On-Chip Evaluation of DNA Methylation with Electrochemical

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On-Chip Evaluation of DNA Methylation with Electrochemical Combined Bisulfite Restriction Analysis utilizing a Carbon Film Containing a Nanocrystalline Structure Ryoji Kurita, Hiroyuki Yanagisawa, Tomoyuki Kamata, Dai Kato, and Osamu Niwa Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 03 May 2017 Downloaded from http://pubs.acs.org on May 7, 2017

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Analytical Chemistry

On-Chip Evaluation of DNA Methylation with Electrochemical Combined Bisulfite Restriction Analysis utilizing a Carbon Film Containing a Nanocrystalline Structure Ryoji Kurita*, Hiroyuki Yanagisawa, Tomoyuki Kamata, Dai Kato and Osamu Niwa† Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) and DAILAB, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan 305-8566 Tel; +81-29-861-6158, E-mail; [email protected]

ABSTRACT: This paper reports an on-chip electrochemical assessment of the DNA methylation status in genomic DNA on a conductive nanocarbon film electrode realized with combined bisulfite restriction analysis (COBRA). The film electrode consists of sp2 and sp3 hybrid bonds and is fabricated with an unbalanced magnetron (UBM) sputtering method. First, we studied the effect of the sp2/sp3 ratio of the UBM nanocarbon film electrode with p-aminophenol, which is a major electro-active product of the labeling enzyme from p-aminophenol phosphate. The signal current for p-aminophenol increases as the sp2 content in the UBM nanocarbon film electrode increases because of the π-π interaction between aromatic p-aminophenol and the graphene-like sp2 structure. Furthermore, the capacitative current at the UBM nanocarbon film electrode was successfully reduced by about one order of magnitude thanks to the angstrom-level surface flatness. Therefore, a high signal-to-noise ratio was achieved compared with that of conventional electrodes. Then, after performing an ELISA-like hybridization assay with a restriction enzyme, we undertook an electrochemical evaluation of the cytosine methylation status in DNA by measuring the oxidation current derived from p-aminophenol. When the target cytosine in the analyte sequence is methylated (unmethylated), the restriction enzyme of HpyCH4IV is able (unable) to cleave the sequence, i.e. the detection probe cannot (can) hybridize. We succeeded in estimating the methylation ratio at a site-specific CpG site from the peak current of a cyclic voltammogram obtained from a PCR product solution ranging from 0.01 to 1 nM.

INTRODUCTION Cytosine methylation at the fifth position in DNA is the most studied epigenetic modification. It plays a crucial role in controlling genetic expression1, including that in cancer2-5, genomic imprinting6, cellular differentiation and Alzheimer’s disease7. A growing number of human diseases have been found to be associated with aberrant cytosine methylation.5,6 Therefore, a quick, simple, and highly sensitive way of obtaining, quantitative information about cytosine methylation in DNA would be valuable as regards diagnosing human diseases 8 . A bisulfite based determination method is widely used to distinguish between cytosine and methylcytosine9-14. Treatment with bisulfite converts cytosine to uracil, while methylcytosine remains unaffected. Therefore, information about methylcytosine in DNA can be obtained by comparing bisulfite-treated and untreated samples. For example, bisulfitesequencing15, combined bisulfite restriction analysis (COBRA)16, methylation-specific PCR17 and pyrosequencing18 can provide the methylation status of a specific sequence with a single CpG level. These results are generally obtained from variations in fluorescence or chemiluminescence intensity. Electrochemical methods for methylcytosine determination have also been investigated by a number of researchers. This is because electrochemical detection is expected to be a simple

and inexpensive system with small equipment. Electrochemical methods are classifiable into two main types; one is the direct oxidation of methylcytosine utilizing the difference between the oxidation potentials of cytosine and methylcytosine19-23, and the other is selective labeling with electroactive molecules24-29. For example, some methods have been reported for labeling methylcytosine with an electroactive metal complex25,30. An electrochemical hybridization assay was reported with ferrocenylnaphthalene diimide as an electro-active intercalator31. We have reported an electrochemical method for methylcytosine determination that involves labeling with nanoparticles containing cadmium for anodic stripping voltammetry32. However, the method requires a step in which the nanoparticles are dissolved with an acidic solution. Furthermore, a neutralization step with an alkaline solution is also required prior to anodic stripping voltammetry. Therefore, the assay procedure becomes complicated, and this induces a relatively high error variance (the relative standard deviation was 8.9 %). Moreover, the assay results have to be diluted to 5 mL with acetate buffer because the anodic stripping voltammetry is performed in a large-volume electrochemical cell. As regards electroanalysis, the careful selection of the working electrode material is essential in terms of such aspects of electrochemical performance as sensitivity, detection limit, reproducibility, stability and selectivity for biomolecular determination. In the past, metal electrodes were preferred for electroanalysis, however well ordered nanostructured carbon

