Evaluation of Electrokinetic Parameters for All DNA Bases with Sputter

Nov 19, 2012 - Division of Materials Science and Engineering, Graduate School of Science and Engineering, Yamaguchi ... E-mail: [email protected]...
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Evaluation of Electrokinetic Parameters for All DNA Bases with Sputter Deposited Nanocarbon Film Electrode Dai Kato,† Michinori Sumimoto,‡ Akio Ueda,†,∥ Shigeru Hirono,†,§ and Osamu Niwa*,† †

National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan Division of Materials Science and Engineering, Graduate School of Science and Engineering, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611, Japan § MES-Afty Corporation, 2-35-2 Hyoe, Hachioji, Tokyo 192-0918, Japan ‡

ABSTRACT: The electrokinetic parameters of all the DNA bases were evaluated using a sputter-deposited nanocarbon film electrode. It is very difficult to evaluate the electrokinetic parameters of DNA bases with conventional electrodes, and particularly those of pyrimidine bases, owing to their high oxidation potentials. Nanocarbon film formed by employing an electron cyclotron resonance sputtering method consists of a nanocrystalline sp2 and sp3 mixed bond structure that exhibits a sufficient potential window, very low adsorption of DNA molecules, and sufficient electrochemical activity to oxidize all DNA bases. A precise evaluation of rate constants (k) between all the bases and the electrodes is achieved for the first time by obtaining rotating disc electrode measurements with our nanocarbon film electrode. We found that the k value of each DNA base was dominantly dependent on the surface oxygencontaining group of the nanocarbon film electrode, which was controlled by electrochemical pretreatment. In fact, the treated electrode exhibited optimum k values for all the mononucleotides, namely, 2.0 × 10−2, 2.5 × 10−1, 2.6 × 10−3, and 5.6 × 10−3 cm s−1 for GMP, AMP, TMP, and CMP, respectively. The k value of AMP was sufficiently enhanced by up to 33 times with electrochemical pretreatment. We also found the k values for pyrimidine bases to be much lower than those of purine bases although there was no large difference between their diffusion coefficient constants. Moreover, the theoretical oxidation potential values for all the bases coincided with those obtained in electrochemical experiments using our nanocarbon film electrode.

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bases such as thymine and cytosine, and a graphene electrode exhibits higher electrode activity that enables it to oxidize these pyrimidine bases. However, these electrodes are still insufficient as regards the quantitative detection of all DNA bases and the evaluation of their electrokinetic parameters. This is because a BDD electrode has low electrochemical activity compared with sp2-based carbon materials, and commonly used graphene has a flakelike structure making it difficult to fabricate a suitable electrode, such as a rotating disk electrode (RDE), for evaluating electrokinetic parameters. Recently, we have developed a nanocarbon film with a nanocrystalline sp2 and sp3 hybrid structure formed by employing the electron cyclotron resonance (ECR) sputtering method.7 This film provides excellent electrochemical characteristics including a wide potential window, a low background current, and little surface fouling by analytes after oxidation.8,9 These characteristics allow the detection of all the DNA bases with a higher Eox region with excellent reproducibility to distinguish a single base difference in the target sequence. As a

any groups have studied the electrochemical detection of DNA and related materials because this method is very simple and inexpensive compared with optical detection. Since the oxidation potential (Eox) of each base in DNA is high, electrochemical intercalators or redox species labeled probe DNA have often been used for DNA sensors or chips in combination with sequence specific hybridization. However, in terms of DNA electroanalysis, the direct oxidation and reduction of DNA is the simplest with various kinds of electrochemical methods and has traditionally been investigated. Paleček was the first to report DNA sensing based on the electrochemical reduction at a mercury electrode.1 Steenken has also extensively reported on the reduction mechanisms of DNA bases.2 In contrast, the direct electrochemical oxidation of DNA has recently been studied more intensively.3−6 For such purposes, carbon-based electrodes are widely used for detecting DNA because they have a wider potential window than metal electrodes and exhibit good electrochemical activity especially against aromatic compounds such as dopamine and DNA bases. Some groups have already reported that glassy carbon (GC), boron-doped diamond (BDD), and graphene electrodes exhibit oxidation currents for free bases.4−6 In particular, the potential window of a BDD electrode is sufficient to oxidize pyrimidine © 2012 American Chemical Society

