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Direct electrochemical detection of DNA methylation for retinoblastoma and CpG fragments using a nanocarbon film
Analytical Biochemistry 405 (2010) 59–66
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Direct electrochemical detection of DNA methylation for retinoblastoma and CpG fragments using a nanocarbon film Keisuke Goto a,b, Dai Kato a, Naoyuki Sekioka a,b, Akio Ueda a,c, Shigeru Hirono d, Osamu Niwa a,b,c,* a
National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan University of Tsukuba, 1-1-1 Tenno-dai, Tsukuba, Ibaraki 305-8571, Japan c Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan d MES-Afty Corporation, 2-35-2 Hyoe, Hachioji, Tokyo 192-0918, Japan b
a r t i c l e
i n f o
Article history: Received 12 March 2010 Received in revised form 13 May 2010 Accepted 2 June 2010 Available online 4 June 2010 Keywords: Electrochemistry Nanocarbon film DNA methylation Direct oxidation sp2 and sp3 nanocrystallite
a b s t r a c t We describe the direct electrochemical detection of DNA methylation in relatively long sequences by using a nanocarbon film electrode. The film was formed by employing the electron cyclotron resonance sputtering method and had a nanocrystalline sp2 and sp3 mixed bond structure. Our methylation detection technique measures the differences between the oxidation currents of both 5-methylcytosine and cytosine without a bisulfite reaction or labeling. This was possible because this film electrode has a wide potential window while maintaining the high electrode activity needed to quantitatively detect both bases by direct oxidation. By optimizing the electrode surface conditions using electrochemical pretreatment, we used this film to quantitatively detect single cytosine methylation regardless of the methylation position in the sequence including retinoblastoma gene fragments (24mers). This was probably due to the high stability of this film electrode, which we achieved by controlling the surface hydrophilicity to suppress the fouling, and by maintaining electrode activity against all the bases. The pH optimization of the oligonucleotide measurements was also useful for distinguishing both bases separately. Under the optimized conditions, this film electrode allowed us to realize the quantitative detection of DNA methylation ratios solely by measuring methylated 50 -cytosine-phosphoguanosine (CpG) repetition oligonucleotides (60mers) with different methylation ratios. Ó 2010 Elsevier Inc. All rights reserved.
Carbon materials have attracted much interest because of their structural differences (e.g., graphene, graphite, fullerene, carbon nanotube (CNT),1 diamond, and diamond-like carbon) and their wide variety of structurally dependent electronic and electrochemical properties [1–3]. Many studies have focused on electroanalysis for biomolecules using carbon materials since electrochemical techniques offer the advantages of versatility and sensitivity as regards constructing a sensor format. The glassy carbon (GC) electrode has been widely used for detecting biomolecules over several decades [1]. Recently, new carbon materials, and in particular doped diamond, CNT, and graphene, have been actively developed as sensing platforms for biomolecules such as DNA and proteins [4–8]. In terms of DNA electroanalysis, many concepts have been reported including a hybridization-based technique with electroactive tags or intercala* Corresponding author at: National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan. Fax: +81 29 861 6177. E-mail address: [email protected] (O. Niwa). 1 Abbreviations used: CpG, 50 -cytosine-phosphoguanosine; G, guanine; A, adenine; T, thymine; C, cytosine; mC, 5-methylcytosine; CNT, carbon nanotube; GC, glassy carbon; BDD, boron doped diamond; ECR, electron cyclotron resonance; SWV, square wave voltammogram; Ep, peak potential. 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.06.004
tors [9–18], and the direct oxidation of DNA [19–27]. With the hybridization-based techniques, electrode materials including gold and indium tin oxide to immobilize DNA probes have been reported [9–18]. DNA immobilization on a boron doped diamond (BDD) substrate surface using a photo-attachment technique has also recently been reported despite its chemical inertness [7]. In contrast, the direct oxidation of DNA is the simplest of these methods. Some groups have already reported that GC and BDD electrodes exhibit oxidation currents for all the free bases [23,24]. However, with these direct oxidation-based measurements, it is difficult to discriminate single-base substitutions in the oligonucleotides because of the large background current at the GC electrode, and because of the low electrochemical activity at the BDD electrode despite its wide potential window [26,27]. Some groups have reported electrochemical DNA sensors that employ electroinactive inosine substituted capture probes instead of guanine (G) [12]. Once the target DNA has been hybridized with the probes immobilized on the electrode surface, the complemented G oxidation signal can be specifically detected without labeling. Recently, we demonstrated a nanocarbon film electrode that has a wide potential window with sufficiently high electrode activity for the quantitative detection of all the bases in an oligonucleotide
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by direct electrochemical oxidation [25–27]. The nanocarbon film that we developed using the electron cyclotron resonance (ECR) sputtering method consists of a stable and homogeneous nanocrystalline sp2 and sp3 hybrid structure with an atomically flat surface, and the sp3/sp2 ratios in the film can be widely controlled by controlling the sputtering conditions [28,29]. Our previous work clearly indicated that ECR nanocarbon film with an sp2/(sp2 + sp3) ratio of 0.6 could provide excellent electrochemical characteristics compared with BDD and GC electrodes. This is because it has similar advantages to BDD electrodes, namely a wide potential window, a low background current, and little surface fouling, but provides high electrode activity as found with GC electrodes. These characteristics allow the detection of biomolecules with slower electron transfer rates, such as NADPH and pyrimidine bases, while maintaining a wide potential window and a low background current. As a result, we succeeded in the direct detection of single nucleotide polymorphisms, which enabled us to oxidize all the DNA bases individually, by using the ECR nanocarbon film electrode [26,27]. Such properties are advantageous for distinguishing DNA bases and their derivatives, for example cytosine (C) and its derivative 5-methylcytosine (mC), which have recently attracted attention in the field of epigenetics. Genomic DNA methylation has also attracted considerable attention because it plays an important role in the regulation of gene transcription, embryogenesis, and various diseases such as cancer, without any change in the DNA sequence [30–33]. In DNA methylation assays, sequencing techniques cannot be used since both bases exhibit identical Watson–Crick base-pair behavior. Thus, current DNA methylation assays involve some technologies designed to distinguish both mC and C in DNA, including a bisulfite [34–39], restriction enzymes [40,41], and molecules with a biological/chemical affinity [42–46]. In bisulfite-based assays, C is deaminated and then converted to uracil, whereas mC remains almost unchanged due to its extremely low reactivity [34]. Restriction enzymes that catalyze the scission reaction of a specific base sequence have also been used. When the C in the specific sequence is methylated, the scission is inhibited [40,41]. The use of the above techniques commonly provides a C-positive assay. Nevertheless, the bisulfite-based methods are currently