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materials (diamond, graphene, carbon nanotubes) have recently attracted interest including as electrode materials for biomolecular determination. We have developed a carbon material consisting of sp2 and sp3 hybrid bonds formed by using an unbalanced magnetron (UBM) sputtering method33. One of the features of the UBM sputtering method is that the sp2/sp3 ratio in a carbon film can be controlled by tuning the ion irradiation energy between a substrate and a carbon target. The carbon film has sufficient conductivity as an electrode material as deposited, and has a nanocrystalline structure consisting of sp2 and sp3 bonds with an extremely flat surface (Ra= 0.07 nm). In this paper, we report an on-chip electrochemical cytosine methylation assessment of genomic DNA with a UBM nanocarbon film electrode. First, the UBM nanocarbon film was fabricated on a silicon wafer suitable as a detection electrode for p-aminophenol (p-AP), which is a major enzyme product in an electrochemical enzyme-linked immunosorbent assay (ELISA)34,35. We then applied the UBM nanocarbon film to an on-chip evaluation of cytosine methylation. This was achieved by measuring the oxidation current derived from pAP, which was catalyzed from a detection probe labeled with alkaline phosphatase, following digestion by a restriction enzyme.

trode, an Ag-AgCl reference electrode and a platinum counter electrode. The UBM nanocarbon film electrode was prepared as described in a previous report 33. The 40 nm thick film was deposited on a boron-doped silicon substrate with UBM sputtering equipment (USP330, Universal Systems Co., Ltd. (Tokyo, Japan)) at room temperature. Sintered carbon was used as a target. During the sputter deposition, the argon gas pressure was 6.0×10-1 Pa. The substrate bias voltage used for the ion irradiation was 20, 75 or 100 V. The sp2/sp3 ratio of the carbon was confirmed with X-ray photoelectron spectroscopy (XPS) (Kratos Axis Ultra, Shimadzu Corporation, (Kyoto, Japan)) by comparing the magnitudes of peaks observed at 284.5 eV (sp2) and 285.5 eV (sp3). An 80 µm thick adhesive sheet (Toyo Adtec, Japan) was used for the passivation. The sheet with a 2 mm-diameter hole was attached to the sputtered carbon film to form an electrode.

EXPERIMENTAL SECTION Chemicals Polymerase, dNTP and unmethylated lambda DNA were purchased from Takara-Bio (Japan). Methylated lambda DNA was prepared as follows, 1 µg of unmethylated lambda DNA was incubated with 4 U CpG Methyltransferase (New England Biolabs, Beverly, MA) in a supplied 20 µl of 1×methyltransferase reaction buffer solution containing 80 µM S-adenosylmethionine for 2 hours at 37°C. Dulbecco’s phosphate buffered saline (PBS) (pH7.4) and streptavidinlabeled alkaline phosphatase were purchased from SigmaAldrich (Japan). HpyCH4IV was purchased from New England Biolabs (Beverly, MA). p-AP and hydrochloric acid were purchased from Kanto Kagaku (Japan). p-aminophenyl phosphate (p-APP) was purchased from LKT Laboratories (St. Paul, MN). Urea and 0.2 M acetate buffer (pH 5.0) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Biotin was purchased from Wako Pure Chemical Industries, Ltd. A streptavidin-immobilized microplate was purchased from Sumitomo-Bakelite (Japan). Ultrapure water (Milli-Q) was used in all of the experiments. An Agilent DNA 1000 kit and a DNA chip for an Agilent 2100 bioanalyzer were purchased from Agilent Technologies (Germany). Conventional glassy carbon and gold electrodes were purchased from BAS (Tokyo, Japan). We prepared two buffer solutions for our assay. Tris buffer (pH 8.0) was prepared containing 10 mM tris (hydroxymethyl) aminomethane, 0.15 M NaCl and 5 mM MgCl2. Tris buffer containing 0.05 (v/v)% Tween 20 was used as a washing buffer, and tris buffer containing 0.05 (v/v)% Tween 20 and 0.1 (w/v)% bovine serum albumin was used as an incubation buffer. Fabrication of carbon films containing a nanocrystalline structure and a microchip Figure 1 is a schematic showing the setup of our electrochemical chip for DNA methylation analysis. Our electrochemical setup consists of a UBM nanocarbon working elec-

Figure1 (a) Schematic and (b) TEM image of a nanocarbon film electrode. (c) Schematic of experimental setup for electrochemical measurement.

