Number of graphene layers exhibiting an influence on oxidation of DNA bases: Analytical parameters

doi:10.1016/j.aca.2011.10.054

Analytica Chimica Acta

Volume 711, 20 January 2012, Pages 29-31

https://doi.org/10.1016/j.aca.2011.10.054 Get rights and content

Abstract

This article investigates the analytical performance of double-, few- and multi-layer graphene upon oxidation of adenine and guanine. We observed that the sensitivity of differential pulse voltammetric response of guanine and adenine is significantly higher at few-layer graphene surface than single-layer graphene. We use glassy carbon electrode as substrate coated with graphenes. Our findings shall have profound influence on construction of graphene based genosensors.

Graphical abstract

Highlights

► Double-layer, few-layer and multilayer graphene sheets were used as electrochemical surfaces. ► Guanine and adenine electrooxidation was studied in the presence of cytosine and thymine. ► The number of graphene layers have profound influence on electrochemical oxidation of DNA bases. ► Few-layer graphene provides the most sensitive response.

Introduction

In the current post-genomic era, the development of fast and efficient DNA genosensors continue to be of enormous interest and such advancements are motivated towards improving point-of-care diagnosis as well as on-spot forensic analysis [1], [2], [3]. Detection platforms have also transited from fluorescent-based devices towards the current trend of electrochemical devices [4]. Electrochemical detection of DNA is based on two broad categories: (i) label free oxidation of target bases where signal of guanine (G) or adenine (A) is measured; or (ii) label-employing assays where signal from the label is measured after hybridization [5]. A large variety of electrode materials such as mercury, gold or carbon have been employed in the construction of label-free electrochemical genosensors [1], [2], [3] and recently, nanomaterials such as carbon nanotubes (CNTs) [6] were also used.

Graphene is a novel nanomaterial which exhibit outstanding performance in biosensing and electrochemical sensing [7], [8]. The effectiveness of this nanomaterial is due to a large number of intrinsic properties [9], [10], [11], [12], of which high electrical conductivity [13] and large surface area [14] are especially advantageous for electrochemistry. The most common product of graphite exfoliation is few-layer graphene [15] but in general, graphene-based materials also occur in single-layer (G-SL), double-layer (G-DL), few-layer (G-FL) or multi-layer (stacked) (G-ML) configurations [16]. Recently, numerous research has employed graphene surfaces in DNA sensing [17], [18], [19], [20], [21] and one noteworthy study was done by Loh et al., where they demonstrated the use of anodized epitaxial graphene (EG) for direct DNA detection. Anodized EG material contains oxygenated defects, which according to the authors, were able to improve detection and signal resolution of free bases, individual bases of both single stranded and hybridized DNA [21], [22]. However, in best of our knowledge, there was no study done to compare the influences of number of graphene sheets in graphene-based nanomaterials on oxidation of DNA bases. Previously, we had performed comparison of single-, few- and multilayer graphene for electro-oxidation of dopamine, uric acid and ascorbic acid [23], [24] as well as for the reduction of nitroaromatic explosives [25]. Here, we wish to show for the first time, the influence of number of graphene layers on electrochemical oxidation of DNA bases because it has significant implication for electrochemical sensing of DNA.

Section snippets

Experimental

G-DL and G-FL were obtained from NanoIntegris, IL, USA, while multilayer graphene (product number 698830, base dimensions 100 nm × 100 nm, length 5 μm) and graphite (product number 282863, size of particles 10–20 μm) were purchased from Sigma–Aldrich, USA. According to AFM analysis, the area of the G-DL and G-FL flakes was ∼10,000 nm² and the average length of the edge of the graphene was ∼100 nm. The content of the G-DL was 27% of single-layer, 48% of double-layer, 20% of triple-layer, and 5% of four-

Results and discussion

We investigated the electrochemical oxidation of guanine and adenine in a mixture of four DNA bases (guanine (G); adenine (A); thymine (T); cytosine (C)). G and A are typically chosen in DNA detection studies due to its relative ease of oxidation at potentials ∼0.7 and ∼1 V, respectively, while T and C are also included in the reaction mixture since these bases are naturally presented in DNA molecules. We studied the oxidation of A and G at G-DL, G-FL, G-ML, graphite microparticles, EPPG

Conclusion

In conclusion, we have demonstrated on the example of DNA bases that the use of single-layer graphene is not always beneficial over few-layer graphene. In contrary, few-layer graphene exhibits higher sensitivity towards oxidation of adenine and guanine. These findings have profound impact on the development of graphene based genosensors as graphene fabrication methods commonly lead to the production of few-layer graphene.