considered gold standard assay techniques because of their accuracy and applicability, but they are complicated and require several steps for mC detection and this is time consuming. By contrast, certain recent studies have used antibodies [42], binding proteins [43], and metal complexes [44–46] with high affinity for mC that can be read out as an mC-positive assay, as well as some analysis with HPLC. In a previous study, we found that the ECR nanocarbon film electrode could distinguish both mC and C bases individually, by measuring the peak potential differences (ca. 150 mV vs. Ag/AgCl) caused by C methylation. This is because this electrode has a wide potential window while maintaining the high electrode activity needed to oxidize both bases. As a result, we succeeded in the direct detection of DNA methylation from a short oligonucleotide (6mer) without any treatment [26]. This concept for DNA methylation constitutes the first discrimination of both mC and C base differences solely by using electrochemical oxidation without bisulfite or labeling processes. However, we must undertake a further investigation of the quantitative ability and applicability with respect to significant actual sequences or much longer sequences if we are to construct a simple, rapid, and low cost assay technique for DNA methylation. This is because analytes with a higher molecular weight usually foul the electrode surface very quickly. In this work, we studied the electrochemical detection of DNA methylation, based on the direct oxidation of both mC and C in much longer actual sequences by employing ECR nanocarbon film electrodes. We examined the nanocarbon film electrode for its ability to detect the DNA methylation site from the synthetic oligo-
nucleotides including methylation sites of retinoblastoma (RB1) gene fragments (up to 24mers) and the CpG repeat sequences (60mers), which are much longer sequences than those described in previous report [26]. We studied the effect of electrochemical pretreatment on the stability of an ECR nanocarbon film electrode to obtain the optimum conditions when oxidizing longer oligonucleotides. We also investigated the dependence of the pH condition on electrochemical measurements using the ECR nanocarbon film electrode with a view to achieving the optimum peak separation among adenine (A), mC, and C in the methylated oligonucleotides, and we compared the obtained electrochemical results with UV spectrum results for the same samples. We also investigated the quantitative performance of the DNA methylation ratios by measuring CpG samples with different methylation ratios. Materials and methods Materials ECR nanocarbon films suitable for use in DNA electroanalysis were prepared in accordance with previous reports [25–27,29]. Briefly, the nanocarbon films were deposited on highly doped silicon (1 0 0) substrates with ECR sputtering equipment (AFTEX3200, 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 5.0 102 Pa. During deposition, the irradiation ion current density was 5.8 mA/cm2, and the ion acceleration voltage was 75 V. The nanocarbon films were obtained in about 8 min and were typically 40 nm thick. Deoxynucleotide monophosphates, 5methyl-20 -deoxycytidine 50 -monophosphate, disodium salt, and 20 -deoxycytidine 50 -monophosphate, sodium salt, were purchased from USB Corporation and Sigma-Aldrich, respectively. The synthetic oligonucleotides were purchased from Hokkaido System Science, Sapporo, Japan. All other chemicals were of analytical grade. Electrochemical experiments All electrochemical experiments were performed using an ALS/ CHI 760B electrochemical analyzer (CH Instruments, Inc., USA). A platinum wire and an Ag/AgCl (3 M NaCl) electrode were used as auxiliary and reference electrodes, respectively. ECR nanocarbon film was used as the working electrode. A GC disk electrode (diameter = 3 mm, BAS, Tokyo, Japan) was used as a control electrode in the experiment. The ECR nanocarbon film electrode area was defined by using masking tape with a 2.5-mm-diameter hole in it. A 50 mM acetate buffer with/without 2 M sodium nitrate (pH 3.3–5.0, Sigma) was used as the electrolyte solution for the electrochemical measurements. Sodium nitrate was used to realize highly sensitive square-wave voltammogram (SWV) measurements by reducing the intermolecular/intramolecular interaction of the oligonucleotides. The ECR nanocarbon film electrodes were electrochemically pretreated to control the activity of their electrode surfaces with cyclic voltammetry by changing the scan rate, number of cycles, and potential region. We obtained optimized conditions by scanning the potential between 0 and 2.3 V 15 times at a scan rate of 0.1 V/s in 50 mM, pH 7.0, phosphate buffer solution [26]. All the SWV measurements were performed with an amplitude of 25 mV and a DE of 5 mV at 10 Hz. UV experiments The UV signals of all the mononucleotides and oligonucleotides were recorded simultaneously on a NanoDrop 1000 spectrophotometer (Thermo Scientific).
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Results and discussion Comparison of direct discrimination of mC and C deoxynucleotides using electrochemical and optical methods Fig. 1A and B show the background-subtracted SWV at the ECR nanocarbon film electrode and the UV spectra of mC and C deoxynucleotides, respectively. The change in the molecular structure reflected the electrochemical oxidation sufficiently to allow us to distinguish the two bases. As shown in Fig. 1A, the electrochemical oxidation peak potential (Ep) of mC is about 160 mV lower than that of C as we reported previously [26]. On the other hand, the maximum absorption wavelength (kmax) of the mC peak became only 8 nm larger than that of C (272 nm) (Fig. 1B), owing to the electron donor property of the methyl group [26,47]. Then both bases were distinguishable. However, the mixture of the two mononucleotides overlapped to form one peak since their peak differences were too small to separate them. Therefore, none of the bases in the mixture and/or oligonucleotides can be distinguished by using UV experiments without separation systems such as HPLC or electrophoresis. The above result indicates that electrochemical discrimination between mC and C is much clearer than that of UV detection, which is also a nonlabeling measurement. Effect of electrochemical pretreatment of ECR nanocarbon film electrode We have already reported that our film electrode could also detect both mC and C from synthetic short oligonucleotides (up to 6mers) [26]. As regards longer oligonucleotides, fouling affected the resulting oxidation currents even when using the ECR nanocarbon film electrode. To suppress the fouling, and detect much longer oligonucleotides quantitatively, we studied the effect of electrochemical pretreatment on the ECR nanocarbon film electrode stability. This was because we were able to modify the surface of the ECR nanocarbon film easily using electrochemical pretreatment without losing high electrode activity and surface flatness [48,49]. Indeed, the modification of oxygen-containing groups at the ECR nanocarbon film surface enabled us to reduce the fouling by small biomolecules while maintaining the intrinsic properties [48]. Fig. 2 compares the peak current changes for G and A in oligonucleotide 1 (50 -CGAACACCCAGGC-30 ) at GC, as-deposited and
Fig. 2. Stability of three electrodes in successive oligonucleotide (13mers) measurements. Ratio of the peak currents for a base content of 5 lM oligonucleotide 1 oxidation (G, A) in each measurement on the carbon electrode to that in the first measurement as a function of time. The peak currents at each electrode were obtained from SWV measurements in 50 mM, pH 3.3, acetate buffer containing 2 M sodium nitrate. The electrode surface was rinsed with pure water before each measurement.