Cytosine methylation assessment Figure 2 is a schematic showing the detection principle of our technique for the electrochemical evaluation of cytosine methylation. The detection principle is a variation of the COBRA technique16, where the result is conventionally obtained by separation techniques such as poly-acrylamide gel electrophoresis (PAGE). In this study, the result was obtained electrochemically on a carbon electrode chip containing nanocrystalline by using a hybridization assay. Briefly, methylated and unmethylated lambda genomic DNAs were bisulfitetreated, and then the target sequence was amplified by PCR. Next, the PCR product was treated with a restriction enzyme,


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Analytical Chemistry HpyCH4IV, which cleaved the 5’-ACGT- 3’ region. HpyCH4IV had one site in the bisulfite-unconverted sequence when the original sequence was methylated. Therefore, no hybridization occurred with a biotinylated probe in figure 2(3). After labeling with alkaline phosphatase, an electrochemical signal derived from p-AP was observed after an injection of paminophenol phosphate. The procedure shown in figure 2 is detailed below.

Figure 2 Detection principle of our electrochemical DNA methylation analysis technique. 1) Bisulfite-treatment of genomic DNA. 2) PCR and cleavage with a restriction enzyme (HpyCh4IV), which cleaves the 5’-ACGT-3’ region. 3) Immobilization of biotinylated PCR product onto a streptavidin surface. The PCR product is denatured by urea and then hybridized with a biotinylated probe, which has the same sequence as the reverse primer for PCR. 4) The biotinylated probe is labeled with streptavidin-modified alkaline phosphatase. Finally, an electrochemical signal is obtained. When alkaline phosphatase is labeled, the oxidation current derived from p-AP catalyzed from p-APP is measured by cyclic voltammetry.

1) Bisulfite-treatment Bisulfite treatment was performed using a commercial kit (MethylEasy Xceed kit, catalog no. GR004, Takara-Bio) in accordance with the manufacturer’s manual. In this process, unmethylated cytosine is converted to uracil, however methylcytosine is unaffected as shown in figure 2(1). 2) PCR and cleavage by HpyCH4IV PCR was carried out in a 50 µL sample comprising 20 ng of bisulfite treated DNA, 1×Takara ex taq buffer, 2.5 mM MgCl2, 0.2 mM dNTP mixture, 1 mM each of forward and reverse primers and 1.25 U DNA polymerase. PCR amplification was used to perform 30 denaturing cycles for 30 s at 94 °C, annealing for 30 s at 55 °C, elongation for 30 s at 72 °C, with a final step at 4° C for 10 min with a thermal cycler (Takara-Bio, model TP600). The primers were designed to generate a 253bp PCR product from lambda DNA. The 5’ end of the forward primer was biotinylated, therefore the PCR product can be captured on a streptavidin surface when we later employ our electrochemical detection technique. The forward and reverse primer sequences are shown in table S1 in the supporting in-

formation. The concentration of the PCR product was confirmed with an Agilent 2100 bioanalyzer. 100 nM of PCR product was treated with 10 U of restriction enzyme HpyCH4IV (New England Labs) in a total volume of 10 µl at 37 °C for 1 h. HpyCH4IV cleaved the ACGT sequence when the target cytosine was methylated but not when it was unmethylated, as shown in figure 2(2). 3) Hybridization with biotinylated probe Each well of a streptavidin-modified microtiter plate was filled with 100 µL of HpyCH4IV treated PCR product, which was kept at room temperature for 30 min. Unbound PCR product was removed by rinsing the plate three times with washing buffer. Next, 200 µL of 8 M urea was added to denature the PCR product for 30 min at 37 °C, and then 200 µL of 1 mg/mL biotin was added for 30 min at 37 °C to block excess avidin sites. After rinsing the plate three times with washing buffer, 50 µL of 50 nM biotinylated detection probe DNA was added and incubated for 30 min at 37 °C. The biotinylated detection probe hybridized when the target cytosine was methylated but not when the target cytosine was unmethylated as shown in figure 2(3). 4). Enzyme labeling and determination 50 µL of 1 µg/mL streptavidin-labeled alkaline phosphatase was added to each well for 0.5 h at 37 °C. After rinsing, 100 µL of 5 mM p-APP (Tris buffer (pH 8.0)) was added as an alkaline phosphatase substrate, and incubated for 0.5 h at 37 °C. 50 µL of supernatant was transferred to our electrochemical chip shown in figure 1. Finally, a cyclic voltammogram was obtained by scanning the potential of a working electrode from -0.3 to 0.4 V at a scan rate of 0.5 V/sec.

