Label-free electrochemical analysis of purine nucleotides and nucleobases at disposable carbon electrodes in microliter volumes
Introduction
Besides techniques using a variety of nucleic acids labels (e.g., enzymes, nanoparticles including quantum dots or transition metal complexes) developed to improve sensitivity and specificity of the DNA electrochemical analysis [[1], [2], [3], [4], [5]] label-free electrochemical approaches for the detection and determination of nucleic acid components are still attractive for the researchers due to their analytical performance, inexpensiveness and simplicity [2,4,6]. Carbon-based electrodes in combination with the label-free DNA detection has been based on the direct electro-oxidation of purine (guanine and adenine) nucleobases and corresponding nucleosides and nucleotides (deoxyguanosine, deoxyadenosine and corresponding nucleoside 5′-monophosphates – dGMP and dAMP, respectively) [2,6,7]. Quite recently, it has been shown by our group that pyrolytic graphite in basal orientation can also be advantageously used to study cathodic reduction of canonical nucleobases in ODNs [8].
A detailed description of the electro-oxidation mechanisms of purine nucleobases and nucleosides/nucleotides at the pyrolytic graphite electrode was carried out by the Dryhurst laboratory at the beginning of the 1970s [9]. In general, both guanine and adenine on carbon-based surfaces give well-separated irreversible oxidation waves with associated peak potentials near +0.7 V (Gox) and +1.0 V (Aox) versus Ag|AgCl|3 M KCl, respectively [2,6,7]. The peak Gox can be ascribed to −4e−, −4H+ oxidation of guanine while the peak Aox reflects a complex oxidation process of adenine involving the transfer of −6e− and −6H+ [[9], [10], [11]]. Electro-oxidation of corresponding nucleosides or nucleotides occur at remarkably more positive potentials, compared to the parent free nucleobases (dGMP at +0.9 V and dAMP at +1.2 V) [2,6,7]. The observed potential shifts have been explained by inductive effect caused by the N-glycosidic bond on the π-system of the aromatic purine rings, making it more difficult to remove electrons from the sugar-bound nucleobases [12,13]. Moreover, lower diffusion coefficients of the nucleosides/nucleotides compared to those of the free nucleobases are reflected in significantly lower oxidation peak currents of the former compounds. In addition, electrostatic interactions between the negatively charged phosphate groups and the positively charged electrode surface during potential scanning may cause orientation of the nucleobase residues in nucleotides away from the electrode surface toward the solution, thus increasing the energy necessary for reorganization of the nucleotide at the surface after adsorption and before the charge transfer and causing a further decrease in the oxidation peak heights of nucleotides compared to the uncharged nucleosides [2,6,7,12]. Additionally, the positions and heights of oxidation peaks of nucleobases, nucleosides and nucleotides are influenced by properties of solution, such pH [2,7,9].
Firm adsorption of nucleic acids (NA), including DNA and RNA, at the graphite-based electrodes allows to use adsorptive transfer stripping procedures [2]. Firstly, NA is adsorbed on the electrode surface from a few-microliter drop followed by transfer of NA-modified electrode to the background electrolyte. This approach leading to sensitive detection of small quantities of various NA samples, including short synthetic oligodeoxynucleotides (ODN), has extensively been used during the past three decades. In contrast to long DNA or ODN molecules, nucleobases are relatively weakly-adsorbed [2]. Sensitivity of their electroanalysis can be improved after the complexation with copper ions [[14], [15], [16]].
In the middle of the 1990s, about 20 years after the discovery of the oxidation signals of purine nucleobases on graphite surfaces, the oxidation of pyrimidine nucleobases thymine and cytosine on the carbon electrodes was described for the first time [17]. Corresponding oxidation signals Tox and Cox appear around +1.2 V or +1.3 V, respectively, while oxidation peaks for pyrimidine nucleotides are shifted about 200 mV to more positive potentials [2,6,7,17]. Oxidation of cytosine occurs close to the oxidation of the supporting electrolyte at many types of the carbon-based surfaces. In addition, at least 10-fold higher concentrations of pyrimidine nucleobases are required to obtain peak currents comparable to those of the purine ones [7,17,18], which may be partly caused by lower number of transferred electrons [18].