pretreated ECR nanocarbon film electrodes. The as-deposited ECR nanocarbon film electrode exhibited large G and A oxidation currents in the first measurement, but in the second measurement the respective peak currents decreased greatly to about 15% and 17% of their initial values, despite the fact that the electrode surface was rinsed with pure water between each measurement. This is probably because the oxidized oligonucleotides were strongly adsorbed on the electrode surface. The obtained currents had fallen to less than 10% of their initial values after five measurements. The GC electrode exhibited a similar tendency in that there was an obvious decrease in the peak currents of G and A in 1 (a decrease of more than 95% after five measurements). In contrast, the nanocarbon film pretreated by scanning the potential between 0 and 2.3 V 15 times in 50 mM, pH 7.0, phosphate buffer solution exhibited the stable current responses of oligonucleotide 1. This is
Fig. 1. Background-subtracted SWVs (A) and UV spectra (B) of 100 lM mononucleotides and their mixture measured in 50 mM, pH 5.0, acetate buffer.
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because the hydrophilicity and the number of oxygen-containing groups on the ECR nanocarbon film electrode surface increased without it losing much of its surface flatness or many of its sp2 bonds after the pretreatment [48]. As a result, this treatment provided electrode stability against longer oligonucleotides than those described in previous reports [25–27]. Subsequent experiments were therefore performed with the pretreated ECR nanocarbon film electrode.
Table 1 Normalized current values of A, mC, and C of oligonucleotides 1–4 obtained from Fig. 3. Oligonucleotide Sequence 1
50 CGAACACCCAGGC-30
2
50 -(mC)GAACACCCAGGC-30
3
50 -CGAACACCCAGG(mC)-30
4
50 -(mC)GAACACCCAGG(mC)-30
Electrochemical methylation detection of RB1 oligonucleotides The improved stability realized by electrochemical pretreatment meant that we could employ this film electrode to detect DNA methylation in sequences that constitute frequently occurring methylation hotspots from the tumor suppressor RB1 gene. The RB1 gene encodes an important RB1 protein that prevents excessive cell growth by inhibiting cell cycle progression. When this gene is damaged or silenced by DNA methylation, the RB1 protein is inactivated and this induces retinoblastoma, which is a childhood tumor of the eye [50]. In retinoblastoma tumors, methylation frequently occurs at CGA codons (arginine), and there are three main hotspots (codons 251, 255, and 262) in exon 8 [45,50]. We directly detected different types of methylation status in codons 251 and 255 of the RB1 gene by using the ECR nanocarbon film electrode. The methylation events at codons 251 and 255 are now frequently observed in various tissues including retinas, fibroblasts, and lymphocytes [50]. Fig. 3 shows background-subtracted SWVs of a wild-type oligonucleotide 1 (50 -CGAACACCCAGGC-30 ), and three kinds of methylated oligonucleotides 2 (50 -(mC)GAACACCCA GGC-30 ), 3 (50 -CGAACACCCAGG(mC)-30 ), and 4 (50 -(mC)GAACACCC AGG(mC)-30 ), whose methylation status and positions are different, including the sequence from codons 251 and 255 of the RB1 gene. When these voltammograms were normalized by the peak current of the G that is independent of the detection of DNA methylation, we were able to quantitatively differentiate the voltammograms for these oligonucleotides as shown in Fig. 3B. The DNA bases in the oligonucleotides tend to be oxidized at lower potentials than those of mononucleotides under the same conditions, as also observed in another report [51]. Table 1 summarizes normalized current values of A, mC, and C of oligonucleotides 1–4 obtained based on the current response of G in each oligonucleotide
A (RSD%)
mC (RSD%)
C (RSD%)
1.63 (2.06) 1.69 (2.07) 1.68 (3.56) 1.73 (3.18)
0.45 (2.70) 0.55 (3.88) 0.56 (2.80) 0.65 (2.08)
1.11 (5.12) 1.04 (3.63) 1.03 (4.36) 0.97 (5.27)
The normalized values were calculated based on the current response of G in each oligonucleotide. The relative standard deviation (RSD) of each current was obtained from five measurements.
from Fig. 3B. The magnitude of the current increase for a single mC (the normalized current value of 0.09–0.11 observed at 1.45 V vs. Ag/AgCl) coincided precisely with that of the current decrease of a single C (the normalized current value of 0.06–0.08 observed at 1.55–1.60 V vs. Ag/AgCl) at the ECR nanocarbon film electrode. These results indicate that the influence of the methylation position of the sequences used in this study was almost negligible as regards electrochemical DNA methylation detection. Moreover, we measured RB1 oligonucleotides longer than 1 and 2. Fig. 4A shows background-subtracted SWVs of oligonucleotides 5 (50 -AATGGTTCAC CTCGAACACC CAGG-30 ) and 6 (50 -AATGGTTCAC CT(mC)GAACACC CAGG-30 ), whose oligonucleotide lengths are extended. As regards these longer oligonucleotides, which include double amounts of A and T bases, the mC and C peaks obtained from the voltammograms are somewhat changed but difficult to evaluate quantitatively, even when using the ECR nanocarbon film electrode. In contrast, UV spectra for the oligonucleotides 5 and 6 exhibited no separation peaks for mC and C (Fig. 4B). Furthermore, a closer inspection revealed that there was a subtle current increase assigned to A as the amount of mC increased as summarized in Table 1 because the obtained Ep of mC is very close to that of A. If the oxidation peak potential difference (DE) between A and mC is sufficiently large for them to be distinguished from each other, we are able to detect sharp mC and C peaks in the oligonucleotides with higher selectivity and sensitivity. That is, a wider DE between
Fig. 3. Electrochemical responses of the methylated oligonucleotides (13mers) including the sequence from codons 251 and 255 of the RB1 gene obtained using the ECR nanocarbon film electrode. Background-subtracted SWVs of 1 lM nonmethylated oligonucleotide 1 and its methylated oligonucleotides 2–4, measured in 50 mM, pH 3.3, acetate buffer containing 2 M sodium nitrate.
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Fig. 4. DNA methylation detection of oligonucleotides (24mers) including the sequence from codons 251 and 255 of the RB1 gene. (A) Background-subtracted SWVs of 3 lM oligonucleotides 5 and 6 by using ECR nanocarbon film electrodes in 50 mM, pH 3.3, acetate buffer containing 2 M sodium nitrate. (B) UV spectra of 10 lM 5 and 6 in 50 mM, pH 5.0, acetate buffer.