RESULTS AND DISCUSSION Electrochemical performance of UBM nanocarbon film for pAP determination First, we investigated the electrochemical performance of our UBM nanocarbon film electrode with p-AP, which is one of the best-known enzyme products of electrochemical immunoassay.36-40 Three kinds of UBM nanocarbon film were deposited on a silicon wafer with different bias voltages of 20, 75 and 100 V. From XPS measurement, the ratios of the sp2 and sp3 bonds on the UBM nanocarbon films were confirmed to be 80:20, 64:36 and 50:50, respectively. Therefore, these UBM nanocarbon electrodes are abbreviated as UBM (80:20), (64:36) and (50:50), respectively. Figure 3 shows calibration curves for p-AP obtained with our UBM nanocarbon film electrodes, and conventional glassy carbon (GC) and with gold electrodes as references because GC and gold electrodes have been preferred for measuring pAP in previous reports.37-40 Linear increases in oxidation current are observed as the p-AP concentration increases. However the slopes are different for different electrodes. The slope becomes gentle as the sp2 content on the UBM nanocarbon film decreases. This is because the shape of the cyclic voltammogram becomes distorted as the sp2 content decreases as shown in figure S1 in the supporting information. A wellshaped and reversible voltammogram for p-AP was observed on a sp2-rich UBM (80:20).



Figure 3 Calibration curves obtained with various electrodes for p-AP measurement. ○; UBM(80:20), □; UBM(64:36), ◇ ; UBM(50:50), ●; Glassy carbon (GC), △; Gold. Scan rate is 50 mV/sec.

Table 1 shows a summary of the electrochemical performance of the electrodes for p-AP determination used in this study. Sensitivity (signal current) tended to increase as the sp2 content in the carbon electrodes increased. This is because of the π-π interaction between aromatic p-AP and the graphenelike sp2 carbon structure. It has been reported that graphenemodified electrodes exhibit a large oxidation current for aromatic compounds because of the π-π interaction41-43. We have already reported33 that our UBM nanocarbon film contains a graphene-like structure as revealed by TEM observation and shown in figure 1(b). Therefore, the sp2 content helps us to obtain a large signal current for p-AP. Table 1 Summary of electrode performance for electrochemical p-AP determination. Signal-to-noise ratio and reproducibility were calculated when 50 µM p-AP was measured. ∆Ep was calculated from a cyclic voltammogram at a scan rate of 50 mV/sec.

Although the sensitivities of the UBM (80:20) and the conventional GC electrode were comparable, the capacitance at the UBM (80:20) was smaller than that at the GC electrodes shown in figure S1. For examples, the capacitative current at the GC electrode was 5.1 µA/cm-2 at -0.1 V, which was successfully reduced to 0.59 µA/cm-2 for our UBM (80:20). Therefore, for example, the signal-to-noise ratio with the UBM (80:20) electrode was 9.5 times better than that with the GC electrode. The small capacitance at our UBM nanocarbon film results from its flat surface. An extremely flat UBM carbon surface at the angstrom level was confirmed with an AFM observation33. We also tried to fabricate more sp2 rich UBM

