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Label-free electrochemical analysis of purine nucleotides and nucleobases at disposable carbon electrodes in microliter volumes
Journal of Electroanalytical Chemistry 847 (2019) 113252
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Label-free electrochemical analysis of purine nucleotides and nucleobases at disposable carbon electrodes in microliter volumes
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Stanislav Hasoň, Miroslav Fojta, Veronika Ostatná
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Institute of Biophysics of the Czech Academy of Sciences, Královopolská 135, 61265 Brno, Czech Republic
ARTICLE INFO
ABSTRACT
Dedicated to Professor Claudine Buess-Herman on the occasion of her 65th birthday.
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.
Keywords: Voltammetric analysis Screen printed graphite electrodes Pencil graphite electrode Purine nucleotides Purine nucleobase Microliter volumes
1. 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 [1–5] 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
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reflects a complex oxidation process of adenine involving the transfer of −6e− and −6H+ [9–11]. 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
Corresponding author at: Institute of Biophysics CAS, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic. E-mail address: [email protected] (V. Ostatná).
https://doi.org/10.1016/j.jelechem.2019.113252 Received 27 April 2019; Received in revised form 6 June 2019; Accepted 19 June 2019 Available online 21 June 2019 1572-6657/ © 2019 Elsevier B.V. All rights reserved.
Journal of Electroanalytical Chemistry 847 (2019) 113252
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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 [14–16]. 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 [6,20–23]. 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 [32–34] 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) acidachieved 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. 2. Experimental 2.1. Materials Synthetic oligodeoxynucleotides (ODNs) were purchased from VBCBIOTECH (Austria). ODNs were dissolved in triply distilled water. Following ODN sequences were used: homo-ODNs: 5′-GGGGG-3′(d(G5)), 5′-AAAAAAAAAAAAAAAAAAA AAAAAA-3' (d(A25)); hetero-ODNs: 5′-GGGAAAGGGAAAGGGAAAGGG AAAGGGAAA-3′ (d(G3A3)5), and 5′-TGGGTTTTTTCTCTTTCTCTTCCTT CCTCTCTTTCTCTGGAAAAAAAAAAAAAAAAAAAAAAAAA-3′(d (G5A25Py35)). Concentration of ODN was determined spectrophotometrically using a Libra S22 spectrophotometer (Biochrom Ltd., United Kingdom). The extinction coefficients of ODNs were determined using a configurator based on the nearest-neighbors model [37]. Aliquots of dissolved ODNs were stored at −20 °C in a freezer. Acid hydrolysis of ODNs. Hydrolysis of the ODNs was performed by mixing of the ODN sample with the same volume of 1.0 M HClO4 followed by incubation at 75 °C for 30 min under shaking at 1000 rpm [16]. After that, the samples were cooled down, neutralized with an equal volume of 0.5 M NaOH, and diluted by the background electrolyte solution to the desired final concentration. 2.2. Methods Electrochemical measurements were performed using an Autolab PGStat12 potentiostat (Metrohm, Switzerland) equipped with a frequency-response analyzer module (FRA 2) connected to a three-electrode system consisting of either a disposable unmodified and modified screen-printed graphite electrodes (SPGEs, DropSens, see Scheme 1A) or a disposable pencil graphite electrode (PeGE, see Scheme 1B) which, together with an Ag|AgCl|3 M KCl reference electrode and a platinum plate (area 25 mm2) auxiliary electrode, forms an 50-μL electrochemical drop micro-cell (Scheme 1B). All measurements were carried out at room temperature. In the case of SPGEs (unmodified (cat. No. C110) and also those Scheme 1. Electrochemical drop micro-cell (50-μL) consisting of a three electrode system containing either (A) a screen-printed graphite electrode platform or (B) a disposable pencil graphite electrode (PeGE) together with an Ag|AgCl|3 M KCl reference electrode and a platinum plate auxiliary electrode.
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modified by graphene (DRP-110GPH), single- (DRP-110SWCNT) and multi-walled (DRP-110CNT) carbon nanotubes) from DropSens, the working carbon ink electrode has a diameter of 4 mm (Scheme 1A). At a distance of 1 mm from the working electrode there is a circular auxiliary electrode from carbon ink of 0.8 mm width. This concentric carbon ink auxiliary electrode is discontinued, and the residual area (about 1/7) is covered with a silver paste that serves as a pseudo-reference electrode. This screen-printed three-electrode system was thoroughly wetted with a 50-μL drop of the analyzed solution forming the electrochemical drop micro-cell (Scheme 1A). The PeGE was laboratory-made from a 0.5-mm-diameter Rotring Hipolymer leads (HB grade), which were clamped into the metallic Rotring pencil Tikky II holder so that the 15-mm lengths of the leads were immersed in analyzed solution. The PeGE's electrical contact was obtained by soldering a copper wire to the metallic holder. The electrode areas were determined from cyclic voltammograms of 1 mM Fe(CN)64− in 1 M KCl using Randles-Ševčik equation, α = assuming = 0.5 and D = 6.67 × 10−6 cm2 s−1 [38]. The peak-topeak separation (ΔEp) of 1 mM Fe(CN)64− in 1 M KCl was used to determination of Nicholson's kinetic parameter Ψ [39]. A linear fit of Ψ versus v-1/2 was used to determine the standard heterogeneous rate constant (k0) of each used surface. Adsorptive stripping-differential pulse voltammetry (AdS-DPV) were performed in 0.1 M acetate buffer (pH ~5) with the following settings: pulse amplitude of 50 mV; pulse width of 50 ms; scan rate of 10 mV s−1; potential of accumulation, EA of +0.3 V; accumulation time, tA of 2 min. All voltammograms were smoothed using a SavickyGolay algorithm and baseline-corrected by the moving average method (peak width of 1 mV), using GPES software (Metrohm, Switzerland). Raman spectroscopy was performed on a confocal Raman Microscope Mono Vista CRS+ (S and I, Germany) with a 300 grooves mm−1 grating and a 50× objective at laser excitation energy of 2.33 eV (532.0 nm). The experimental spectra were normalized with the G band intensity and were baseline corrected. SEM pictures were collected on a MAGELLAN 400 L (FEI, USA), operating in gentle-beam mode at 5 kV at a working distance of 4.18 mm. The samples were transferred onto a carbon tape that was held onto a SEM holder for analysis.