A and mC (defined as DE1 = EpmC – EpA) and mC and C (defined as DE2 = EpC – EpmC) will enable us to discriminate both mC and C more clearly even in the longer methylated oligonucleotides. pH dependence on electrochemical response of DNA bases in RB1 oligonucleotide In this study, we investigate the pH dependence of Ep for the oxidation of A, mC, and C in oligonucleotide 6 since the Ep values of the DNA bases are known to be dependent on the pH conditions [23,24]. Fig. 5A shows the pH dependence of the three bases (A, mC, and C) in oligonucleotide 6 by measuring the SWV. Some groups have already reported the pH dependence of individual bases [23,24]. For example, Brett and co-workers reported a slope (dEp/dpH) of 58 mV for A base at a GC electrode [23]. Also in our case, we obtained a value of 63 mV for A in the Ep – pH curves (Fig. 5A), which is almost the theoretical value of a 60 mV/pH
unit [24]. It is noteworthy that this slope value was obtained from the A base in oligonucleotide 6 (24mer). In contrast, the mC and C values were 46 and 54 mV, respectively. These values are relatively small compared with those of purine bases. The subtle difference between the slopes of mC/C bases and A is presumably due to their different pKa values (4.6 for mC/C and 4.1 for A) as previously reported [23]. Such differences lead to two kinds of defined DE variation in pH as shown in Fig. 5B. Indeed, DE1 decreased with decreasing pH, whereas DE2 exhibited its maximum value in a pH range of 3.8–4.0. As noted above, the wider DE1 and DE2 are suitable for the clearer discrimination of both mC and C in methylated oligonucleotides. From these results, we decided that the most appropriate pH in the SWV measurement of methylated oligonucleotide 6 at an ECR nanocarbon film electrode is in a range of approximately pH 3.8–4.4, which provided sufficient values for both DE1 and DE2. With the above optimization, an independent peak assigned to single mC was observed at 1.47 V in the
Fig. 5. Ep – pH plots of the bases in oligonucleotide 6 (A), and pH dependence on DE between A and mC (DE1) and mC and C (DE2) (B).
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Fig. 6. Background-subtracted SWV of 3 lM oligonucleotide 6 in 50 mM, pH 4.4, acetate buffer containing 2 M sodium nitrate.
voltammogram of oligonucleotide 6 when measured at pH 4.4 (Fig. 6). We also measured oligonucleotides 5 and 6 by using UV spectroscopy, and we observed no separation peaks of mC and C despite a kmax value shift of the both oligonucleotides as noted in the above figures. This is probably because the kmax shifts with the UV adsorption were little influenced by the pH environment [47] compared with the Ep shift with electrochemical oxidation. These results clearly indicate that electrochemical direct detection distinguishes both mC and C in the longer sequences more selectively than UV detection. Quantification of methylated CpG oligonucleotides We also measured the longer oligonucleotides (60mers) that constitute a nonmethylated and a methylated CpG dinucleotide using our ECR nanocarbon film electrode. The CpG unit constitutes the major part of the promoter region of the gene (CpG island), and the CpG methylation density is closely associated with the tran-
scription level of a gene [30–33]. In normal cells, most CpG islands spanning the promoter regions are unmethylated, and their downstream genes are transcriptionally active. In contrast, when CpG island promoters in cancer cells are methylated, their downstream genes such as tumor suppressor genes are consistently silenced [30–33]. These characteristics clearly demonstrate that CpG methylation density is a critical marker for research in this field. Therefore, the methylation quantification of the CpG repetition sequence is important with respect to understanding DNA methylation related diseases and developing future diagnosis tools. Fig. 7A shows SWVs of nonmethylated oligonucleotide 7 (50 -(CG)30-30 ) and its methylated oligonucleotide 8 (50 -(CG)15(mCG)15-30 , the methylation ratio: 50%). In the voltammogram for 7, the two peaks assigned to G (1.13 V) and C (1.63 V) were observed (red curve). The voltammogram for 8 (blue curve) exhibited another peak corresponding to mC at 1.48 V, whereas the peak current height for C decreased. At the same time, the two oxidation current responses of G in these voltammograms were coincident. These results suggest that we can expect to achieve a simple assay for the DNA methylation ratio solely by performing electrochemical measurements. We estimate the quantitative performance of the DNA methylation ratios from the voltammograms obtained for CpG samples with different methylation ratios. Fig. 7B shows the relationship between the methylation ratio in the oligonucleotides and the current response assigned to mC of the oligonucleotides on a given electrode. It is noteworthy that the current responses were linear in the region of the higher methylation ratio (over 30%) whose correlation coefficient was 0.9955. In the promoter region of the gene, the CpG methylation ratio is known to be 60–90% [30–33]. Therefore, our film electrode is particularly advantageous with regard to the quantitative detection of hypermethylated DNA. Further improvement of the electrochemical sensitivity of each base can be expected to achieve the quantification of the DNA methylation assay with a wide methylation status range. Therefore, we are now studying ways to improve the sensitivity of each base in the samples. Conclusion We used an ECR nanocarbon film electrodes to develop a simple electrochemical DNA methylation analysis technique without
Fig. 7. Typical responses of the methylated oligonucleotides (60mers) consisting of the CpG repetition unit at the ECR nanocarbon film electrode. SWVs of 2 lM nonmethylated CpG oligonucleotide 7 and its methylated CpG oligonucleotides 8 in 50 mM, pH 4.4, acetate buffer containing 2 M sodium nitrate (A), and calibration of methylation ratio obtained from SWV of some oligonucleotides with different methylation ratios (9 (50 -(CG)21(mCG)9-30 ), 10 (50 -(CG)9(mCG)21-30 ), and 11 (50 -(mCG)30-30 )) (B).
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bisulfite or labeling processes. The electrochemically pretreated ECR nanocarbon film electrode enabled us to detect DNA methylation quantitatively, irrespective of the methylation position in longer sequences of the RB1 oligonucleotides, unlike UV experiments. Moreover, the selectivity for detecting both mC and C bases in the oligonucleotides can be successfully optimized by controlling the pH conditions. We also succeeded in the simple and quantitative detection of DNA methylation ratios from the peak current heights of mC in methylated CpG repetition oligonucleotides by using our ECR nanocarbon film electrode. Since several improvements are required if we are to detect DNA methylation in real samples, we are now developing this film to achieve a more sophisticated DNA methylation electroanalysis platform by combining sample pretreatment approaches such as enzymatic reaction or separation technologies. Our carbon film electrodes are suitable for integrating in devices with such functions because this film can be deposited at low temperatures and is easy to microfabricate for applications to a micro-total analysis system or lab-on-a-chip.