Received: July 13, 2012 Accepted: November 18, 2012 Published: November 19, 2012 10607

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Carbon Film Characterization. Atomic force microscopy (AFM) measurements were performed with an SPI4000 (SII NanoTechnology, Inc., Japan). All measurements were made using a silicon cantilever (SI-DF40: spring constant = 40 N m−1, resonance frequency = 330 kHz, SII NanoTechnology, Inc.) in air at room temperature. Images were recorded in the dynamic force mode at scan rates of 0.3 Hz with 256 × 256 pixels. X-ray photoelectron spectroscopy (XPS) was conducted with a Quantum 2000 (ULVAC-PHI, monochromatic Al Kα source at 1486.6 eV) to determine the elemental composition of the film surface. O/C was calculated from the intensities of C1s and O1s (n = 3). Electrochemical Experiments. All electrochemical experiments were performed using an ALS/CHI 760B electrochemical analyzer (CH Instruments, Inc.). A platinum wire and a Ag/AgCl (3 M NaCl) electrode were used as auxiliary and reference electrodes, respectively. ECR nanocarbon film was used as the working electrode. The ECR nanocarbon film electrode area was defined by using masking tape in which there was a 2 mm diameter hole. A 50 mM acetate buffer (pH 5.0) or 50 mM phosphate buffer (pH 7.0) were used as the electrolyte solution for the electrochemical measurements. The ECR nanocarbon film electrodes were electrochemically pretreated to control the activity of their electrode surfaces with cyclic voltammetry (CV) by changing the number of cycles for 8 or 15 between 0 and 2.3 V at a scan rate of 0.1 V s−1 in a 50 mM phosphate buffer (pH 7.0) solution. All the squarewave voltammogram (SWV) measurements were performed with an amplitude of 25 mV and a ΔE of 5 mV at 10 Hz. The RDE of the ECR nanocarbon film was fabricated as follows: Square cut nanocarbon film was fixed on a commercially available GC electrode for the RDE system (BAS, Japan) by using Ag paste. After drying, the fixed nanocarbon film electrode area was defined in the same manner as described above. The rotation conditions for the RDE were controlled by using an RRDE-3 (BAS, Japan) apparatus combined with the above electrochemical analyzer. Computational Details. The geometry optimization of the DNA bases was carried out using the DFT method, where the B3PW91 functional22−27 was used as the exchange-correlation term. Analytical vibrational frequency computations at the optimized structure were then performed to confirm that the optimized structure was at an energy minimum. The 6311+G(d,p) basis sets were employed for all atoms. The electrode Eox values were calculated by the Nernst equation using the free energy changes in the gas phase (ΔGgas) and solution (ΔGsol).28 ΔG calculations were performed at the SCS-MP2/6-311+G(d,p)29−31 levels for the DNA bases on the B3PW91 geometries. In relation to the solvent effect of water, we employed the PCM method using the Pauling atomic radii. All these calculations were performed with the Gaussian 09 program package.32

result, we achieved the direct quantitative measurement of all the DNA bases individually by using a nanocarbon film electrode.10−13 Our nanocarbon films will help us to understand the mechanism of the electron transfer of DNA due to the electrochemical performance described above including the wide potential window and the sufficiently high electron transfer. Although, the electrokinetic parameters of DNA have been thoroughly studied by many groups,2−4,14−17 it is difficult to evaluate the electrokinetic parameters of pyrimidine bases because their Eox values are higher than those of purine bases. In fact, only the electrochemical parameters including the diffusion coefficient constant (D) values for purine bases such as guanine were determined.16,17 Previous reports clearly suggest that the potential window of conventional electrodes such as GC electrodes is insufficient. Although a BDD electrode has a much wider potential window, its electrochemical activity with respect to DNA is not high.11 Since each DNA base exhibits a very different current magnitude as a result of electrochemical oxidation, we can expect the evaluation of these electrokinetic parameters using a nanocarbon film electrode to allow us to achieve a more quantitative analysis of DNA.2,14,15,18−20 Moreover, the k value is strongly dependent on the molecular structure of the bases and their interactions with electrode materials and their surface functional groups. These facts demonstrate that the evaluation of the k value enables us to provide good feedback with respect to the design of electrodes for electrochemical DNA sensing. In this work, we evaluated electrokinetic parameters such as rate constant (k) and D for each DNA base using ECR nanocarbon films. Since surface hydrophilicity will affect the electron transfer of each base, we compared the properties of the electrodes, such as the surface O/C ratio and roughness (average roughness: Ra) with results obtained for an asdeposited nanocarbon film electrode. We controlled the surface O/C ratio by using electrochemical pretreatment (ECP) because we previously found that the ECP of a nanocarbon film electrode is an effective way of enhancing the electrode activity against certain biomolecules such as glutathione without losing the wide potential window and low background current. Moreover, we evaluated the reliability of the obtained electrochemical parameter including the Eox values by comparison with the energy level of an electronic orbital for all the bases by using the density functional theory (DFT) method.