nucleobases than the same residues bound in oligonucleotide strands [2,6,7]. Similarly to PeGE, all SPGE electrodes produced well-developed oxidation peaks Aox, Gox, dAox, dGox of the respective analytes with the exception of GPH-SPGE (Fig. 1M). At the latter electrode, the oxidation peak Gox was overlapped by peak of background electrolyte in the range of potentials from +0.55 to +1.0 V. Not only at GPH-SPGE but also at other electrodes with nanostructured surface, responses of background electrolyte were significantly higher than those observed for unmodified surfaces probably as a result of electrode preparation or composition of the nanomaterial [40–42]. Notably, purine oxidation signals measured at individual electrodes (where distinguishable) differed in their heights and potentials (Fig. 1A, D, G, J). All oxidation signals (peaks Aox, Gox, dAox and dGox) obtained at the SPGEs were apparently shifted by about 150 to 200 mV to more negative potentials compared to carbon electrodes (such as PeGE in our case, Fig. 1A, D, G, J, M) connected in conventional threeelectrode setups. These shifts were due to utilization of Ag|AgCl paste as a pseudo-reference electrode in the printed electrodes. Since the pseudoreference electrodes of the individual SPGEs might somewhat differ, we did not further deal with the apparent peak positions. The ratios of peak heights Aox/Gox or dAox/dGox obtained in individual measurements at different electrodes (but with the same analyte sample) also showed certain variations, probably due to varying surface properties of the given SPGEs. Based on previous studies [6,10,43], we suggest that voltammetric responses of free guanine and adenine residues and those of the same bases bound in the ODN chain on graphite-based electrodes are influenced by at the least two factors: by adsorption at the given electrode surface and by the kinetics of electron transfer which both are influenced by the electrode surface morphology and/or chemistry. 3.2. Raman spectra and SEM imaging of various SPGE surfaces We characterized electrodes used in this study by means of Raman spectroscopy and SEM imaging (Fig. 2). The main features in the Raman spectra of carbons are the so-called G (graphite) and D (disorder) bands [44]. Analysis of D and G bands ratio (ID/IG) in Raman spectra, as well as the SEM images revealed remarkable differences among individual surfaces. Fig. 2A shows quite large graphene sheets in basal orientation for PeGE [45] well-correlated with relatively low ratio ID/IG of 0.19 [46]. The SPGE surface with clearly separated, smaller copious graphene-shaped sheets (e.g. rectangles, sometimes ellipses) contained higher amount edges and defects (Fig. 2B) [42,47]. This finding is in good agreement with Raman data, where remarkably higher ID/IG ratio was obtained for SPGE (0.89) than for PeGE (0.19). SPGE modification by multi-walled carbon nanotubes caused an increase of D and G band ratio from 0.88 for unmodified SPGE to 1.13 for MWCNT-SPGE, indicating the latter to be richer in edges and defects. In contrast to MWCNT-SPGE, ratio ID/IG for SWCNT-SPGE decreased to 0.42, probably due to relatively higher representation of side walls in comparison to the ends of the nanotubes [40,48]. Also SEM exhibited significant differences between surfaces modified by either single- or multi-walled nanotubes (Fig. 2C, D). Thick fibers of the MWCNTs created sparser network with accessible areas of supportive material, while soft thin fibers SWCNT formed dense net. GPH-SPGE surface was rich in graphene sheets, larger than those observed for PeGE, in parallel orientation (with basal planes exposed to the solution). Existence of a small number of edges or defects was proved by the lowest ID/IG value (0.09, Fig. 2E).
3. 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 pencil graphite electrode (PeGE) was used as another type of disposable carbon electrode for comparison with the SPGEs. 3.1. Analysis of purines at various SPGE Fig. 1(A, D, G, J, M) shows differential pulse voltammetric responses of purine nucleotides in 500 nM intact d(G3A3)5 ODN (yielding oxidation peaks dGox and dAox, respectively), and acid hydrolysate of the same ODN containing free nucleobase residues (guanine and adenine giving peaks - Gox and Aox, respectively) measured in 0.1 M acetate buffer (pH ~5). For a better comparison of responses obtained at different electrodes, the current responses were converted to the current densities by calculation per unit area (more details in Section 2.2). At the PeGE, guanine was electrooxidized at a potential of +0.77 V (Gox) and adenine at +1.03 V (Aox) (Fig. 1A). Peaks dGox and dAox of the d(G3A3)5 ODN were shifted by about 200 mV to more positive potentials, compared to peaks Gox and Aox, respectively. These results were in good agreement with previous ones showing easier oxidation of free purine
3.3. Kinetics of electron transfer for various SPGE surfaces Ratio of the D and G bands in Raman spectra clearly indicated different relative representation basal planes and edge planes for individual electrodes, which can influence orientation of purine nucleotides/nucleobases residues during interaction with the electrode surface. Nevertheless, surface properties were affected by (i) the inks, containing 3
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Fig. 1. Baseline-corrected AdS-DPVs of (A, D, G, J, M) 500 nM, (B, E, H, K, N) 50 nM and (C, F, I, L, O) 22 nM intact (orange and green curve) and 5 nM acid hydrolyzed (red and blue curve) oligodeoxynucleotide (ODN) d(G3A3)5 obtained in 0.1 M acetate buffer (pH ~ 5) at (A–C) pencil graphite electrode (PeGE), (D–F) screen-printed graphite electrode (SPGE), (G–I) multi-walled carbon nanotube modified SPGE (MWCNT-SPGE), (J–L) single-walled carbon nanotube modified SPGE (SWCNT-SPGE) and (M–O) graphene modified SPGE (GPH-SPGE). The green and orange parts of AdS-DPVs correspond to the deoxyguanosine monophosphate oxidation signal (dGox) and deoxyadenosine monophosphate oxidation signal (dAox), respectively. The red and blue parts of AdS-DPVs correspond to the guanine oxidation signal (Gox) and adenine oxidation signal (Aox), respectively. Black curves represent the blank electrolyte. Accumulation conditions: potential of accumulation, EA of +0.3 V; accumulation time, tA of 2 min. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the loading and functionalized graphite, the amount of organic binder and various impurities, (ii) curing regime and (iii) wettability of the electrode surface [42,47,49]. Different distribution of graphene basal planes and edges influenced also kinetics of electron transfer of the studied electrodes [6,10,42,49]. The electrode surfaces were tested by a commonly used redox probe [Fe(CN)6]4−/3− in 1 M KCl using CVs at different scan rates (Fig. 2F–J). The magnitude of the oxidation peak current densities of Fe(CN)64− oxidation linearly increased with the square root of scan rate (v1/2), yielding similarly dependencies for all surfaces (and indicating a diffusion-controlled process). However, plots of peak-to-peak separation (ΔEp) and Nicholson's kinetic parameter Ψ versus the reciprocal of the scan rate square root (v−1/2) for the Fe (CN)64− differed among individual surfaces (Fig. 2F–O). We used linear fit of Ψ versus v−1/2 to determine the standard heterogeneous rate constant k0. Obtained values k0 of 0.004 cm.s−1 were similar for PeGE and SPGE. Nanoobject-modified electrodes yielded k0 values higher by about 4-times for GPH-SPGE, 5-times for SWNT-SPGE and even 7-times for MWNT-SPGE than those observed for the unmodified SPGE in agreement with earlier observations [40,42,50,51]. Such order correlates
with relative abundance of graphene edges at the given modified electrode surface (see Section 3.2). 3.4. Purine nucleotide and nucleobase residues analysis at low concentrations In next experiments we decreased the concentration of d(G3A3)5 ODN and purine nucleobase residues from 500 nM (used in Fig. 1A, D, G, J, M) to 50 nM. Under these conditions we observed well-developed oxidation peaks Gox and Aox (of free nucleobases) at the all electrode surfaces with the exception of GPH-SPGE as was mentioned before (Fig. 1B, E, H, K, N). However, oxidation peaks dGox and dAox of the intact d(G3A3)5 ODN were observed only for the unmodified SPGE and PeGE (Fig. 1B, E). Absence of the peaks dGox and dAox at voltammograms measured at the MWCNT- and SWCNT-SPGE may be due to (i) limited accessibility of bases in the intact ODN strand, compared to the free purine nucleobase residues and/or (ii) disadvantageous orientation of nucleotides in the ODN and/or (iii) various surface wettability [2,6,10]. It seems that these effects did not play such important role at higher concentrations (such as 4
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Fig. 2. (A–E) SEM pictures of (A) PeGE, (B) SPGE, (C) MWCNT-SPGE, (D) SWCNT-SPGE, and (E) GPH-SPGE. Red lines represent 500 nm. (F–J) The current densities of the oxidation peak of 1 mM Fe(CN)64− and peak-to-peak separation (ΔEp) for 1 mM Fe(CN)64− in 1 M KCl as a function of the square root of scan rate (v1/2). (K–O) Nicholson's kinetic parameter Ψ versus the reciprocal of the scan rate's square root (v-1/2) for 1 mM Fe(CN)64− in 1 M KCl. The current densities, ΔEp and Ψ are means (standard deviations of 10 experiments). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
500 nM) of the analytes (including the ODN), where almost both free bases and the ODN produced well-developed peaks at almost all tested surface (see Section 3.1 and Fig. 1A, D, G, J, M). However, at lower concentrations of the analytes (including the free bases) occurrence of “parasitic” background signal affected analytical usefulness of some of the electrodes tested. Especially the nanomaterial-modified SPCEs produced considerable background signals at potentials close to those of purine, particularly guanine electrooxidation. These parasitic current signals either fully obscured the purine signals or at least decreased the signal-to-noise ratio (see below). ODN detection at concentrations in the range of tens of nM using SWCNT- or MWCNT-SPGE was only possible using the peak Aox. Detection of the peak Gox at all nanomaterial-modified surfaces was either impossible or markedly affected by the background currents (Fig. 1H, K, N). Relatively usable peaks Aox and Gox were obtained at MWCNT-SPGE (Fig. 1H). At 50 nM concentration of each of the purines, the peak Aox and Gox current densities were about 7-fold higher on the MWCNT surface than on unmodified SPGE (Fig. 1E). For 5 nM adenine, Aox peak at MWCNT-SPGE was more than 10-times higher than that at SPGE (Fig. 1F, I). However it should be emphasized that the signal-noise ratio was significantly in favor of the unmodified SPGE (S/ NSPGE~6, S/NMWCNT ~2), at which the background response was significantly smaller. The electrode modification with nanomaterials thus paradoxically worsened analytical figures of merit of the SPGE. Regarding the comparison of SPGE with PeGE, in the case of the Aox, the S/
N ratios were very similar (S/NSPGE~6.0 and S/NPeGE~4.6), whereas the Gox was affected by the background currents on the PeGE (S/NPeGE~1.7) in contrast to well-developed Gox on SPGE (S/NSPGE~6.7). These results indicate that the unmodified SPGE is the most suitable electrode for the label-free detection of low concentrations of ODN/DNA hydrolysates in small volumes. In the case of 22 nM intact d(G3A3)5 ODN, very wellresolved peaks dAox and dGox were observed at both PeGE (insert in Fig. 1C) and SPGE (insert in Fig. 1F). 3.5. Influence of competitive pyrimidine adsorption on purine electrooxidation 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. Based on the above results, unmodified SPGE and PeGE were chosen for these experiments (Fig. 3). For the experiments we used ODN differing in the contents of individual nucleotides, namely: 5G (dG5), 25A (dA25) and 5G + 25A + 23 T + 12C (d(G5A25Py35), see Experimental for sequences). Acid hydrolysate of the d(G5A25Py35) ODN consisted of 5 guanine and 25 adenine free nucleobases and the apurinic ODN
Fig. 3. (A) Baseline-corrected AdS-DPVs of 150 nM intact (solid yellow and green curve) and acid-hydrolyzed (solid red and blue curve) ODN d (G5A25Py35), and an equimolar mixture of 150 nM ODNs d(G5) and d(A25) in both intact (dashed yellow and green curve) and acid-hydrolyzed (dashed red and blue curve) forms obtained in 0.1 M acetate buffer (pH ~5) at SPGE. Black curves represent the blank electrolyte. (B) The effect of competitive adsorption of pyrimidine (Py) fraction within the ODN chain on the intensities of dGox (7-fold higher Py content) and dAox (1.4-fold higher Py content) oxidation signals of purine nucleotides present in the ODN sequence and/or the effect of the presence of Py nucleobase residues on the intensities of Gox and Aox oxidation signals of purine nucleobases removed from the ODN chain during acid hydrolysis, respectively, obtained in 0.1 M acetate buffer (pH ~5) at SPGE, PeGE and basal-oriented pyrolytic graphite electrode (BGPE). Current densities of both dGox and dAox peaks and the corresponding Gox and Aox signals obtained for an equimolar mixtures of d(G5) and d(A25) in both intact and acid-hydrolyzed forms were taken as 100%, respectively. The current densities are average values calculated from 10 independent experiments). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 5
