, we developed an electrochemical aptasensor for the determination of thrombin based on decrease of guanine oxidation signal after interaction between nucleotides and thrombin [40].

In this work, we have proposed a platform for the fabrication of electrochemical biosensors by using GO as electrode modifier and short sequence oligonucleotides related to hepatitis C virus selected as model oligonucleotides. Unlike the graphene, oxidized form of graphene, GO, has negative surface charge which result in desorption of probe DNA from electrode surface after hybridization with its target DNA. As shown in Scheme 1, the probe ss-DNA can be easily immobilized on the surface of GO/PGE due to the π-π* hydrophobic physical adsorption and van der Waals attraction between the purine/pyrimidine ring structure in the nucleobases and the hexagonal cells of GO and target molecules will alter the structure of probe ss-DNA and leads to desorption of formed duplex from the surface of GO and decreasing of guanine oxidation signal. Consequently, electrochemical response obtained at the electrode will be changed, thus the target can be detected. By this approach, more practical biosensor was obtained because of the simple electrode preparation and further signal change due to the ds-DNA desorption from electrode surface. Besides, there is no need for inosine substituted probe as used in previous work. This strategy was demonstrated as a convenient, sensitive and selective detection platform for a range of target analytes.