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MATERIALS AND METHODS Materials. ECR nanocarbon films suitable for use in DNA electroanalysis were prepared in accordance with previous reports.8−10,21 Briefly, the nanocarbon films were deposited on highly doped silicon (100) substrates with ECR sputtering equipment (AFTEX-3200, MES-Afty, Japan) at room temperature. The microwave power and DC voltage applied to the carbon target were 500 W and 500 V, respectively. The argon gas pressure for the sputtering was 5.0 × 10−2 Pa. During deposition, the irradiation ion current density was 5.8 mA cm−2, and the ion acceleration voltage was 75 V. The nanocarbon films were obtained in about 8 min and were typically 40 nm thick. Four kinds of nucleoside 5′-monophosphates (NMPs), guanosine 5′-monophosphate (GMP), adenosine 5′-monophosphate (AMP), thymidine 5′-monophosphate (TMP), and cytidine 5′-monophosphate (CMP), were purchased from Sigma-Aldrich. All other chemicals were of analytical grade.

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RESULTS AND DISCUSSION Activation of Nanocarbon Film Surface. The measurement of the electrokinetic parameter of each DNA base requires electrochemical pretreatment to make the surface hydrophilic and suppress fouling with biomolecules. We characterized the surface properties of the nanocarbon film before and after ECP. As shown in Figure 1, we obtained a typical average roughness (Ra, n = 5) of 0.05 nm for asdeposited ECR nanocarbon film determined by scanning along the line with an image size of 1 × 1 μm2. This is much flatter 10608

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electrochemical parameter of DNA bases since the film has a wide potential window, sufficient electrode activity, a low background current, and low adsorption of biomolecules after ECP. Subsequent experiments were therefore performed with the treated ECR nanocarbon film electrode. Electrode Activity for All the Bases. We investigated the electrochemical activity of all the bases before and after ECP. Figure 2 show the background subtracted SWVs of all the bases

Figure 1. AFM images of the nanocarbon film surface (a) before ECP, (b) after ECP for 8 cycles, and (c) after ECP for 15 cycles. (d) Relationship between surface O/C ratio, Ra, and number of ECP cycles. Figure 2. Background-subtracted SWVs of 100 μM of NMP at nanocarbon film electrode before (- - -) and after () ECP, measured in 50 mM pH 5.0 acetate buffer. Amplitude = 25 mV, ΔE = 5 mV, f = 10 Hz.

than that of GC (2.4−4.7 nm)21,33 and nanocrystalline BDD (typically around 34 nm)34 and almost the same as that of ultraflat carbon films prepared by the pyrolysis of a photoresist or by electron beam evaporation as reported by McCreery and McDermott et al. (subnanometer flatness: 0.07−0.5 nm).35,36 We fixed the ECP condition within 15 cycles so that the treated nanocarbon film electrodes could maintain their surface flatness with the same order of roughness as the untreated one as described later. After the ECP, the Ra and the O/C ratio of the nanocarbon surfaces were gradually increased by increasing the scanning cycle for the ECP. These results agreed well with our previous results.21,33 Note that the increased surface roughness of our nanocarbon film was very small compared with that of the conventional GC. Indeed, after a 15-cycle potential scan, the Ra of the nanocarbon film surface was about 1.4 Å, which was still smoother than that of untreated GC described above.21,33 This was because the nanocarbon film electrode consists of both sp2- and sp3-bonded carbon, which is unlike the GC electrode, which consists largely of sp2-bonded carbon. This is highly advantageous in terms of obtaining the electrokinetic parameters since electrokinetic parameters such as the k value of the analyte are generally dependent on the surface structure of the electrode rather than the property of the analyte. Moreover, the increased Ra of the treated nanocarbon film electrode was at most 1.4 Å, which is less than the analyte size (e.g., even the cytosine, which is the smallest molecule of the four bases, has a molecular length of about 5.6 Å). Therefore, the flat surface of the nanocarbon film after ECP was suitable for use in evaluating the electrochemical parameters of DNA bases by suppressing the increase in the k value caused by increases in surface area. In addition, we also confirmed that the current decrease after ECP was much smaller than that of other electrodes when measuring relatively long DNA.12 These results indicate that a treated nanocarbon film electrode is particularly advantageous for evaluating the