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heights purine nucleotide signals (peaks dGox and dAox) measured at the SPGE increased linearly with the d(G3A3)5 ODN concentration from 22 nM to 500 nM. For equimolar mixture of purine nucleobases the linear dynamic ranges were found between 5 nM and to 500 nM concentration. At higher concentrations all peaks showed a tendency to levelling-off (Fig. 4. LODs, calculated from the standard deviation of the response and the slope of the calibration curve, were determined as 0.6 nM for dG,0.9 nM for dA, 0.5 nM for adenine and 0.3 nM for guanine. 4. 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 concentrations of purine nucleotides in model ODNs and of free purine nucleobases in few tens-of-microliter samples. We compared the purine oxidation signals at unmodified SPGE and PeGE with those obtained at SPGEs modified by graphene, single- and multiwalled carbon nanotubes. At relatively high analyte concentration, welldeveloped oxidation peaks were observed for all tested electrodes. At 50 nM and lower concentration, the analysis at the nanomaterial-modified graphite surface with higher content of edges/defects was limited to detection of free nucleobases, particularly adenine because oxidation peaks of guanine overlapped with strong signals of the background. Modification of the electrodes by the nanomaterials caused a remarkable decrease of signal/noise ratio due to increase of the background currents. The best signal/noise ratio for free purine nucleobases at lower concentrations we observed for unmodified SPGE, followed by PeGE. The SPCE was identified as a comparable electrode with the PeGE for labelfree analysis of nucleotides in ODN at low concentrations. Competitive adsorption of pyrimidine fraction of the ODN (both intact and acid-hydrolyzed) did not significantly influence purines oxidation signals at SPGE, while at the PeGE it caused remarkable decrease of signals of the purine components. Differences in surface properties of the graphitebased electrodes, such as presence of basal planes, edges and defects as well we kinetics of the electron transfer have a significant impact on the electrode properties in the ODN analysis. Thus, our results show the SPGE as the disposable electrode of choice for the analysis of purine-containing ODNs and nucleobase residues in microliter volumes with a negligible interference of the pyrimidine fraction.
Fig. 4. Concentration dependences of the current oxidation signals' densities of (A) (●) dGox and (▲) dAox or (B) (○) Gox and (△) Aox obtained in 0.1 M acetate buffer (pH ~5) containing either intact d(G3A3)5 or acid-hydrolysed d (G3A3)5 at SPGE. Accumulation conditions: EA of +0.3 V; tA of 2 min.
residuum containing 23 thymine and 12 cytosine nucleotides per starting ODN molecule. As a control, the equimolar mixture of two homopurine ODNs: short homoguanine (dG5) and homoadenine (dA25), each at concentration of 150 nM was used. Fig. 3A shows that the presence of 3fold excess of the pyrimidine content had almost no effect (only within statistical errors (Fig. 3B)) on the peaks dGox, dAox of the intact d (G5A25Py35) ODN measured at the SPGE when compared to the corresponding signals of equimolar mixture of homopurine dG5 and dA25 ODNs lacking the pyrimidine fraction (Fig. 3A). The same applies to the comparison of responses obtained for acid-hydrolyzed ODNs (Fig. 3A). In contrast, similar experiment performed with PeGE revealed significant decrease of oxidation peak dAox and Aox by about 70% and 47%, respectively, caused by the presence of the pyrimidine fraction (Fig. 3B). Interestingly, presence of the pyrimidine content in the ODN did not affect the oxidation peak dGox in the intact ODN. However, the current density of oxidation signal Gox of guanine residues in the hydrolysate decreased by about 37%. Similar effects of the pyrimidine fraction on the purine nucleotide/nucleobase electro-oxidation signals were also observed for the basal plane oriented pyrolytic graphite electrode (BPGE), commonly used to study of electro-oxidation of DNA components [2,6,10,11,43]. For both BPGE and PeGE, individual oxidation signals were affected by the pyrimidine content the order of dGox ≪ Gox ≪ Aox ≤ dAox (Fig. 3B). In general, purines are known to be stronger adsorbed at carbonbased electrodes in comparison to pyrimidines [54,55]. Nevertheless, in our study the pyrimidines exhibited higher affinity to electrodes with relatively large abundance of graphite sheets in the basal orientation than to surfaces with higher content of edges and defects (as assessed from relative interference with the purine oxidation signals) [56]. Thus at graphite-based electrodes with prevailing basal planes pyrimidines may show compete stronger competition with the purines [57].
Acknowledgment This work was supported by the Czech Science Foundation 1708971S 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). References [1] J. Galandová, J. Labuda, Polymer interfaces used in electrochemical DNA-based biosensors, Chem. Pap. 63 (2009) 1–14, https://doi.org/10.2478/s11696-0080083-2. [2] E. Paleček, M. Bartošík, Electrochemistry of nucleic acids, Chem. Rev. 112 (2012) 3427–3481, https://doi.org/10.1021/cr200303p. [3] M. Fojta, A. Daňhel, L. Havran, V. Vyskočil, Recent progress in electrochemical sensors and assays for DNA damage and repair, TrAC Trends Anal. Chem. 79 (2016) 160–167, https://doi.org/10.1016/j.trac.2015.11.018. [4] E.E. Ferapontova, DNA electrochemistry and electrochemical sensors for nucleic acids, Annu. Rev. Anal. Chem. 11 (2018) 197–218, https://doi.org/10.1146/ annurev-anchem-061417-125811. [5] S. Campuzano, P. Yáñez-Sedeño, J.M. Pingarrón, Nanoparticles for nucleic-acidbased biosensing: opportunities, challenges, and prospects, Anal. Bioanal. Chem. 411 (2019) 1791–1806, https://doi.org/10.1007/s00216-018-1273-6.