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The main reason is that the total current density of individual electrooxidation signals is a sum of current contributions associated with the electrooxidation of both diffusing (in solution phase) and surface-bound (adsorbed) molecules [34,35]. These individual contributions are highly dependent on the distribution of both basal- and edge-oriented graphene sheets and their sizes for individual graphite-based surfaces [34–39]. Moreover, the process of electrochemical oxidation is not only a matter of reversible electric charge transfer between the molecules and the electrode surface but is often accompanied by irreversible chemical reactions [40–43].
In this work we experimentally show the formation of a planarly-arranged guanine multilayer on a pyrolytic graphite surface, which was predicted by theoretical calculations at the ideal graphene surface. In the case of adenine, a multilayered architecture like that of guanine was not confirmed. Changes in the purine bases’ arrangement on charged surface in dependence on their concentration is studied using electrooxidation peaks of guanine and adenine, together with time changes in capacitance of electric double layer. Existence of predicted guanine multilayers is supported by electrochemical findings and confirmed by atomic force microscopy. Moreover, our data suggest a link between an additional guanine electrooxidation peak and supramolecular guanine assembly formation.
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Studies on functionalized graphene exhibiting good solubility and biocompatibility have been reported [27,28]. DNA nucleobases have been reported as proper functional moieties with graphene nanomaterials for electrochemical biosensors that reveals good sensitivity and selectivity [29–31]. Experimental studies on nanographene oxide of few nanometers lateral width functionalized through covalent grafting with polyethylene glycol, folic acid and drug complexes have been reported for the effective solubility of functionalized graphene complexes [32–34].
The density functional theory (DFT) calculations on the chemical functionalization of zigzag graphene nanoribbon (ZGNR) with DNA (Deoxyribonucleic acid) nucleobases were carried out using the local density approximation (DFT-LDA). The binding energies, charge transfer, equilibrium geometries and band gap variations of bare ZGNR (b-ZGNR) and nucleobase functionalized ZGNR (nb-ZGNR), were examined at an electron temperature of 300 K. The functionalization of ZGNR with nucleobases adenine (Anb-ZGNR) and cytosine (Cnb-ZGNR) reveals a significant structural deformation compared to bare ZGNR(b-ZGNR).Our findings suggest that functionalization at the edge of ZGNR is effective in tuning its electronic and structural properties through significant enhancement of dipole moment, reduction in %s character and structural asymmetry, thereby suggesting the possibilities of using functionalized ZGNR as nanocarriers in biomedical applications.
Label-free electrochemical analysis of purine nucleotides and nucleobases at disposable carbon electrodes in microliter volumes
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In the case of 22 nM intact d(G3A3)5 ODN, very well-resolved peaks dAox and dGox were observed at both PeGE (insert in Fig. 1C) and SPGE (insert in Fig. 1F). It has been found that the presence of pyrimidine nucleotides in ODN as well as the incompletely hydrolyzed ODN (DNA) fraction after the acid treatment to release purine bases (apurinic acid) - affects the electrooxidation signals of the purine components [16,52,53]. In the following section, the effect of competitive adsorption of the pyrimidine fraction was studied for 150 nM concentrations of the purine components.
We analyzed purine nucleotides in oligodeoxynucleotides (ODN) and nucleobase residues acid-hydrolyzed ODN at nanomolar concentrations in few tens of microliters of solution at commercially available unmodified graphite pencil electrodes, screen printed graphite electrodes (SPGEs) and SPGEs modified by graphene, single- and multi-walled carbon nanotubes. Our results suggest that specific properties of different graphite-based electrodes have significant impact on ODN analysis. Nanostructure-modified electrodes produced higher signals due to purine nucleobase oxidation but also higher background currents. The unmodified disposable carbon electrodes allowed analysis of both free nucleobases and ODNs. Our results show that unmodified SPGEs represent a good compromise for signal/noise ratio. Electrooxidation of purines at these electrodes was only little affected by the presence of pyrimidines in contrast to the pencil graphite electrode.
Carbon electrodes in electrochemical analysis of biomolecules and bioactive substances: Roles of surface structures and chemical groups