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Acknowledgments This work was supported in part by a Grand-in-Aid for Scientific Research (O.N. No. 20350042) from the Ministry of Education, Culture, Science, Sports, and Technology of Japan. References
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Direct electrochemical detection of DNA methylation for retinoblastoma and CpG fragments using a nanocarbon film Keisuke Goto a,b, Dai Kato a, Naoyuki Sekioka a,b, Akio Ueda a,c, Shigeru Hirono d, Osamu Niwa a,b,c,* a
National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan University of Tsukuba, 1-1-1 Tenno-dai, Tsukuba, Ibaraki 305-8571, Japan c Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan d MES-Afty Corporation, 2-35-2 Hyoe, Hachioji, Tokyo 192-0918, Japan b
a r t i c l e
i n f o
Article history: Received 12 March 2010 Received in revised form 13 May 2010 Accepted 2 June 2010 Available online 4 June 2010 Keywords: Electrochemistry Nanocarbon film DNA methylation Direct oxidation sp2 and sp3 nanocrystallite
a b s t r a c t We describe the direct electrochemical detection of DNA methylation in relatively long sequences by using a nanocarbon film electrode. The film was formed by employing the electron cyclotron resonance sputtering method and had a nanocrystalline sp2 and sp3 mixed bond structure. Our methylation detection technique measures the differences between the oxidation currents of both 5-methylcytosine and cytosine without a bisulfite reaction or labeling. This was possible because this film electrode has a wide potential window while maintaining the high electrode activity needed to quantitatively detect both bases by direct oxidation. By optimizing the electrode surface conditions using electrochemical pretreatment, we used this film to quantitatively detect single cytosine methylation regardless of the methylation position in the sequence including retinoblastoma gene fragments (24mers). This was probably due to the high stability of this film electrode, which we achieved by controlling the surface hydrophilicity to suppress the fouling, and by maintaining electrode activity against all the bases. The pH optimization of the oligonucleotide measurements was also useful for distinguishing both bases separately. Under the optimized conditions, this film electrode allowed us to realize the quantitative detection of DNA methylation ratios solely by measuring methylated 50 -cytosine-phosphoguanosine (CpG) repetition oligonucleotides (60mers) with different methylation ratios. Ó 2010 Elsevier Inc. All rights reserved.
Carbon materials have attracted much interest because of their structural differences (e.g., graphene, graphite, fullerene, carbon nanotube (CNT),1 diamond, and diamond-like carbon) and their wide variety of structurally dependent electronic and electrochemical properties [1–3]. Many studies have focused on electroanalysis for biomolecules using carbon materials since electrochemical techniques offer the advantages of versatility and sensitivity as regards constructing a sensor format. The glassy carbon (GC) electrode has been widely used for detecting biomolecules over several decades [1]. Recently, new carbon materials, and in particular doped diamond, CNT, and graphene, have been actively developed as sensing platforms for biomolecules such as DNA and proteins [4–8]. In terms of DNA electroanalysis, many concepts have been reported including a hybridization-based technique with electroactive tags or intercala* Corresponding author at: National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan. Fax: +81 29 861 6177. E-mail address: [email protected] (O. Niwa). 1 Abbreviations used: CpG, 50 -cytosine-phosphoguanosine; G, guanine; A, adenine; T, thymine; C, cytosine; mC, 5-methylcytosine; CNT, carbon nanotube; GC, glassy carbon; BDD, boron doped diamond; ECR, electron cyclotron resonance; SWV, square wave voltammogram; Ep, peak potential. 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.06.004
tors [9–18], and the direct oxidation of DNA [19–27]. With the hybridization-based techniques, electrode materials including gold and indium tin oxide to immobilize DNA probes have been reported [9–18]. DNA immobilization on a boron doped diamond (BDD) substrate surface using a photo-attachment technique has also recently been reported despite its chemical inertness [7]. In contrast, the direct oxidation of DNA is the simplest of these methods. Some groups have already reported that GC and BDD electrodes exhibit oxidation currents for all the free bases [23,24]. However, with these direct oxidation-based measurements, it is difficult to discriminate single-base substitutions in the oligonucleotides because of the large background current at the GC electrode, and because of the low electrochemical activity at the BDD electrode despite its wide potential window [26,27]. Some groups have reported electrochemical DNA sensors that employ electroinactive inosine substituted capture probes instead of guanine (G) [12]. Once the target DNA has been hybridized with the probes immobilized on the electrode surface, the complemented G oxidation signal can be specifically detected without labeling. Recently, we demonstrated a nanocarbon film electrode that has a wide potential window with sufficiently high electrode activity for the quantitative detection of all the bases in an oligonucleotide
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by direct electrochemical oxidation [25–27]. The nanocarbon film that we developed using the electron cyclotron resonance (ECR) sputtering method consists of a stable and homogeneous nanocrystalline sp2 and sp3 hybrid structure with an atomically flat surface, and the sp3/sp2 ratios in the film can be widely controlled by controlling the sputtering conditions [28,29]. Our previous work clearly indicated that ECR nanocarbon film with an sp2/(sp2 + sp3) ratio of 0.6 could provide excellent electrochemical characteristics compared with BDD and GC electrodes. This is because it has similar advantages to BDD electrodes, namely a wide potential window, a low background current, and little surface fouling, but provides high electrode activity as found with GC electrodes. These characteristics allow the detection of biomolecules with slower electron transfer rates, such as NADPH and pyrimidine bases, while maintaining a wide potential window and a low background current. As a result, we succeeded in the direct detection of single nucleotide polymorphisms, which enabled us to oxidize all the DNA bases individually, by using the ECR nanocarbon film electrode [26,27]. Such properties are advantageous for distinguishing DNA bases and their derivatives, for example cytosine (C) and its derivative 5-methylcytosine (mC), which have recently attracted attention in the field of epigenetics. Genomic DNA methylation has also attracted considerable attention because it plays an important role in the regulation of gene transcription, embryogenesis, and various diseases such as cancer, without any change in the DNA sequence [30–33]. In DNA methylation assays, sequencing techniques cannot be used since both bases exhibit identical Watson–Crick base-pair behavior. Thus, current DNA methylation assays involve some technologies designed to distinguish both mC and C in DNA, including a bisulfite [34–39], restriction enzymes [40,41], and molecules with a biological/chemical affinity [42–46]. In bisulfite-based assays, C is deaminated and then converted to uracil, whereas mC remains almost unchanged due to its extremely low reactivity [34]. Restriction enzymes that catalyze the scission reaction of a specific base sequence have also been used. When the C in the specific sequence is methylated, the scission is inhibited [40,41]. The use of the above techniques commonly provides a C-positive assay. Nevertheless, the bisulfite-based