before and after ECP (15 cycles). The Eox values of all the NMPs before ECP are summarized in Table 1. After ECP, the Table 1. Electrochemical Responses of All the DNA Bases Obtained from Figure 2 Ip (μA cm−2)

EOX (V) ECP

−

+

−

+

GMP AMP TMP CMP

1.12 1.50 1.76 1.85

1.10 1.39 1.62 1.72

12.6 12.8 2.91 3.42

19.1 36.3 13.1 27.0

Eox values at all the NMPs shifted to lower potentials, and their currents became larger than those at the as-deposited nanocarbon film electrode. Specifically, the AMP current increased 2.8 times, suggesting that the treated electrode showed the highest electrode activity against AMP oxidation, and its Eox value was 110 mV lower than those obtained at the as-deposited nanocarbon film electrode. These results clearly demonstrated that the electron transfer of the oxidation of all the NMPs was greatly improved. In fact, we have already reported that the peak current (Ip) values of GMP and AMP were larger at our nanocarbon film electrode than those of a BDD electrode but smaller than those at a GC electrode.10,11 These results indicate that an electrode surface with a sufficient number of sp2 bonds is important for the oxidation of each NMP. In contrast, the number of surface oxygen-functional groups had increased after ECP as noted above, but the sp2 ratio had decreased slightly after ECP.21 These results indicate that both surface oxygen (or wettability) and sp2 bonds are 10609

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Figure 4a shows the linear sweep voltammograms (LSVs) of GMP with different rotation speeds at the nanocarbon film electrode. Also in this case, the ECP led to an increase in the current response of GMP. As shown in Figure 4b, Koutecký− Levich plots of GMP were also obtained from the limited current (Ilim) at the applied potential defined in Figure 4a and the following equation

required to obtain high electrode activity with respect to the NMPs oxidation. A similar result was reported for dopamine or NADPH measurement.21 Although sp2 carbon such as GC, which hardly contains any sp3 bonds, exhibits superior electrochemical activity for NMPs, these sp2 carbons are not suitable because such carbon electrodes have narrower potential windows that are insufficient to measure pyrimidine bases. The above results clearly demonstrate that our nanocarbon film provides a suitable electrode for evaluating and comparing the electrochemical parameters of all DNA bases. This has not been reported by any other groups owing to electrode performance limitations. Effect of Surface Status on Rate Constant. To conduct a more quantitative study of DNA bases with the nanocarbon film after ECP, we performed RDE experiments using nanocarbon film. The RDE experiment is widely used in a variety of fields including for evaluating fuel cell electrodes.37,38 In this study, we evaluated the ECP effect in terms of the electron transfer rate of each NMP at the nanocarbon film electrode by RDE experiments. Before conducting the RDE experiment for DNA bases, we confirmed the electrochemical response of Fe(CN)64‑ with an RDE experiment using both nanocarbon film-based and original GC rotating electrodes. Figure 3a shows a schematic drawing of the nanocarbon film