nanocarbon film by tuning the bias voltage less than 20 V. However the surface roughness becomes large, and this induces the increase of background noise level. This would be because weak electron irradiation during the UBM nanocarbon film deposition33.” Several researchers have investigated new carbon electrode materials for p-AP measurement because p-AP is also routinely detected in pharmaceutical preparations as a product of paracetamol degradation or as a synthetic intermediate44,45. Fan et al46 and Filik et al 47 reported an electrochemical p-AP measurement with a graphene-modified carbon electrode. These graphene or carbon nanotube-modified carbon electrodes exhibited a signal current that was two or three orders of magnitudes larger than that of our carbon, mainly due to their large electrode surface. Unfortunately, the graphenemodified electrodes also caused an increase in the background current owing to the large capacitance at the same time. Therefore, the detectable linear range for p-AP is comparable to that of our carbon electrode. One significant advantage is that our nanocarbon film can be used for electroanalysis as deposited without any further modification. Moreover we can easily tune the sp2/sp3 ratio in the nanocarbon film by changing the bias voltage while maintaining its ultraflat surface. The lowest detection limit of 0.5 µM p-AP was obtained with UBM (80:20) and UBM (64:36) electrodes thanks to the large signal-to-noise ratio. In our previous report on heavy metal ion determination, an sp3-rich UBM (50:50) showed the lowest detection limit for cadmium determination by anodic stripping voltammetry owing to the wide cathodic potential window of the robust sp3-rich electrode. For determining aromatic compounds such as p-AP as described in this manuscript, an sp2rich UBM electrode would be advantageous because it is wellbalanced with both a high sensitivity for p-AP and a low noise current caused by a small capacitance. Furthermore, an excellent reproducibility of 1.2 % ~ 2.8 % was achieved with our nanocarbon electrodes as shown in Table 1. This was sufficient for quantitative evaluation of DNA methylation in the following experiments. Electrochemical determination of methylated and unmethylated DNA Methylated and unmethylated genomic DNAs were electrochemically evaluated on the basis of the procedure shown in figure 2. Figure 4 (a, b) show cyclic voltammograms for the methylated and unmethylated DNA samples on our nanocarbon film when we varied the PCR product concentration from 0 to 1000 pM. When the target cytosine in genomic DNA is methylated, the 5’-ACGT-3’ sequence is maintained after the bisulfite treatment because methylcytosine is not affected by the treatment, and the restriction enzyme cleaves the 5’ACGT-3’ sequence into two fragments. Therefore, no hybridization occurs with a biotinylated detection probe, i.e. p-AP is not produced by the alkaline phosphatase reaction. As expected, no significant increase in the redox current of p-AP can be seen in figure 4 (a) when the PCR product concentration increased to 1000 pM. The slight increase in redox current around 0 and 0.2 V was considered to be the effect of the nonspecific adsorption of alkaline phosphatase. This is because the slight redox current was independent of the PCR product concentration, and was constantly observed. The slight peak is negligible in terms of background noise.


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Figure 4 Cyclic voltammograms for (a) methylated and (b) unmethylated lambda DNA when varying the concentration of each PCR product. (c) Variations in anodic peak currents estimated from (a) and (b).

In contrast, in figure 4 (b) a large increase in redox current derived from p-AP can be seen for unmethylated DNA as the concentration of the PCR product increased. This is because cytosine was converted to uracil during the bisulfite treatment, therefore the restriction enzyme did not cleave the PCR product and the biotinylated detection probe hybridized. Figure 4 (c) shows the variation in the anodic current of cyclic voltammograms for methylated and unmethylated DNAs. A linear relationship was found between the PCR product and the an-

odic current when the target cytosine was unmethylated (R2= 0.9945). By contrast, a small, steady background current was observed for methylated DNA. The detection limit of the PCR product for unmethylated DNA was 1.9 pM (S/N=3) Table 2 shows a summary of the performance of Combined Bisulfite Restriction Analysis, which is called COBRA. The detection limit for the bisulfite-treated PCR product improved by about three orders of magnitude compared with that obtained with on-chip electrophoretic analysis with fluorescence intercalator labeling. Moreover the detection limit is more than two orders of magnitude better than that realized by our recent electrochemical approach with CdSe labeling. The reasons for achieving this low detection limit in this manuscript are considered to be as follows. First, the signal current was enhanced because a number of redox species (p-AP) were produced by the alkaline phosphatase. Furthermore, a high concentration of p-AP was produced in a short time because the enzymatic reaction was performed in a small volume solution (100 µL). In our previous study, a commercial electrochemical cell with a volume of 5 mL was used for electrochemical measurements. Therefore, the electro-active molecules had to be diluted. In this study, the high concentration of p-AP was electrochemically measured without dilution on-chip. Note that this method, unlike that used in our previous study, is a simple procedure where we neither dilute nor change the treatment solution. This simplicity allowed us to obtain better analytical performance including a small R.S.D. value as well as a low detection limit as summarized in Table 2. Second, we have developed a nanocarbon film electrode suitable for p-AP determination with a high signal-to-noise ratio. Of course, the PCR product concentration becomes high as a result of increasing the number of PCR cycles. However, COBRA is usually performed for 30 PCR cycles because non-specific amplification occurs simultaneously, and DNA band smearing is observed in electrophoretic analysis. Therefore, increasing the number of PCR cycles does not improve the sensitivity of COBRA. It is important to undertake a quantitative analysis to reveal whether or not the PCR product is cleaved by a restriction enzyme with a small sample volume. With the conventional COBRA method, the DNA band shift in PAGE is used to confirm cleavage. Furthermore, highly sensitive measurement in a small volume sample was achieved with microfluidic electrophoresis, for example, using an Agilent bioanalyzer48,49. We have further demonstrated highly sensitive electrochemical COBRA with a nanocarbon film electrode combined with a hybridization assay. The highly sensitive measurement of the PCR product with COBRA should be useful for reducing the input DNA because a lot of input DNA is required for COBRA because more than 99 % of the input DNA is destroyed50 during the bisulfite-treatment on account of its severe acidic and thermal conditions.51 Table 2 Comparison of performance of COBRA based DNA methylation assay.