Significant amplification of the electro-oxidation signals of both purine and pyrimidine DNA components has been observed after introduction of carbon-based nanomaterials, such as carbon nanotubes, carbon nanofibers, graphenes and their oxides as the electrode surface modifiers [1,2,6,19]. New carbon-based materials stimulated a number of researchers to further develop electrochemical studies of DNA, ODNs and their monomeric components. Thanks to these nanostructured sp2 materials, it has been possible to detect purine nucleobases and/or purine residues of DNA/ODN at nanomolar and subnanomolar concentrations [6,[20], [21], [22], [23]]. The effort to achieve the best detection limit has led the authors to combine nanostructured sp2 carbon surfaces with different nanoparticles and/or polymer films [1,5,24,25].
Mastering the preparation of surfaces containing different carbon nanoobjects has made it possible to focus on the miniaturization of the electroanalytical system and to perform routine electrochemical analyses in tens-of-microliter volumes. In the literature there are presented mainly two concepts: one uses the so-called inverted electrochemical micro-cells [26,27] and the other involves screen-printed electrodes [28,29]. In order to make the analysis as fast as possible and potentially applicable in clinical laboratories, disposable types of these nanostructured sp2 carbon materials have been introduced. Among the most widely used are epitaxial prepared graphene, pencil graphite electrode (PeGE), or screen printed graphite electrode (SPGE) [19,30,31].
Numerous electrochemical experiments have been performed with both PeGE and SPGEs to date, including DNA hybridization detection [[32], [33], [34]] or detection of all four nucleobases/nucleotides in different DNA samples [18,20,35,36]. Nucleotides in ODN or DNA were usually determined in micromolar concentrations, while detection of both nucleobases (including those in DNA or ODN acid hydrolysates) acid- achieved subnanomolar levels.
This work is focused on the comparison of the properties of disposable PeGE, unmodified SPGE and modified SPGEs with various carbon nanomaterials (namely single- or multi-walled carbon nanotubes and graphene) in voltammetric analysis of purine nucleotides in ODN and nucleobases in acid-hydrolyzed ODNs at nanomolar concentrations in 50-μL drops. Our results show that all tested commercially available electrodes can be used for the detection of submicromolar concentrations of monomeric purine NA components, while only unmodified PeGE and SPGE are suitable for the analysis of ODNs in nanomolar concentrations. On one hand the modification of electrodes by the nanomaterials enhanced the purine oxidation responses, as was described earlier. On the other hand these electrodes exhibited significantly higher background currents resulting in a decrease of the signal/noise ratio. Further, we compared the effect of competitive adsorption of pyrimidine residues in ODN on the electro-oxidation signals of the purine nucleotides and nucleobases on PeGE and SPGEs. Presence of the pyrimidines influences purine oxidation signals only negligibly at SPGE but significantly at the PeGE.
Section snippets
Materials
Synthetic oligodeoxynucleotides (ODNs) were purchased from VBC-BIOTECH (Austria). ODNs were dissolved in triply distilled water. Following ODN sequences were used:
homo-ODNs: 5′-GGGGG-3′(d(G5)), 5′-AAAAAAAAAAAAAAAAAAAAAAAAA-3' (d(A25)); hetero-ODNs: 5′-GGGAAAGGGAAAGGGAAAGGGAAAGGGAAA-3′ (d(G3A3)5), and 5′-TGGGTTTTTTCTCTTTCTCTTCCTTCCTCTCTTTCTCTGGAAAAAAAAAAAAAAAAAAAAAAAAA-3′(d(G5A25Py35)). Concentration of ODN was determined spectrophotometrically using a Libra S22 spectrophotometer (Biochrom Ltd.,
Results and discussion
In this work, we focused on electrochemical detection of nanomolar concentrations of purine containing ODNs and of free purine nucleobases in the ODN hydrolysates. For this purpose we tested 5 different disposable carbon-based electrodes, including commercially available unmodified screen printed graphite electrode (SPGE) and SPGE electrodes modified by carbon nanomaterials (graphene - GPH-SPGE, single-walled carbon nanotubes - SWCNT-SPGE or multi-walled carbon nanotubes - MWCNT-SPGE). The
Conclusion
Medicine, environmental science, biodefence, and agriculture still require sensitive, specific, and fast analysis of nucleic acids. Electrochemistry, as an accurate, simple, inexpensive method, offers robust tools for the development of analytical platforms. Additionally, electrochemical instruments and devices can be easy miniaturized utilizing disposable electrodes suitable for parallel analysis.
Here we tested various commercially available electrodes for the analysis of nanomolar
Acknowledgment
This work was supported by the Czech Science Foundation 17-08971S project, the SYMBIT project reg. no. CZ.02.1.01/0.0/0.0/15_003/0000477 financed from the ERDF, and the research support of IBP CAS (No. 68081707).
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