3.6. Purine nucleotide and nucleobase residues detection at SPGE Our results show that SPGE represents in terms of signal/noise ratio and other analytical figures of merit, a good tool for the detection of nanomolar concentrations of purine-containing ODNs and free purine nucleobases. Additionally, the presence of pyrimidine (apurinic) fraction in the samples only negligibly affected the purine oxidation peaks. The 6
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Label-free electrochemical analysis of purine nucleotides and nucleobases at disposable carbon electrodes in microliter volumes
T
Stanislav Hasoň, Miroslav Fojta, Veronika Ostatná
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Institute of Biophysics of the Czech Academy of Sciences, Královopolská 135, 61265 Brno, Czech Republic
ARTICLE INFO
ABSTRACT
Dedicated to Professor Claudine Buess-Herman on the occasion of her 65th birthday.
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.
Keywords: Voltammetric analysis Screen printed graphite electrodes Pencil graphite electrode Purine nucleotides Purine nucleobase Microliter volumes
1. 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 [1–5] 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
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reflects a complex oxidation process of adenine involving the transfer of −6e− and −6H+ [9–11]. 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
Corresponding author at: Institute of Biophysics CAS, v.v.i., Královopolská 135, 612 65 Brno, Czech Republic. E-mail address: [email protected] (V. Ostatná).
https://doi.org/10.1016/j.jelechem.2019.113252 Received 27 April 2019; Received in revised form 6 June 2019; Accepted 19 June 2019 Available online 21 June 2019 1572-6657/ © 2019 Elsevier B.V. All rights reserved.
Journal of Electroanalytical Chemistry 847 (2019) 113252
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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 [14–16]. 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 [6,20–23]. 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 [32–34] 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) acidachieved 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. 2. Experimental 2.1. Materials Synthetic oligodeoxynucleotides (ODNs) were purchased from VBCBIOTECH (Austria). ODNs were dissolved in triply distilled water. Following ODN sequences were used: homo-ODNs: 5′-GGGGG-3′(d(G5)), 5′-AAAAAAAAAAAAAAAAAAA AAAAAA-3' (d(A25)); hetero-ODNs: 5′-GGGAAAGGGAAAGGGAAAGGG AAAGGGAAA-3′ (d(G3A3)5), and 5′-TGGGTTTTTTCTCTTTCTCTTCCTT CCTCTCTTTCTCTGGAAAAAAAAAAAAAAAAAAAAAAAAA-3′(d (G5A25Py35)). Concentration of ODN was determined spectrophotometrically using a Libra S22 spectrophotometer (Biochrom Ltd., United Kingdom). The extinction coefficients of ODNs were determined using a configurator based on the nearest-neighbors model [37]. Aliquots of dissolved ODNs were stored at −20 °C in a freezer. Acid hydrolysis of ODNs. Hydrolysis of the ODNs was performed by mixing of the ODN sample with the same volume of 1.0 M HClO4 followed by incubation at 75 °C for 30 min under shaking at 1000 rpm [16]. After that, the samples were cooled down, neutralized with an equal volume of 0.5 M NaOH, and diluted by the background electrolyte solution to the desired final concentration. 2.2. Methods Electrochemical measurements were performed using an Autolab PGStat12 potentiostat (Metrohm, Switzerland) equipped with a frequency-response analyzer module (FRA 2) connected to a three-electrode system consisting of either a disposable unmodified and modified screen-printed graphite electrodes (SPGEs, DropSens, see Scheme 1A) or a disposable pencil graphite electrode (PeGE, see Scheme 1B) which, together with an Ag|AgCl|3 M KCl reference electrode and a platinum plate (area 25 mm2) auxiliary electrode, forms an 50-μL electrochemical drop micro-cell (Scheme 1B). All measurements were carried out at room temperature. In the case of SPGEs (unmodified (cat. No. C110) and also those Scheme 1. Electrochemical drop micro-cell (50-μL) consisting of a three electrode system containing either (A) a screen-printed graphite electrode platform or (B) a disposable pencil graphite electrode (PeGE) together with an Ag|AgCl|3 M KCl reference electrode and a platinum plate auxiliary electrode.
2
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modified by graphene (DRP-110GPH), single- (DRP-110SWCNT) and multi-walled (DRP-110CNT) carbon nanotubes) from DropSens, the working carbon ink electrode has a diameter of 4 mm (Scheme 1A). At a distance of 1 mm from the working electrode there is a circular auxiliary electrode from carbon ink of 0.8 mm width. This concentric carbon ink auxiliary electrode is discontinued, and the residual area (about 1/7) is covered with a silver paste that serves as a pseudo-reference electrode. This screen-printed three-electrode system was thoroughly wetted with a 50-μL drop of the analyzed solution forming the electrochemical drop micro-cell (Scheme 1A). The PeGE was laboratory-made from a 0.5-mm-diameter Rotring Hipolymer leads (HB grade), which were clamped into the metallic Rotring pencil Tikky II holder so that the 15-mm lengths of the leads were immersed in analyzed solution. The PeGE's electrical contact was obtained by soldering a copper wire to the metallic holder. The electrode areas were determined from cyclic voltammograms of 1 mM Fe(CN)64− in 1 M KCl using Randles-Ševčik equation, α = assuming = 0.5 and D = 6.67 × 10−6 cm2 s−1 [38]. The peak-topeak separation (ΔEp) of 1 mM Fe(CN)64− in 1 M KCl was used to determination of Nicholson's kinetic parameter Ψ [39]. A linear fit of Ψ versus v-1/2 was used to determine the standard heterogeneous rate constant (k0) of each used surface. Adsorptive stripping-differential pulse voltammetry (AdS-DPV) were performed in 0.1 M acetate buffer (pH ~5) with the following settings: pulse amplitude of 50 mV; pulse width of 50 ms; scan rate of 10 mV s−1; potential of accumulation, EA of +0.3 V; accumulation time, tA of 2 min. All voltammograms were smoothed using a SavickyGolay algorithm and baseline-corrected by the moving average method (peak width of 1 mV), using GPES software (Metrohm, Switzerland). Raman spectroscopy was performed on a confocal Raman Microscope Mono Vista CRS+ (S and I, Germany) with a 300 grooves mm−1 grating and a 50× objective at laser excitation energy of 2.33 eV (532.0 nm). The experimental spectra were normalized with the G band intensity and were baseline corrected. SEM pictures were collected on a MAGELLAN 400 L (FEI, USA), operating in gentle-beam mode at 5 kV at a working distance of 4.18 mm. The samples were transferred onto a carbon tape that was held onto a SEM holder for analysis.