DNA methylation evaluation by electrochemical COBRA CpG methylated and unmethylated lambda DNAs were prepared, and they were blended to form a model genomic DNA with 0, 25, 50, 75 and 100 % methylation at a target CpG site. We then estimated the methylation ratio at the target cytosine with our electrochemical COBRA system. Figure 5(a) shows cyclic voltammograms that we obtained when varying the methylation ratio. The peak height for p-AP increased as the methylation ratio decreased due to the decrease in the cleavage ratio by the restriction enzyme. Figure 5(b) shows results for the DNA methylation assessment of the target. We successfully obtained a linear relationship (R2=0.988) between the methylation ratio and the anodic current of the voltammograms on our nanocarbon electrode.

pressed to within ±4.3 %, which is comparable to conventional electrophoretic results (5.0 %) as summarized in table 2. It is difficult to compare the value with recent reports related to new methylation assay development because most previous reports do not mention it. However, the value is superior to that for recently reported on-chip DNA methylation analysis when we compare the sizes of the error bars in the figures. 52,53 As shown in figure S2, an almost steady current was observed independent of the methylation ratio without restriction enzyme treatment. This is useful as a positive control for confirming that the assay has been carried out successfully, and the current value can be used as the maximum current signal for an unmethylated DNA sample. Although we achieved improvement of sensitivity with high accuracy, throughput becomes rather limited in comparison with conventional COBRA detected by PAGE. This is because PAGE can be performed in parallel on multiple samples including standard DNA ladder, whereas we can obtain one result on a chip at the present. For simultaneous measurement of multiple samples in a high throughput, integration of multi-electrodes is a future challenge.

CONCLUSION We proposed the on-chip evaluation of cytosine methylation in genomic DNA with electrochemical COBRA using a nanocarbon film. A high signal-to-noise ratio was obtained for electroactive p-AP on a nanocarbon chip by optimizing the ratio of the sp2 and sp3 content. This is thanks to both the large oxidation current derived from the π-π interaction between aromatic p-AP and the graphene-like structure of our nanocarbon, and the low capacitive current made possible by the flat angstrom-level surface. Our fine nanocarbon material fabricated with a vacuum apparatus can be reproducibly used for electroanalysis as deposited without any further modification. The methylation ratio of a target cytosine was accurately evaluated from the hybridization assay result for a bisulfite-treated PCR product with a restriction enzyme. Our electrochemical approach using a nanocarbon electrode is beneficial for DNA methylation evaluation in a small amount of DNA.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional information including cyclic voltammograms when measuring p-AP with various electrodes (S-1), electrochemical responses without restriction enzyme treatment (S-2), and primer sequences used in this study (S-3).

AUTHOR INFORMATION Corresponding Author * [email protected] Figure 5 (a) Cyclic voltammograms for genomic DNA with various methylation ratios at a target cytosine. (b) Relationship between methylation ratio and signal current with our electrochemical COBRA system.

The standard deviations were 0.17, 0.08, 0.12, 0.05 and 0.02 µA/cm for 0, 25, 50, 75 and 100 % methylated samples, respectively. From the slope of the regression line in figure 5(b), the margin of error for the DNA methylation ratio was sup-

Present Addresses †Advanced Science Research Laboratory, Saitama Institute of Technology, Fukaya, Saitama 369-0293, Japan

ACKNOWLEDGMENT Our study was financially supported by JSPS KAKENHI, Grant No. 26410168. We thank Mr. D. Meacock for revising the language of the manuscript.