Ilim−1 = Ik −1 + IL−1 = (nFAkC)−1 + (0.62nFAD2/3ν−1/6Cω1/2)−1

where Ik is the kinetically controlled current and other symbols have their usual meanings. This plot was used to analyze the electrochemical properties of each base at electrodes with different surface states.16,17,39,40 As shown in Figure 4b, we observed a linear correlation between an inverse of Ilim and an inverse of the square root of the rotating speed (ω1/2). From the y-intercept of these plots, we determined the k value between the analytes and the used electrode. For example, a k value of 3.1 × 10−3 cm s−1 was obtained at the as-deposited nanocarbon film electrode. Brett et al. reported the k value of guanine at a bare GC electrode (6.2 × 10−3 cm s−1) obtained by the electrochemical impedance method,41 which is very close to that obtained at our as-deposited nanocarbon film electrode. These results revealed that the k values at the asdeposited (untreated) nanocarbon film electrode estimated by using Koutecký−Levich plots were reasonable compared with that of the untreated sp2-based carbon electrode GC. With the treated nanocarbon film electrode, the y-intercept of the plot for GMP was decreased compared with that at the as-deposited nanocarbon film electrode, indicating that the k value increased (3.1 × 10−3 and 2.0 × 10−2 cm s−1 at the as-deposited and treated nanocarbon film electrode, respectively). That is, ECP made it possible to enhance the electrode activity for the DNA base. Such a trend was also observed in the AMP measurement. With the AMP measurement at the treated nanocarbon film electrode, the k value (2.5 × 10−1 cm s−1) exhibited as much as a 33-fold enhancement compared with that at an untreated nanocarbon film electrode (7.5 × 10−3 cm s−1). These results show that the k value is dominantly dependent on the electrode surface functional groups despite the fact that we maintained the surface roughness of the nanocarbon film as mentioned above. Therefore, it is important to evaluate k when we investigate the relationship between the electrode surface property and electron transfer. Likewise, we conducted RDE experiments for all NMPs at the treated nanocarbon film electrode and precisely determined the k values for all bases from the Koutecký−Levich plots (Figure 5), as summarized in Table 2. The k values at purine bases were much higher than those at pyrimidine bases. This was presumably because more aromatic purines exhibited a stronger π−π interaction with the nanocarbon film electrode than the pyrimidines. The π−π interaction between the aromatic DNA bases and the carbon surface is very important with respect to electrode activity including the k values, as we previously reported.10,11 That is, the strength of the π−π interaction of the DNA bases is strongly related to the k values. Indeed, Cysewski estimated the “aromaticity” of the DNA bases, whose order is A > G > C >T.42 Interestingly, this order agrees well with the k value order in this study (A > G > C >T, in Table 2). We also determined the D value of each NMP from the slope in Figure 5. As summarized in Table 2, the D value of GMP under this condition was 8.2 × 10−6 cm2 s−1 when two-electron

Figure 3. (a) Schematic drawing of the fabricated nanocarbon film electrode used for the RDE experiment. (b) Levich plots of 100 μM Fe(CN)64‑ at the nanocarbon film (■) and GC (□) electrodes used for the RDE experiment, respectively, measured in 1 M KCl at various rotating speeds over a range of 500−4000 rpm (500, 750, 1000, 2000, and 4000 rpm, the potential scan rate was 50 mV s−1).

electrode for an RDE experiment, as described in the Materials and Methods section. In RDE, the mass transfer limited current depends on the square root of the rotation speed according to the Levich equation IL = 0.62nFAD2/3ν−1/6Cω1/2

where ν is the kinematic viscosity of the solution (0.01 cm2 s−1), ω is the angular velocity, F is the Faraday constant, and other symbols have their usual meanings. Figure 3b shows Levich plots of Fe(CN)64‑ at both the nanocarbon film-based and commercially available GC electrodes for the RDE experiment, demonstrating that the nanocarbon film-based rotating electrode was very stable and produced comparable results to those obtained with the GC rotating electrode. Therefore, we concluded that the nanocarbon film-based rotating electrode works correctly in the same manner as the GC rotating electrode. 10610