nucleobases than the same residues bound in oligonucleotide strands [2,6,7]. Similarly to PeGE, all SPGE electrodes produced well-developed oxidation peaks Aox, Gox, dAox, dGox of the respective analytes with the exception of GPH-SPGE (Fig. 1M). At the latter electrode, the oxidation peak Gox was overlapped by peak of background electrolyte in the range of potentials from +0.55 to +1.0 V. Not only at GPH-SPGE but also at other electrodes with nanostructured surface, responses of background electrolyte were significantly higher than those observed for unmodified surfaces probably as a result of electrode preparation or composition of the nanomaterial [40–42]. Notably, purine oxidation signals measured at individual electrodes (where distinguishable) differed in their heights and potentials (Fig. 1A, D, G, J). All oxidation signals (peaks Aox, Gox, dAox and dGox) obtained at the SPGEs were apparently shifted by about 150 to 200 mV to more negative potentials compared to carbon electrodes (such as PeGE in our case, Fig. 1A, D, G, J, M) connected in conventional threeelectrode setups. These shifts were due to utilization of Ag|AgCl paste as a pseudo-reference electrode in the printed electrodes. Since the pseudoreference electrodes of the individual SPGEs might somewhat differ, we did not further deal with the apparent peak positions. The ratios of peak heights Aox/Gox or dAox/dGox obtained in individual measurements at different electrodes (but with the same analyte sample) also showed certain variations, probably due to varying surface properties of the given SPGEs. Based on previous studies [6,10,43], we suggest that voltammetric responses of free guanine and adenine residues and those of the same bases bound in the ODN chain on graphite-based electrodes are influenced by at the least two factors: by adsorption at the given electrode surface and by the kinetics of electron transfer which both are influenced by the electrode surface morphology and/or chemistry. 3.2. Raman spectra and SEM imaging of various SPGE surfaces We characterized electrodes used in this study by means of Raman spectroscopy and SEM imaging (Fig. 2). The main features in the Raman spectra of carbons are the so-called G (graphite) and D (disorder) bands [44]. Analysis of D and G bands ratio (ID/IG) in Raman spectra, as well as the SEM images revealed remarkable differences among individual surfaces. Fig. 2A shows quite large graphene sheets in basal orientation for PeGE [45] well-correlated with relatively low ratio ID/IG of 0.19 [46]. The SPGE surface with clearly separated, smaller copious graphene-shaped sheets (e.g. rectangles, sometimes ellipses) contained higher amount edges and defects (Fig. 2B) [42,47]. This finding is in good agreement with Raman data, where remarkably higher ID/IG ratio was obtained for SPGE (0.89) than for PeGE (0.19). SPGE modification by multi-walled carbon nanotubes caused an increase of D and G band ratio from 0.88 for unmodified SPGE to 1.13 for MWCNT-SPGE, indicating the latter to be richer in edges and defects. In contrast to MWCNT-SPGE, ratio ID/IG for SWCNT-SPGE decreased to 0.42, probably due to relatively higher representation of side walls in comparison to the ends of the nanotubes [40,48]. Also SEM exhibited significant differences between surfaces modified by either single- or multi-walled nanotubes (Fig. 2C, D). Thick fibers of the MWCNTs created sparser network with accessible areas of supportive material, while soft thin fibers SWCNT formed dense net. GPH-SPGE surface was rich in graphene sheets, larger than those observed for PeGE, in parallel orientation (with basal planes exposed to the solution). Existence of a small number of edges or defects was proved by the lowest ID/IG value (0.09, Fig. 2E).
3. 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 pencil graphite electrode (PeGE) was used as another type of disposable carbon electrode for comparison with the SPGEs. 3.1. Analysis of purines at various SPGE Fig. 1(A, D, G, J, M) shows differential pulse voltammetric responses of purine nucleotides in 500 nM intact d(G3A3)5 ODN (yielding oxidation peaks dGox and dAox, respectively), and acid hydrolysate of the same ODN containing free nucleobase residues (guanine and adenine giving peaks - Gox and Aox, respectively) measured in 0.1 M acetate buffer (pH ~5). For a better comparison of responses obtained at different electrodes, the current responses were converted to the current densities by calculation per unit area (more details in Section 2.2). At the PeGE, guanine was electrooxidized at a potential of +0.77 V (Gox) and adenine at +1.03 V (Aox) (Fig. 1A). Peaks dGox and dAox of the d(G3A3)5 ODN were shifted by about 200 mV to more positive potentials, compared to peaks Gox and Aox, respectively. These results were in good agreement with previous ones showing easier oxidation of free purine
3.3. Kinetics of electron transfer for various SPGE surfaces Ratio of the D and G bands in Raman spectra clearly indicated different relative representation basal planes and edge planes for individual electrodes, which can influence orientation of purine nucleotides/nucleobases residues during interaction with the electrode surface. Nevertheless, surface properties were affected by (i) the inks, containing 3
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Fig. 1. Baseline-corrected AdS-DPVs of (A, D, G, J, M) 500 nM, (B, E, H, K, N) 50 nM and (C, F, I, L, O) 22 nM intact (orange and green curve) and 5 nM acid hydrolyzed (red and blue curve) oligodeoxynucleotide (ODN) d(G3A3)5 obtained in 0.1 M acetate buffer (pH ~ 5) at (A–C) pencil graphite electrode (PeGE), (D–F) screen-printed graphite electrode (SPGE), (G–I) multi-walled carbon nanotube modified SPGE (MWCNT-SPGE), (J–L) single-walled carbon nanotube modified SPGE (SWCNT-SPGE) and (M–O) graphene modified SPGE (GPH-SPGE). The green and orange parts of AdS-DPVs correspond to the deoxyguanosine monophosphate oxidation signal (dGox) and deoxyadenosine monophosphate oxidation signal (dAox), respectively. The red and blue parts of AdS-DPVs correspond to the guanine oxidation signal (Gox) and adenine oxidation signal (Aox), respectively. Black curves represent the blank electrolyte. Accumulation conditions: potential of accumulation, EA of +0.3 V; accumulation time, tA of 2 min. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the loading and functionalized graphite, the amount of organic binder and various impurities, (ii) curing regime and (iii) wettability of the electrode surface [42,47,49]. Different distribution of graphene basal planes and edges influenced also kinetics of electron transfer of the studied electrodes [6,10,42,49]. The electrode surfaces were tested by a commonly used redox probe [Fe(CN)6]4−/3− in 1 M KCl using CVs at different scan rates (Fig. 2F–J). The magnitude of the oxidation peak current densities of Fe(CN)64− oxidation linearly increased with the square root of scan rate (v1/2), yielding similarly dependencies for all surfaces (and indicating a diffusion-controlled process). However, plots of peak-to-peak separation (ΔEp) and Nicholson's kinetic parameter Ψ versus the reciprocal of the scan rate square root (v−1/2) for the Fe (CN)64− differed among individual surfaces (Fig. 2F–O). We used linear fit of Ψ versus v−1/2 to determine the standard heterogeneous rate constant k0. Obtained values k0 of 0.004 cm.s−1 were similar for PeGE and SPGE. Nanoobject-modified electrodes yielded k0 values higher by about 4-times for GPH-SPGE, 5-times for SWNT-SPGE and even 7-times for MWNT-SPGE than those observed for the unmodified SPGE in agreement with earlier observations [40,42,50,51]. Such order correlates
with relative abundance of graphene edges at the given modified electrode surface (see Section 3.2). 3.4. Purine nucleotide and nucleobase residues analysis at low concentrations In next experiments we decreased the concentration of d(G3A3)5 ODN and purine nucleobase residues from 500 nM (used in Fig. 1A, D, G, J, M) to 50 nM. Under these conditions we observed well-developed oxidation peaks Gox and Aox (of free nucleobases) at the all electrode surfaces with the exception of GPH-SPGE as was mentioned before (Fig. 1B, E, H, K, N). However, oxidation peaks dGox and dAox of the intact d(G3A3)5 ODN were observed only for the unmodified SPGE and PeGE (Fig. 1B, E). Absence of the peaks dGox and dAox at voltammograms measured at the MWCNT- and SWCNT-SPGE may be due to (i) limited accessibility of bases in the intact ODN strand, compared to the free purine nucleobase residues and/or (ii) disadvantageous orientation of nucleotides in the ODN and/or (iii) various surface wettability [2,6,10]. It seems that these effects did not play such important role at higher concentrations (such as 4
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Fig. 2. (A–E) SEM pictures of (A) PeGE, (B) SPGE, (C) MWCNT-SPGE, (D) SWCNT-SPGE, and (E) GPH-SPGE. Red lines represent 500 nm. (F–J) The current densities of the oxidation peak of 1 mM Fe(CN)64− and peak-to-peak separation (ΔEp) for 1 mM Fe(CN)64− in 1 M KCl as a function of the square root of scan rate (v1/2). (K–O) Nicholson's kinetic parameter Ψ versus the reciprocal of the scan rate's square root (v-1/2) for 1 mM Fe(CN)64− in 1 M KCl. The current densities, ΔEp and Ψ are means (standard deviations of 10 experiments). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
500 nM) of the analytes (including the ODN), where almost both free bases and the ODN produced well-developed peaks at almost all tested surface (see Section 3.1 and Fig. 1A, D, G, J, M). However, at lower concentrations of the analytes (including the free bases) occurrence of “parasitic” background signal affected analytical usefulness of some of the electrodes tested. Especially the nanomaterial-modified SPCEs produced considerable background signals at potentials close to those of purine, particularly guanine electrooxidation. These parasitic current signals either fully obscured the purine signals or at least decreased the signal-to-noise ratio (see below). ODN detection at concentrations in the range of tens of nM using SWCNT- or MWCNT-SPGE was only possible using the peak Aox. Detection of the peak Gox at all nanomaterial-modified surfaces was either impossible or markedly affected by the background currents (Fig. 1H, K, N). Relatively usable peaks Aox and Gox were obtained at MWCNT-SPGE (Fig. 1H). At 50 nM concentration of each of the purines, the peak Aox and Gox current densities were about 7-fold higher on the MWCNT surface than on unmodified SPGE (Fig. 1E). For 5 nM adenine, Aox peak at MWCNT-SPGE was more than 10-times higher than that at SPGE (Fig. 1F, I). However it should be emphasized that the signal-noise ratio was significantly in favor of the unmodified SPGE (S/ NSPGE~6, S/NMWCNT ~2), at which the background response was significantly smaller. The electrode modification with nanomaterials thus paradoxically worsened analytical figures of merit of the SPGE. Regarding the comparison of SPGE with PeGE, in the case of the Aox, the S/
N ratios were very similar (S/NSPGE~6.0 and S/NPeGE~4.6), whereas the Gox was affected by the background currents on the PeGE (S/NPeGE~1.7) in contrast to well-developed Gox on SPGE (S/NSPGE~6.7). These results indicate that the unmodified SPGE is the most suitable electrode for the label-free detection of low concentrations of ODN/DNA hydrolysates in small volumes. In the case of 22 nM intact d(G3A3)5 ODN, very wellresolved peaks dAox and dGox were observed at both PeGE (insert in Fig. 1C) and SPGE (insert in Fig. 1F). 3.5. Influence of competitive pyrimidine adsorption on purine electrooxidation 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. Based on the above results, unmodified SPGE and PeGE were chosen for these experiments (Fig. 3). For the experiments we used ODN differing in the contents of individual nucleotides, namely: 5G (dG5), 25A (dA25) and 5G + 25A + 23 T + 12C (d(G5A25Py35), see Experimental for sequences). Acid hydrolysate of the d(G5A25Py35) ODN consisted of 5 guanine and 25 adenine free nucleobases and the apurinic ODN
Fig. 3. (A) Baseline-corrected AdS-DPVs of 150 nM intact (solid yellow and green curve) and acid-hydrolyzed (solid red and blue curve) ODN d (G5A25Py35), and an equimolar mixture of 150 nM ODNs d(G5) and d(A25) in both intact (dashed yellow and green curve) and acid-hydrolyzed (dashed red and blue curve) forms obtained in 0.1 M acetate buffer (pH ~5) at SPGE. Black curves represent the blank electrolyte. (B) The effect of competitive adsorption of pyrimidine (Py) fraction within the ODN chain on the intensities of dGox (7-fold higher Py content) and dAox (1.4-fold higher Py content) oxidation signals of purine nucleotides present in the ODN sequence and/or the effect of the presence of Py nucleobase residues on the intensities of Gox and Aox oxidation signals of purine nucleobases removed from the ODN chain during acid hydrolysis, respectively, obtained in 0.1 M acetate buffer (pH ~5) at SPGE, PeGE and basal-oriented pyrolytic graphite electrode (BGPE). Current densities of both dGox and dAox peaks and the corresponding Gox and Aox signals obtained for an equimolar mixtures of d(G5) and d(A25) in both intact and acid-hydrolyzed forms were taken as 100%, respectively. The current densities are average values calculated from 10 independent experiments). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 5
