Electrochemical biosensing method for the detection of DNA methylation and assay of the methyltransferase activity

Sensors and Actuators B 178 (2013) 412–417

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Electrochemical biosensing method for the detection of DNA methylation and assay of the methyltransferase activity Zhenning Xu, Mo Wang, Tingting Zhou, Huanshun Yin ∗ , Shiyun Ai ∗ College of Chemistry and Material Science, Shandong Agricultural University, Taian, Shandong 271018, China

a r t i c l e

i n f o

Article history: Received 16 October 2012 Received in revised form 30 December 2012 Accepted 31 December 2012 Available online 11 January 2013 Keywords: DNA methylation Methyltransferase Methylene blue Differential pulse voltammetry

a b s t r a c t In this article, we presented an electrochemical method for detection of DNA methylation and assay of DNA methyltransferase (MTase) activity using methylene blue (MB) as electrochemical indicator. After the DNA hybrid was methylated by T.aqI MTase, it could not be cleaved by HincII endonuclease. On the contrary, the double DNA without methylation could be cleaved, which would decrease the amount of intercalated MB. Thus, the DNA methylation status and MTase activity could be determinated based on the voltammetric signal change of MB. The methylation site could be found based on this method. The electrochemical signal of MB increased linearly with increasing T.aqI MTase concentration from 0.1 to 100 U/mL. Moreover, the inhibition investigation demonstrated that fisetin could inhibit the T.aqI MTase activity with the IC50 value of 280 ␮M. Therefore, the screening of the inhibitors of MTase could be accomplished using the novel method. © 2013 Elsevier B.V. All rights reserved.

1. Introduction DNA methylation, the first identified epigenetic modification, can exert an important influence in the gene transcription, embryogenesis and disease [1,2]. It was discovered that aberrant DNA methylation of CpG islands in promoter could change normal cellular functions and phenotypes, which was regard as a new symptom for monitoring cancer development [3–5]. It has long been known that cancer cells undergo changes in 5-methylcytosine distribution including global DNA hypomethylation [6] and the hypermethylation of promoter CpG islands associated with tumor-suppressor genes [7–9]. DNA methylation was regulated by DNA methyltransferase (MTase) in the presence of S-adenosylmethionine (SAM) which catalyzed cytosine or adenine in DNA sequences with a methyl group [10]. The abnormity of DNA MTase activity would affect the level of DNA methylation, thus the investigation of DNA methylation and assay of DNA MTase activity have catalyzed considerable developments in the fields of genomics [5,11]. In recent years, a variety of methods including PCR (polymerase chain reaction)-based techniques [12,13], surface enhanced Raman spectroscopy (SERS) [14], opto-fluidic ring resonator (OFRR) [15], surface plasmon resonance (SPR) [16], microarray [17], bisulfite methods [18], fluorescence methods [19–21], high-performance liquid chromatography (HPLC), colorimetric methods [22,23], electrogenerated chemiluminescence (ECL) method [24,25] and

∗ Corresponding authors. Tel.: +86 538 8249248; fax: +86 538 8242251. E-mail addresses: [email protected] (H. Yin), [email protected] (S. Ai). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2012.12.124

electrochemical method [26–29] have been reported for the determination of DNA methylation and assay of MTase activity. Due to the advantages of convenience, sensitivity, fast response and low production cost, electrochemical method has become a useful method for the detection of DNA methylation and assay of the MTase activity. Wang et al. present an electrochemical method based on a choline chloride monolayer supported multiwalled carbon nanotubes film modified glassy carbon electrode (MWNTs/Ch/GCE) for the investigation of DNA methylation according to the direct electrocatalytic oxidation of DNA bases. However, thymine and 5-methylcytosine were very difficult to recognize because of the nearly same oxidation potential. Thus, this method needs further improvement. Liu et al. developed a method to detect genomic DNA methylation level based on M. SssI methylase-HpaII endonuclease interaction system [27]. He et al. report a signalon electrochemical method for the detection of methylation using AuNPs amplification [30]. However, these methods could not find the methylation site. We presented an electrochemically immune approach to detect DNA methylation and the assay of MTase activity previously [31]. However, Anti-5-methylcytosine antibody and HRP-IgG were cost too much and need the extreme operating conditions and storage. In this article, an electrochemical approach for the detection of DNA methylation and the assay of MTase activity was investigated. The schematic diagram of this proposed strategy has been shown in Scheme 1. The DNA hybrid was methylated by T.aqI MTase and then digested by HincII endonuclease. As known, the T.aqI and HincII can recognize sequence 5 -GTCGAC-3 . Then, methylene blue (MB) was accumulated onto the surface of hybrid-modified Au electrode as an