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Figure 4. (a) LSVs of 300 μM GMP at the nanocarbon film electrode before (- - -) and after () ECP in 50 mM pH 5.0 acetate buffer at various rotating speeds over a range 750−2000 rpm (increasing by 250 rpm at each step and for each voltammogram, the potential scan rate was 20 mV s−1). (b) Koutecký−Levich plots of the obtained voltammograms from part a. The Ilim for the GMP was obtained at 1.3 V vs Ag/AgCl, whose potential was capable of oxidizing the GMP efficiently.16,17

indicate that the large differences in sensitivity between purine and pyrimidine bases are due to the large difference in k, rather than D and the adsorption of NMPs on the electrode surface. This is because it has already been proved that our electrodes suppress the strong adsorption of DNA molecules, which are different from other sp2 carbons such as GC. In contrast, a BDD electrode exhibits low electrode activity with respect to the DNA base oxidation11 despite the fact that BDD has wider potential windows and superior antifouling properties.43 This is presumably due to the small k values of aromatic compounds including dopamine44 and the DNA bases. Therefore, the estimation of these parameters provides both a more quantitative performance and good feedback for electrode design in electrochemical DNA biosensing. Electronic Structures of All Bases Obtained by DFT. The energy levels of electronic orbitals for all the bases were studied theoretically to estimate the reliability of the oxidation reaction for all the bases obtained in the experimental results. Figure 6a compares the energy diagrams of all the free bases in the region of the highest occupied molecular orbitals (HOMO) and the lowest unoccupied molecular orbitals (LUMO). The orders of HOMO energy levels for all the bases correspond to the order of the Eox values obtained by the electrochemical method using our nanocarbon film electrode (CVs measured in 50 mM phosphate buffer (pH 7.0)) (G < A < T < C). Moreover, the theoretical Eox value can be obtained from the free-energy change ΔG° by the Nernst equation. Paukku and Hill extensively investigated the theoretical Eox values for all the bases by using the M06-2X/6-31++G(d,p) method.28 Also, in our case, we obtained the theoretical Eox values of all the bases, which were in good agreement with the experimental values obtained with our nanocarbon film electrode (Figure 6c). The SCS-MP2 calculation in this study provided very similar values to the experimental values compared with a previous report by Paukku et al.,28 which resulted in a suitable method in this case. Moreover, a closer inspection revealed that the theoretical Eox values were subtly larger than those obtained by nanocarbon film electrodes except for GMP. This is because the actual Eox values include the overpotentials. However, the subtle difference between the theoretical and experimental Eox values suggests that the nanocarbon film exhibits a relatively high electron transfer for each base. Therefore, we concluded that

Figure 5. Koutecký−Levich plots of all the NMPs. The Ilim for the NMPs were obtained at 1.3, 1.65, 1.75, and 1.8 V vs Ag/AgCl, respectively, whose potentials were capable of oxidizing the individual NMPs efficiently.16,17

Table 2. Rate Constant of All the NMPs at the Treated Nanocarbon Film Electrode and D Obtained from Koutecký−Levich Plots k (cm s−1) GMP AMP TMP CMP

2.0 2.5 2.6 5.6

× × × ×

−2

10 10−1 10−3 10−3

D (cm2 s−1) 8.2 1.0 8.3 1.7

× × × ×

10−6 10−5 10−6 10−5

mechanisms were considered,16,17 which was the same as the previously reported value.17 Other NMPs including pyrimidine bases such as TMP and CMP exhibited very similar D values. It is noteworthy that we succeeded in determining D for all the bases. Several groups have already reported the electrochemical response of NMPs with GC and BDD electrodes. Most of them reported that the currents of pyrimidine bases are much smaller than those of purine bases.4 Our results, which first compared quantitative evaluations of both the k and D values for all bases, 10611

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Figure 6. (a) Several important orbitals near the HOMO and LUMO of all the free bases obtained by the MP2 calculation, which used the PCM method (solvent = water). The orbitals below the dotted line are occupied. (b) An enlargement of the bottom portion of part a. (c) Relationships between the Eox values obtained in the experiments and obtained by DFT calculation. The Eox values (vs Ag/AgCl) from ref 28 for comparison were converted by subtracting 0.197 V from the reported Eox (vs a normal hydrogen electrode).

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the Eox values obtained at our nanocarbon film electrode were appropriate. In addition, we found that the next HOMO (NHOMO) energy levels of the bases were individually different. In particular, noticeable differences were observed for the energy levels between G and A bases, and T and C bases. Indeed, the NHOMO energy levels of A and C were higher than those of G and T, respectively, despite the fact that they were purine- and pyrimidine-bases. These results suggest that the energy levels might be related to the magnitude of the oxidation current (n of the bases). Assuming that the D values of all the bases were typical (5 × 10−6 cm2 s−1),45,46 we can obtain an n of 2.8, 3.4, 2.8, and 4.5 for GMP, AMP, TMP, and CMP, respectively, which are in accordance with the oxidation current values of the respective bases. These results are essential in terms of further understanding the oxidation mechanism of all the bases at our nanocarbon film electrode. We are now studying ways for the oxidation of each base by using our nanocarbon film electrode in comparison with computational methods.