methods are currently considered gold standard assay techniques because of their accuracy and applicability, but they are complicated and require several steps for mC detection and this is time consuming. By contrast, certain recent studies have used antibodies [42], binding proteins [43], and metal complexes [44–46] with high affinity for mC that can be read out as an mC-positive assay, as well as some analysis with HPLC. In a previous study, we found that the ECR nanocarbon film electrode could distinguish both mC and C bases individually, by measuring the peak potential differences (ca. 150 mV vs. Ag/AgCl) caused by C methylation. This is because this electrode has a wide potential window while maintaining the high electrode activity needed to oxidize both bases. As a result, we succeeded in the direct detection of DNA methylation from a short oligonucleotide (6mer) without any treatment [26]. This concept for DNA methylation constitutes the first discrimination of both mC and C base differences solely by using electrochemical oxidation without bisulfite or labeling processes. However, we must undertake a further investigation of the quantitative ability and applicability with respect to significant actual sequences or much longer sequences if we are to construct a simple, rapid, and low cost assay technique for DNA methylation. This is because analytes with a higher molecular weight usually foul the electrode surface very quickly. In this work, we studied the electrochemical detection of DNA methylation, based on the direct oxidation of both mC and C in much longer actual sequences by employing ECR nanocarbon film electrodes. We examined the nanocarbon film electrode for its ability to detect the DNA methylation site from the synthetic oligo-
nucleotides including methylation sites of retinoblastoma (RB1) gene fragments (up to 24mers) and the CpG repeat sequences (60mers), which are much longer sequences than those described in previous report [26]. We studied the effect of electrochemical pretreatment on the stability of an ECR nanocarbon film electrode to obtain the optimum conditions when oxidizing longer oligonucleotides. We also investigated the dependence of the pH condition on electrochemical measurements using the ECR nanocarbon film electrode with a view to achieving the optimum peak separation among adenine (A), mC, and C in the methylated oligonucleotides, and we compared the obtained electrochemical results with UV spectrum results for the same samples. We also investigated the quantitative performance of the DNA methylation ratios by measuring CpG samples with different methylation ratios. Materials and methods Materials ECR nanocarbon films suitable for use in DNA electroanalysis were prepared in accordance with previous reports [25–27,29]. Briefly, the nanocarbon films were deposited on highly doped silicon (1 0 0) substrates with ECR sputtering equipment (AFTEX3200, 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 5.0 102 Pa. During deposition, the irradiation ion current density was 5.8 mA/cm2, and the ion acceleration voltage was 75 V. The nanocarbon films were obtained in about 8 min and were typically 40 nm thick. Deoxynucleotide monophosphates, 5methyl-20 -deoxycytidine 50 -monophosphate, disodium salt, and 20 -deoxycytidine 50 -monophosphate, sodium salt, were purchased from USB Corporation and Sigma-Aldrich, respectively. The synthetic oligonucleotides were purchased from Hokkaido System Science, Sapporo, Japan. All other chemicals were of analytical grade. Electrochemical experiments All electrochemical experiments were performed using an ALS/ CHI 760B electrochemical analyzer (CH Instruments, Inc., USA). A platinum wire and an Ag/AgCl (3 M NaCl) electrode were used as auxiliary and reference electrodes, respectively. ECR nanocarbon film was used as the working electrode. A GC disk electrode (diameter = 3 mm, BAS, Tokyo, Japan) was used as a control electrode in the experiment. The ECR nanocarbon film electrode area was defined by using masking tape with a 2.5-mm-diameter hole in it. A 50 mM acetate buffer with/without 2 M sodium nitrate (pH 3.3–5.0, Sigma) was used as the electrolyte solution for the electrochemical measurements. Sodium nitrate was used to realize highly sensitive square-wave voltammogram (SWV) measurements by reducing the intermolecular/intramolecular interaction of the oligonucleotides. The ECR nanocarbon film electrodes were electrochemically pretreated to control the activity of their electrode surfaces with cyclic voltammetry by changing the scan rate, number of cycles, and potential region. We obtained optimized conditions by scanning the potential between 0 and 2.3 V 15 times at a scan rate of 0.1 V/s in 50 mM, pH 7.0, phosphate buffer solution [26]. All the SWV measurements were performed with an amplitude of 25 mV and a DE of 5 mV at 10 Hz. UV experiments The UV signals of all the mononucleotides and oligonucleotides were recorded simultaneously on a NanoDrop 1000 spectrophotometer (Thermo Scientific).
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Results and discussion Comparison of direct discrimination of mC and C deoxynucleotides using electrochemical and optical methods Fig. 1A and B show the background-subtracted SWV at the ECR nanocarbon film electrode and the UV spectra of mC and C deoxynucleotides, respectively. The change in the molecular structure reflected the electrochemical oxidation sufficiently to allow us to distinguish the two bases. As shown in Fig. 1A, the electrochemical oxidation peak potential (Ep) of mC is about 160 mV lower than that of C as we reported previously [26]. On the other hand, the maximum absorption wavelength (kmax) of the mC peak became only 8 nm larger than that of C (272 nm) (Fig. 1B), owing to the electron donor property of the methyl group [26,47]. Then both bases were distinguishable. However, the mixture of the two mononucleotides overlapped to form one peak since their peak differences were too small to separate them. Therefore, none of the bases in the mixture and/or oligonucleotides can be distinguished by using UV experiments without separation systems such as HPLC or electrophoresis. The above result indicates that electrochemical discrimination between mC and C is much clearer than that of UV detection, which is also a nonlabeling measurement. Effect of electrochemical pretreatment of ECR nanocarbon film electrode We have already reported that our film electrode could also detect both mC and C from synthetic short oligonucleotides (up to 6mers) [26]. As regards longer oligonucleotides, fouling affected the resulting oxidation currents even when using the ECR nanocarbon film electrode. To suppress the fouling, and detect much longer oligonucleotides quantitatively, we studied the effect of electrochemical pretreatment on the ECR nanocarbon film electrode stability. This was because we were able to modify the surface of the ECR nanocarbon film easily using electrochemical pretreatment without losing high electrode activity and surface flatness [48,49]. Indeed, the modification of oxygen-containing groups at the ECR nanocarbon film surface enabled us to reduce the fouling by small biomolecules while maintaining the intrinsic properties [48]. Fig. 2 compares the peak current changes for G and A in oligonucleotide 1 (50 -CGAACACCCAGGC-30 ) at GC, as-deposited and
Fig. 2. Stability of three electrodes in successive oligonucleotide (13mers) measurements. Ratio of the peak currents for a base content of 5 lM oligonucleotide 1 oxidation (G, A) in each measurement on the carbon electrode to that in the first measurement as a function of time. The peak currents at each electrode were obtained from SWV measurements in 50 mM, pH 3.3, acetate buffer containing 2 M sodium nitrate. The electrode surface was rinsed with pure water before each measurement.