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REFERENCES (1) Doerfler, W. Angew. Chem.-Int. Edit. 1984, 23, 919-931. (2) Esteller, M. In Annu. Rev. Pharmacol. Toxicol., 2005; Vol. 45; pp 629-656. (3) Jones, P. A.; Baylin, S. B. Nat. Rev. Genet. 2002, 3, 415428. (4) Robertson, K. D. Nat. Rev. Genet. 2005, 6, 597-610. (5) Jones, P. A. Cancer Res. 1996, 56, 2463-2467. (6) Monk, M. Dev. Genet. 1995, 17, 188-197. (7) Ledoux, S.; Nalbantoglu, J.; Cashman, N. R. Mol. Brain Res. 1994, 24, 140-144. (8) Kurita, R.; Niwa, O. Lab on a Chip 2016, 16, 3631-3644. (9) Cokus, S. J.; Feng, S.; Zhang, X.; Chen, Z.; Merriman, B.; Haudenschild, C. D.; Pradhan, S.; Nelson, S. F.; Pellegrini, M.; Jacobsen, S. E. Nature 2008, 452, 215-219. (10) Frommer, M.; McDonald, L. E.; Millar, D. S.; Collis, C. M.; Watt, F.; Grigg, G. W.; Molloy, P. L.; Paul, C. L. Proc. Natl. Acad. Sci. USA 1992, 89, 1827-1831. (11) Lister, R.; O'Malley, R. C.; Tonti-Filippini, J.; Gregory, B. D.; Berry, C. C.; Millar, A. H.; Ecker, J. R. Cell 2008, 133, 523-536. (12) Gu, H.; Bock, C.; Mikkelsen, T. S.; Jaeger, N.; Smith, Z. D.; Tomazou, E.; Gnirke, A.; Lander, E. S.; Meissner, A. Nat. Methods 2010, 7, 133-U69. (13) Smallwood, S. A.; Lee, H. J.; Angermueller, C.; Krueger, F.; Saadeh, H.; Peet, J.; Andrews, S. R.; Stegle, O.; Reik, W.; Kelsey, G. Nat. Methods 2014, 11, 817-820. (14) Krueger, F.; Kreck, B.; Franke, A.; Andrews, S. R. Nat. Methods 2012, 9, 145-151. (15) Bock, C.; Reither, S.; Mikeska, T.; Paulsen, M.; Walter, J.; Lengauer, T. Bioinformatics 2005, 21, 4067-4068. (16) Xiong, Z. G.; Laird, P. W. Nucleic Acids Res. 1997, 25, 2532-2534. (17) Herman, J. G.; Graff, J. R.; Myohanen, S.; Nelkin, B. D.; Baylin, S. B. Proc. Natl. Acad. Sci. USA 1996, 93, 9821-9826. (18) Yang, A. S.; Estecio, M. R. H.; Doshi, K.; Kondo, Y.; Tajara, E. H.; Issa, J. P. J. Nucleic Acids Res. 2004, 32, e38. (19) Kato, D.; Sekioka, N.; Ueda, A.; Kurita, R.; Hirono, S.; Suzuki, K.; Niwa, O. J. Am. Chem. Soc. 2008, 130, 3716-3717. (20) Wang, P.; Han, P.; Dong, L.; Miao, X. Electrochem. Commun. 2015, 61, 36-39. (21) Ivandini, T. A.; Honda, K.; Rao, T. N.; Fujishima, A.; Einaga, Y. Talanta 2007, 71, 648-655. (22) Wang, P.; Han, P.; Dong, L.; Miao, X. M. Electrochem. Commun. 2015, 61, 36-39. (23) Wang, P.; Chen, H. B.; Tian, J. Y.; Dai, Z.; Zou, X. Y. Biosens. Bioelectron. 2013, 45, 34-39. (24) Daneshpour, M.; Moradi, L. S.; Izadi, P.; Omidfar, K. Biosens. Bioelectron. 2016, 77, 1095-1103. (25) Tanaka, K.; Tainaka, K.; Kamei, T.; Okamoto, A. J. Am. Chem. Soc. 2007, 129, 5612-5620. (26) Koo, K. M.; Wee, E. J. H.; Rauf, S.; Shiddiky, M. J. A.; Trau, M. Biosens. Bioelectron. 2014, 56, 278-285. (27) Li, W.; Wu, P.; Zhang, H.; Cai, C. X. Anal. Chem. 2012, 84, 7583-7590. (28) Sato, S.; Saeki, T.; Tanaka, T.; Kanezaki, Y.; Hayakawa, M.; Haraguchi, K.; Kodama, M.; Nishihara, T.; Tominaga, K.; Takenaka, S. Appl. Biochem. Biotechnol. 2014, 174, 869-879.