AUTHOR INFORMATION

Corresponding Author

*Phone: +81-29-861-6158. Fax: +81-29-861-6177. E-mail: [email protected]. Present Address ∥

Mitsubishi Chemical Corporation.

Author Contributions

All authors contributed to this manuscript, and approved the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was conducted in part at the Nano-Processing Facility, supported by the IBEC Innovation Platform, AIST.

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REFERENCES

(1) Paleček, E. Nature 1960, 188, 656−657. (2) Steenken, S. Chem. Rev. 1989, 89, 503−520. (3) Boussicault, F.; Robert, M. Chem. Rev. 2008, 108, 2622−2645. (4) Oliveira-Brett, A. M.; Piedade, J. A. P.; Silva, L. A.; Diculescu, V. C. Anal. Biochem. 2004, 332, 321−329. (5) Ivandini, T. A.; Honda, K.; Rao, T. N.; Fujishima, A.; Einaga, Y. Talanta 2007, 71, 648−655. (6) Zhou, M.; Zhai, Y. M.; Dong, S. J. Anal. Chem. 2009, 81, 5603− 5613. (7) Hirono, S.; Umemura, S.; Tomita, M.; Kaneko, R. Appl. Phys. Lett. 2002, 80, 425−427. (8) Niwa, O.; Jia, J.; Sato, Y.; Kato, D.; Kurita, R.; Maruyama, K.; Suzuki, K.; Hirono, S. J. Am. Chem. Soc. 2006, 128, 7144−7145. (9) Jia, J. B.; Kato, D.; Kurita, R.; Sato, Y.; Maruyama, K.; Suzuki, K.; Hirono, S.; Ando, T.; Niwa, O. Anal. Chem. 2007, 79, 98−105. (10) Kato, D.; Sekioka, N.; Ueda, A.; Kurita, R.; Hirono, S.; Suzuki, K.; Niwa, O. J. Am. Chem. Soc. 2008, 130, 3716−3717. (11) Kato, D.; Sekioka, N.; Ueda, A.; Kurita, R.; Hirono, S.; Suzuki, K.; Niwa, O. Angew. Chem., Int. Ed. 2008, 47, 6681−6684. (12) Goto, K.; Kato, D.; Sekioka, N.; Ueda, A.; Hirono, S.; Niwa, O. Anal. Biochem. 2010, 405, 59−66. (13) Kato, D.; Goto, K.; Fujii, S.; Takatsu, A.; Hirono, S.; Niwa, O. Anal. Chem. 2011, 83, 7595−7599. (14) Armistead, P. M.; Thorp, H. H. Anal. Chem. 2001, 73, 558−564. (15) Johnston, D. H.; Thorp, H. H. J. Phys. Chem. 1996, 100, 13837− 13843.

CONCLUSION We successfully evaluated electrokinetic parameters including k and D for all the DNA bases by performing RDE measurements using an ECR nanocarbon film electrode for the first time. The hydrophilic surface of our nanocarbon film electrode, which had undergone electrochemical pretreatment, enabled us to evaluate the k and D values of all the DNA bases precisely thanks to the improved electrode activity and reduced overpotential similar to that found with a GC electrode. As a result, the Eox values of all the DNA bases were in good agreement with the theoretical values. We found that the low electrode activity for the pyrimidine bases could be attributed to the fact that the k values were smaller than those of the purine bases. In contrast, we observed no clear differences between the D values of the purine and pyrimidine bases. The above results indicate that our nanocarbon materials are very promising for use in evaluating the electrokinetic parameters of various biomolecules with high oxidation potentials, which cannot be evaluated by using conventional electrodes. Moreover, this information about DNA bases is very significant for various DNA science fields including work on both Watson− Crick bases and the recently discovered epigenetic DNA bases. 10612

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dx.doi.org/10.1021/ac301964e | Anal. Chem. 2012, 84, 10607−10613