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heights purine nucleotide signals (peaks dGox and dAox) measured at the SPGE increased linearly with the d(G3A3)5 ODN concentration from 22 nM to 500 nM. For equimolar mixture of purine nucleobases the linear dynamic ranges were found between 5 nM and to 500 nM concentration. At higher concentrations all peaks showed a tendency to levelling-off (Fig. 4. LODs, calculated from the standard deviation of the response and the slope of the calibration curve, were determined as 0.6 nM for dG,0.9 nM for dA, 0.5 nM for adenine and 0.3 nM for guanine. 4. 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 concentrations of purine nucleotides in model ODNs and of free purine nucleobases in few tens-of-microliter samples. We compared the purine oxidation signals at unmodified SPGE and PeGE with those obtained at SPGEs modified by graphene, single- and multiwalled carbon nanotubes. At relatively high analyte concentration, welldeveloped oxidation peaks were observed for all tested electrodes. At 50 nM and lower concentration, the analysis at the nanomaterial-modified graphite surface with higher content of edges/defects was limited to detection of free nucleobases, particularly adenine because oxidation peaks of guanine overlapped with strong signals of the background. Modification of the electrodes by the nanomaterials caused a remarkable decrease of signal/noise ratio due to increase of the background currents. The best signal/noise ratio for free purine nucleobases at lower concentrations we observed for unmodified SPGE, followed by PeGE. The SPCE was identified as a comparable electrode with the PeGE for labelfree analysis of nucleotides in ODN at low concentrations. Competitive adsorption of pyrimidine fraction of the ODN (both intact and acid-hydrolyzed) did not significantly influence purines oxidation signals at SPGE, while at the PeGE it caused remarkable decrease of signals of the purine components. Differences in surface properties of the graphitebased electrodes, such as presence of basal planes, edges and defects as well we kinetics of the electron transfer have a significant impact on the electrode properties in the ODN analysis. Thus, our results show the SPGE as the disposable electrode of choice for the analysis of purine-containing ODNs and nucleobase residues in microliter volumes with a negligible interference of the pyrimidine fraction.
Fig. 4. Concentration dependences of the current oxidation signals' densities of (A) (●) dGox and (▲) dAox or (B) (○) Gox and (△) Aox obtained in 0.1 M acetate buffer (pH ~5) containing either intact d(G3A3)5 or acid-hydrolysed d (G3A3)5 at SPGE. Accumulation conditions: EA of +0.3 V; tA of 2 min.
residuum containing 23 thymine and 12 cytosine nucleotides per starting ODN molecule. As a control, the equimolar mixture of two homopurine ODNs: short homoguanine (dG5) and homoadenine (dA25), each at concentration of 150 nM was used. Fig. 3A shows that the presence of 3fold excess of the pyrimidine content had almost no effect (only within statistical errors (Fig. 3B)) on the peaks dGox, dAox of the intact d (G5A25Py35) ODN measured at the SPGE when compared to the corresponding signals of equimolar mixture of homopurine dG5 and dA25 ODNs lacking the pyrimidine fraction (Fig. 3A). The same applies to the comparison of responses obtained for acid-hydrolyzed ODNs (Fig. 3A). In contrast, similar experiment performed with PeGE revealed significant decrease of oxidation peak dAox and Aox by about 70% and 47%, respectively, caused by the presence of the pyrimidine fraction (Fig. 3B). Interestingly, presence of the pyrimidine content in the ODN did not affect the oxidation peak dGox in the intact ODN. However, the current density of oxidation signal Gox of guanine residues in the hydrolysate decreased by about 37%. Similar effects of the pyrimidine fraction on the purine nucleotide/nucleobase electro-oxidation signals were also observed for the basal plane oriented pyrolytic graphite electrode (BPGE), commonly used to study of electro-oxidation of DNA components [2,6,10,11,43]. For both BPGE and PeGE, individual oxidation signals were affected by the pyrimidine content the order of dGox ≪ Gox ≪ Aox ≤ dAox (Fig. 3B). In general, purines are known to be stronger adsorbed at carbonbased electrodes in comparison to pyrimidines [54,55]. Nevertheless, in our study the pyrimidines exhibited higher affinity to electrodes with relatively large abundance of graphite sheets in the basal orientation than to surfaces with higher content of edges and defects (as assessed from relative interference with the purine oxidation signals) [56]. Thus at graphite-based electrodes with prevailing basal planes pyrimidines may show compete stronger competition with the purines [57].
Acknowledgment This work was supported by the Czech Science Foundation 1708971S 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). References [1] J. Galandová, J. Labuda, Polymer interfaces used in electrochemical DNA-based biosensors, Chem. Pap. 63 (2009) 1–14, https://doi.org/10.2478/s11696-0080083-2. [2] E. Paleček, M. Bartošík, Electrochemistry of nucleic acids, Chem. Rev. 112 (2012) 3427–3481, https://doi.org/10.1021/cr200303p. [3] M. Fojta, A. Daňhel, L. Havran, V. Vyskočil, Recent progress in electrochemical sensors and assays for DNA damage and repair, TrAC Trends Anal. Chem. 79 (2016) 160–167, https://doi.org/10.1016/j.trac.2015.11.018. [4] E.E. Ferapontova, DNA electrochemistry and electrochemical sensors for nucleic acids, Annu. Rev. Anal. Chem. 11 (2018) 197–218, https://doi.org/10.1146/ annurev-anchem-061417-125811. [5] S. Campuzano, P. Yáñez-Sedeño, J.M. Pingarrón, Nanoparticles for nucleic-acidbased biosensing: opportunities, challenges, and prospects, Anal. Bioanal. Chem. 411 (2019) 1791–1806, https://doi.org/10.1007/s00216-018-1273-6.
3.6. Purine nucleotide and nucleobase residues detection at SPGE Our results show that SPGE represents in terms of signal/noise ratio and other analytical figures of merit, a good tool for the detection of nanomolar concentrations of purine-containing ODNs and free purine nucleobases. Additionally, the presence of pyrimidine (apurinic) fraction in the samples only negligibly affected the purine oxidation peaks. The 6
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