pretreated ECR nanocarbon film electrodes. The as-deposited ECR nanocarbon film electrode exhibited large G and A oxidation currents in the first measurement, but in the second measurement the respective peak currents decreased greatly to about 15% and 17% of their initial values, despite the fact that the electrode surface was rinsed with pure water between each measurement. This is probably because the oxidized oligonucleotides were strongly adsorbed on the electrode surface. The obtained currents had fallen to less than 10% of their initial values after five measurements. The GC electrode exhibited a similar tendency in that there was an obvious decrease in the peak currents of G and A in 1 (a decrease of more than 95% after five measurements). In contrast, the nanocarbon film pretreated by scanning the potential between 0 and 2.3 V 15 times in 50 mM, pH 7.0, phosphate buffer solution exhibited the stable current responses of oligonucleotide 1. This is
Fig. 1. Background-subtracted SWVs (A) and UV spectra (B) of 100 lM mononucleotides and their mixture measured in 50 mM, pH 5.0, acetate buffer.
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because the hydrophilicity and the number of oxygen-containing groups on the ECR nanocarbon film electrode surface increased without it losing much of its surface flatness or many of its sp2 bonds after the pretreatment [48]. As a result, this treatment provided electrode stability against longer oligonucleotides than those described in previous reports [25–27]. Subsequent experiments were therefore performed with the pretreated ECR nanocarbon film electrode.
Table 1 Normalized current values of A, mC, and C of oligonucleotides 1–4 obtained from Fig. 3. Oligonucleotide Sequence 1
50 CGAACACCCAGGC-30
2
50 -(mC)GAACACCCAGGC-30
3
50 -CGAACACCCAGG(mC)-30
4
50 -(mC)GAACACCCAGG(mC)-30
Electrochemical methylation detection of RB1 oligonucleotides The improved stability realized by electrochemical pretreatment meant that we could employ this film electrode to detect DNA methylation in sequences that constitute frequently occurring methylation hotspots from the tumor suppressor RB1 gene. The RB1 gene encodes an important RB1 protein that prevents excessive cell growth by inhibiting cell cycle progression. When this gene is damaged or silenced by DNA methylation, the RB1 protein is inactivated and this induces retinoblastoma, which is a childhood tumor of the eye [50]. In retinoblastoma tumors, methylation frequently occurs at CGA codons (arginine), and there are three main hotspots (codons 251, 255, and 262) in exon 8 [45,50]. We directly detected different types of methylation status in codons 251 and 255 of the RB1 gene by using the ECR nanocarbon film electrode. The methylation events at codons 251 and 255 are now frequently observed in various tissues including retinas, fibroblasts, and lymphocytes [50]. Fig. 3 shows background-subtracted SWVs of a wild-type oligonucleotide 1 (50 -CGAACACCCAGGC-30 ), and three kinds of methylated oligonucleotides 2 (50 -(mC)GAACACCCA GGC-30 ), 3 (50 -CGAACACCCAGG(mC)-30 ), and 4 (50 -(mC)GAACACCC AGG(mC)-30 ), whose methylation status and positions are different, including the sequence from codons 251 and 255 of the RB1 gene. When these voltammograms were normalized by the peak current of the G that is independent of the detection of DNA methylation, we were able to quantitatively differentiate the voltammograms for these oligonucleotides as shown in Fig. 3B. The DNA bases in the oligonucleotides tend to be oxidized at lower potentials than those of mononucleotides under the same conditions, as also observed in another report [51]. Table 1 summarizes normalized current values of A, mC, and C of oligonucleotides 1–4 obtained based on the current response of G in each oligonucleotide
A (RSD%)
mC (RSD%)
C (RSD%)
1.63 (2.06) 1.69 (2.07) 1.68 (3.56) 1.73 (3.18)
0.45 (2.70) 0.55 (3.88) 0.56 (2.80) 0.65 (2.08)
1.11 (5.12) 1.04 (3.63) 1.03 (4.36) 0.97 (5.27)
The normalized values were calculated based on the current response of G in each oligonucleotide. The relative standard deviation (RSD) of each current was obtained from five measurements.
from Fig. 3B. The magnitude of the current increase for a single mC (the normalized current value of 0.09–0.11 observed at 1.45 V vs. Ag/AgCl) coincided precisely with that of the current decrease of a single C (the normalized current value of 0.06–0.08 observed at 1.55–1.60 V vs. Ag/AgCl) at the ECR nanocarbon film electrode. These results indicate that the influence of the methylation position of the sequences used in this study was almost negligible as regards electrochemical DNA methylation detection. Moreover, we measured RB1 oligonucleotides longer than 1 and 2. Fig. 4A shows background-subtracted SWVs of oligonucleotides 5 (50 -AATGGTTCAC CTCGAACACC CAGG-30 ) and 6 (50 -AATGGTTCAC CT(mC)GAACACC CAGG-30 ), whose oligonucleotide lengths are extended. As regards these longer oligonucleotides, which include double amounts of A and T bases, the mC and C peaks obtained from the voltammograms are somewhat changed but difficult to evaluate quantitatively, even when using the ECR nanocarbon film electrode. In contrast, UV spectra for the oligonucleotides 5 and 6 exhibited no separation peaks for mC and C (Fig. 4B). Furthermore, a closer inspection revealed that there was a subtle current increase assigned to A as the amount of mC increased as summarized in Table 1 because the obtained Ep of mC is very close to that of A. If the oxidation peak potential difference (DE) between A and mC is sufficiently large for them to be distinguished from each other, we are able to detect sharp mC and C peaks in the oligonucleotides with higher selectivity and sensitivity. That is, a wider DE between
Fig. 3. Electrochemical responses of the methylated oligonucleotides (13mers) including the sequence from codons 251 and 255 of the RB1 gene obtained using the ECR nanocarbon film electrode. Background-subtracted SWVs of 1 lM nonmethylated oligonucleotide 1 and its methylated oligonucleotides 2–4, measured in 50 mM, pH 3.3, acetate buffer containing 2 M sodium nitrate.
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Fig. 4. DNA methylation detection of oligonucleotides (24mers) including the sequence from codons 251 and 255 of the RB1 gene. (A) Background-subtracted SWVs of 3 lM oligonucleotides 5 and 6 by using ECR nanocarbon film electrodes in 50 mM, pH 3.3, acetate buffer containing 2 M sodium nitrate. (B) UV spectra of 10 lM 5 and 6 in 50 mM, pH 5.0, acetate buffer.