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In this chapter we focus on the properties of graphitic carbon and boron-doped diamond (BDD) electrodes from the point of view of surface micro- and nanostructures, chemistry of surface termination, effects of these characteristics on electron transfer and adsorption characteristics of the electrodes. Their properties are discussed in relation to electrochemical responses measured for typical groups of analytes, such as nucleic acid and protein constituents or other electroactive compounds. In general, basal planes of graphitic materials are sites of preferential adsorption of organic compounds while the edge planes and defects are sites of fast electron transfer for a number of analytes. At the BDD, fast electron transfer kinetics is observed particularly at hydrogenated and polished surfaces. Surface termination with oxygenous groups causes the electron transfer rate to decrease and renders the carbon surface hydrophilic properties, manifested in decreased adsorption of hydrophobic molecules and involvement of coulombic forces and hydrogen bonds in interactions of relevant analytes with the electrode surfaces. Examples of applications in the area of electroanalysis of biomolecules and their components are briefly discussed.
Vertical graphene nanoflakes for the immobilization, electrocatalytic oxidation and quantitative detection of DNA
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Except for the latest reported chemically reduced graphene oxide [9,10], essentially for all other popular electrodes such as gold [11], glassy carbon (GC) [12], CNT [13,14], polymer modified graphite/GC [15] and pyrolytic graphite electrode [16] only the adenine (A) and guanine (G) residues of the dsDNA can electrochemically be oxidized with a poor signal and a low sensitivity. The reason for the reduced graphene oxide being able to detect the four bases of dsDNA could be either the formation of a mixture of single-layer and few-layer sheets which contain highly actively single-layer atomic edge sites [17,18], or different levels of oxygen contents in the formed single/few-layer sheets which significantly lower the barrier of DNA oxidation [19]. Currently, due to the different electrochemical activity and assays employed, the mechanism of the electrooxidation of dsDNA based on different electrodes is not clear and the interaction of DNA with electrodes has a non consistent behavior.
Vertical graphene nanoflake integrated films having a high density of edge planes have been used as an electrochemical platform to systematically investigate the immobilization, electrochemical oxidation kinetics and direct quantitative determination of native DNA. Consistently, both transmission electron microscopy and atomic force microscopy observations demonstrate the presence of a self-assembled monolayer of native DNA, immobilized on the graphene nanoflakes. Graphene shows excellent electrocatalytic activity for the electrooxidation of double stranded DNA, better than carbon nanotubes and glassy carbon, due to the abundance of electrocatalytic graphene edges present not only at the top but also along the sides of each graphene nanoflake.
Electrochemical behaviors and simultaneous determination of guanine and adenine based on graphene-ionic liquid-chitosan composite film modified glassy carbon electrode
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Therefore, the determination of the bases in organism is important in physiology and clinic fields [3,4]. At present, various methods have been established to investigate the concentration of the guanine and adenine [5–19]. However, it is important to develop a simple and novel method with more reliable, high efficiency and convenience for sensitive analysis of guanine and adenine.
A graphene sheets (GS), ionic liquid (IL) and chitosan (CS) modified electrode was fabricated and the modified electrode displayed excellent electrochemical catalytic activities toward guanine and adenine. The transfer electron number (n) and the charge transfer coefficient (α) were calculated with the result as n = 2, α = 0.58 for guanine, and n = 2, α = 0.51 for adenine, which indicated the electrochemical oxidation of guanine and adenine on GS/IL/CS modified electrode was a two-electron and two-proton process. The oxidation overpotentials of guanine and adenine were decreased significantly compared with those obtained at the bare glassy carbon electrode and multi-walled carbon nanotubes modified electrode. The modified electrode exhibited good analytical performance and was successfully applied for individual and simultaneous determination of guanine and adenine. Low detection limits of 0.75 μM for guanine and 0.45 μM for adenine were obtained, with the linear calibration curves over the concentration range 2.5–150 μM and 1.5–350 μM, respectively. At the same time, the proposed method was successfully applied for the determination of guanine and adenine in denatured DNA samples with satisfying results. Moreover, the GS/IL/CS modified electrode exhibited good sensitivity, long-term stability and reproducibility for the determination of guanine and adenine.

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Number of graphene layers exhibiting an influence on oxidation of DNA bases: Analytical parameters

Abstract

Graphical abstract

Highlights

Introduction

Section snippets

Experimental

Results and discussion

Conclusion

J. Power Sources

Electrochem. Commun.

Electrochem. Commun.

Electroanalysis

Nature

Electroanalysis

Anal. Chem.

Microfluid. Nanofluid.

Electroanalysis

Electroanalysis

Science

Science

Science

Nat. Nanotechnol.

Nano Lett.

Angew. Chem. Int. Ed.

Chem. Soc. Rev.

Chem. Soc. Rev.