(29) Kurita, R.; Arai, K.; Nakamoto, K.; Kato, D.; Niwa, O. Anal. Chem. 2012, 84, 1799-1803. (30) Fojta, M.; Kostecka, P.; Pivonkova, H.; Horakova, P.; Havran, L. Curr. Anal. Chem. 2011, 7, 35-50. (31) Sato, S.; Tsueda, M.; Kanezaki, Y.; Takenaka, S. Anal. Chim. Acta 2012, 715, 42-48. (32) Yanagisawa, H.; Kurita, R.; Yoshida, T.; Kamata, T.; Niwa, O. Sens. Actuator B-Chem. 2015, 221, 816-822. (33) Kamata, T.; Kato, D.; Ida, H.; Niwa, O. Diam. Relat. Mat. 2014, 49, 25-32. (34) Zhou, Y. L.; Yin, H. S.; Li, X.; Li, Z.; Ai, S. Y.; Lin, H. Biosens. Bioelectron. 2016, 86, 508-515. (35) Kurita, R.; Yokota, Y.; Ueda, A.; Niwa, O. Anal. Chem. 2007, 79, 9572-9576. (36) Takahashi, Y.; Miyamoto, T.; Shiku, H.; Asano, R.; Yasukawa, T.; Kumagai, I.; Matsue, T. Anal. Chem. 2009, 81, 27852790. (37) Niwa, O.; Xu, Y.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1993, 65, 1559-1563. (38) Wang, J.; Ibanez, A.; Chatrathi, M. P.; Escarpa, A. Anal. Chem. 2001, 73, 5323-5327. (39) Aguilar, Z. P.; Vandaveer, W. R.; Fritsch, I. Anal. Chem. 2002, 74, 3321-3329. (40) Yasukawa, T.; Yoshimoto, Y.; Goto, T.; Mizutani, F. Biosens. Bioelectron. 2012, 37, 19-23. (41) Kang, X. H.; Wang, J.; Wu, H.; Liu, J.; Aksay, I. A.; Lin, Y. H. Talanta 2010, 81, 754-759. (42) Georgakilas, V.; Tiwari, J. N.; Kemp, K. C.; Perrnan, J. A.; Bourlinos, A. B.; Kim, K. S.; Zboril, R. Chem. Rev. 2016, 116, 54645519. (43) Pandikumar, A.; How, G. T. S.; See, T. P.; Omar, F. S.; Jayabal, S.; Kamali, K. Z.; Yusoff, N.; Jamil, A.; Ramaraj, R.; John, S. A.; Lim, H. N.; Huang, N. M. RSC Advances 2014, 4, 6329663323. (44) Wang, S. F.; Xie, F.; Hu, R. F. Sens. Actuator B-Chem. 2007, 123, 495-500. (45) Rattanarat, P.; Suea-Ngam, A.; Ruecha, N.; Siangproh, W.; Henry, C. S.; Monpichar, S. A.; Chailapakul, O. Anal. Chim. Acta 2016, 925, 51-60. (46) Fan, Y.; Liu, J.-H.; Yang, C.-P.; Yu, M.; Liu, P. Sens. Actuator B-Chem. 2011, 157, 669-674. (47) Filik, H.; Cetintas, G.; Koc, S. N.; Gulce, H.; Boz, I. Russ. J. Electrochem. 2014, 50, 243-252. (48) Brena, R. M.; Auer, H.; Kornacker, K.; Plass, C. Nat. Protoc. 2006, 1, 52-58. (49) Brena, R. M.; Auer, H.; Kornacker, K.; Hackanson, B.; Raval, A.; Byrd, J. C.; Plass, C. Nucleic Acids Res. 2006, 34. (50) Tanaka, K.; Okamoto, A. Bioorg. Med. Chem. Lett. 2007, 17, 1912-1915. (51) Raizis, A. M.; Schmitt, F.; Jost, J. P. Anal. Biochem. 1995, 226, 161-166. (52) Yoon, J.; Park, M. K.; Lee, T. Y.; Yoon, Y.-J.; Shin, Y. Lab on a Chip 2015, 15, 3530-3539. (53) Heimer, B. W.; Shatova, T. A.; Lee, J. K.; Kaastrup, K.; Sikes, H. D. Analyst 2014, 139, 3695-3701.



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On-Chip Evaluation of DNA Methylation with Electrochemical

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