A and mC (defined as DE1 = EpmC – EpA) and mC and C (defined as DE2 = EpC – EpmC) will enable us to discriminate both mC and C more clearly even in the longer methylated oligonucleotides. pH dependence on electrochemical response of DNA bases in RB1 oligonucleotide In this study, we investigate the pH dependence of Ep for the oxidation of A, mC, and C in oligonucleotide 6 since the Ep values of the DNA bases are known to be dependent on the pH conditions [23,24]. Fig. 5A shows the pH dependence of the three bases (A, mC, and C) in oligonucleotide 6 by measuring the SWV. Some groups have already reported the pH dependence of individual bases [23,24]. For example, Brett and co-workers reported a slope (dEp/dpH) of 58 mV for A base at a GC electrode [23]. Also in our case, we obtained a value of 63 mV for A in the Ep – pH curves (Fig. 5A), which is almost the theoretical value of a 60 mV/pH
unit [24]. It is noteworthy that this slope value was obtained from the A base in oligonucleotide 6 (24mer). In contrast, the mC and C values were 46 and 54 mV, respectively. These values are relatively small compared with those of purine bases. The subtle difference between the slopes of mC/C bases and A is presumably due to their different pKa values (4.6 for mC/C and 4.1 for A) as previously reported [23]. Such differences lead to two kinds of defined DE variation in pH as shown in Fig. 5B. Indeed, DE1 decreased with decreasing pH, whereas DE2 exhibited its maximum value in a pH range of 3.8–4.0. As noted above, the wider DE1 and DE2 are suitable for the clearer discrimination of both mC and C in methylated oligonucleotides. From these results, we decided that the most appropriate pH in the SWV measurement of methylated oligonucleotide 6 at an ECR nanocarbon film electrode is in a range of approximately pH 3.8–4.4, which provided sufficient values for both DE1 and DE2. With the above optimization, an independent peak assigned to single mC was observed at 1.47 V in the
Fig. 5. Ep – pH plots of the bases in oligonucleotide 6 (A), and pH dependence on DE between A and mC (DE1) and mC and C (DE2) (B).
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Fig. 6. Background-subtracted SWV of 3 lM oligonucleotide 6 in 50 mM, pH 4.4, acetate buffer containing 2 M sodium nitrate.
voltammogram of oligonucleotide 6 when measured at pH 4.4 (Fig. 6). We also measured oligonucleotides 5 and 6 by using UV spectroscopy, and we observed no separation peaks of mC and C despite a kmax value shift of the both oligonucleotides as noted in the above figures. This is probably because the kmax shifts with the UV adsorption were little influenced by the pH environment [47] compared with the Ep shift with electrochemical oxidation. These results clearly indicate that electrochemical direct detection distinguishes both mC and C in the longer sequences more selectively than UV detection. Quantification of methylated CpG oligonucleotides We also measured the longer oligonucleotides (60mers) that constitute a nonmethylated and a methylated CpG dinucleotide using our ECR nanocarbon film electrode. The CpG unit constitutes the major part of the promoter region of the gene (CpG island), and the CpG methylation density is closely associated with the tran-
scription level of a gene [30–33]. In normal cells, most CpG islands spanning the promoter regions are unmethylated, and their downstream genes are transcriptionally active. In contrast, when CpG island promoters in cancer cells are methylated, their downstream genes such as tumor suppressor genes are consistently silenced [30–33]. These characteristics clearly demonstrate that CpG methylation density is a critical marker for research in this field. Therefore, the methylation quantification of the CpG repetition sequence is important with respect to understanding DNA methylation related diseases and developing future diagnosis tools. Fig. 7A shows SWVs of nonmethylated oligonucleotide 7 (50 -(CG)30-30 ) and its methylated oligonucleotide 8 (50 -(CG)15(mCG)15-30 , the methylation ratio: 50%). In the voltammogram for 7, the two peaks assigned to G (1.13 V) and C (1.63 V) were observed (red curve). The voltammogram for 8 (blue curve) exhibited another peak corresponding to mC at 1.48 V, whereas the peak current height for C decreased. At the same time, the two oxidation current responses of G in these voltammograms were coincident. These results suggest that we can expect to achieve a simple assay for the DNA methylation ratio solely by performing electrochemical measurements. We estimate the quantitative performance of the DNA methylation ratios from the voltammograms obtained for CpG samples with different methylation ratios. Fig. 7B shows the relationship between the methylation ratio in the oligonucleotides and the current response assigned to mC of the oligonucleotides on a given electrode. It is noteworthy that the current responses were linear in the region of the higher methylation ratio (over 30%) whose correlation coefficient was 0.9955. In the promoter region of the gene, the CpG methylation ratio is known to be 60–90% [30–33]. Therefore, our film electrode is particularly advantageous with regard to the quantitative detection of hypermethylated DNA. Further improvement of the electrochemical sensitivity of each base can be expected to achieve the quantification of the DNA methylation assay with a wide methylation status range. Therefore, we are now studying ways to improve the sensitivity of each base in the samples. Conclusion We used an ECR nanocarbon film electrodes to develop a simple electrochemical DNA methylation analysis technique without
Fig. 7. Typical responses of the methylated oligonucleotides (60mers) consisting of the CpG repetition unit at the ECR nanocarbon film electrode. SWVs of 2 lM nonmethylated CpG oligonucleotide 7 and its methylated CpG oligonucleotides 8 in 50 mM, pH 4.4, acetate buffer containing 2 M sodium nitrate (A), and calibration of methylation ratio obtained from SWV of some oligonucleotides with different methylation ratios (9 (50 -(CG)21(mCG)9-30 ), 10 (50 -(CG)9(mCG)21-30 ), and 11 (50 -(mCG)30-30 )) (B).
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bisulfite or labeling processes. The electrochemically pretreated ECR nanocarbon film electrode enabled us to detect DNA methylation quantitatively, irrespective of the methylation position in longer sequences of the RB1 oligonucleotides, unlike UV experiments. Moreover, the selectivity for detecting both mC and C bases in the oligonucleotides can be successfully optimized by controlling the pH conditions. We also succeeded in the simple and quantitative detection of DNA methylation ratios from the peak current heights of mC in methylated CpG repetition oligonucleotides by using our ECR nanocarbon film electrode. Since several improvements are required if we are to detect DNA methylation in real samples, we are now developing this film to achieve a more sophisticated DNA methylation electroanalysis platform by combining sample pretreatment approaches such as enzymatic reaction or separation technologies. Our carbon film electrodes are suitable for integrating in devices with such functions because this film can be deposited at low temperatures and is easy to microfabricate for applications to a micro-total analysis system or lab-on-a-chip.
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Acknowledgments This work was supported in part by a Grand-in-Aid for Scientific Research (O.N. No. 20350042) from the Ministry of Education, Culture, Science, Sports, and Technology of Japan. References
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