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Scheme 1. Schematic illustration of electrochemical method for detection of DNA methylation and assay of DNA methyltransferase activity.

indicator to evaluate the methylation level. The unmethylated DNA hybrid was selectively digested by HincII on the DNA biosensor and the electrochemical signals of MB decreased. However, the methylated DNA cannot be digested by HincII because the cleavage of the endonuclease was blocked. Therefore, the voltammetric signal change after cleavage is used to asaay the T.aqI MTase activity and DNA methylation detection. Moreover, the effect of the inhibitor on MTase activity was also investigated, which would provide a sensitive platform for screening DNA MTase inhibitors.

desired stock concentrations and stored at −20 ◦ C according to the manufacturer’s instructions. The buffer solutions employed in this study are as follows. Oligonucleotide dissolve buffer (TE buffer, pH 8.00) 10 mM Tris–HCl and 1 mM EDTA; probe immobilization buffer: 10 mM Tris–HCl, 1.0 mM EDTA, 1.0 M NaCl, and 1.0 mM TCEP (pH 7.00DNA hybridization buffer: 10 mM Tris–HCl, 1.0 mM EDTA, and 1.0 M NaCl (pH 7.00 electrochemistry determination buffer, 0.1 M PBS (NaH2 PO4 and Na2 HPO4 , pH 7.00). All reagents were analytically pure grade. All of the solution and redistilled deionized water used were autoclaved.

2. Materials and methods 2.2. Electrochemical measurements 2.1. Reagents 6-Mercapto-1-hexanol (MCH) was purchased from Alfa Aesar (Lancashire, England). Hydrogen tetrachloroaurate trihydrate (HAuCl4 • 3H2 O), Tris(hydroxymethyl)aminomethane (Tris), tri(2-carboxyethyl) phosphine hydrochloride (TCEP), disodium ethylenediaminetetraacetic acid (EDTA) and fisetin were purchased from Aladdin (Shanghai, China). Catechol-O-methyltransferase (COMT) was purchased from Sigma-Aldrich (St. Louis, USA). T.aqI and restriction endonuclease HincII are supplied by New England BioLabs (Ipswich, MA) and Fermentas (Maryland, USA), respectively. The methyltransferase T.aqI is stored at −20 ◦ C in a buffer containing 10 mM Tris–HCl (pH 7.4), 50 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl2 . Restriction endonuclease HincII is is stored −20 ◦ C in a buffer containing 10 mM Tris–HCl (pH 7.4), 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.2 mg/ml BSA and 50% glycerol. PAGE-purified DNA was obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). The base sequences are as follows: probe DNA S1, 5 -SH-(CH2 )6 -GTA GCT TCT ATG CAG GTC GAC GCG ACT ATG CAG TCT-3 ; DNA S2, 5 -AGA CTG CAT AGT CGC GTC GAC CTG CAT AGA AGC TAC-3 ; single-based mismatch DNA S3, 5 -AGA CTG CAT AGT CGC GTC GTC CTG CAT AGA AGC TAC-3 . The DNA sequences with different methylation site are follows: probe DNA S4, 5 -SH(CH2)6 -GTC GAC GTA GCT TCT ATG CAG GCG ACT ATG CAG TCT-3 ; DNA S5, 5 -AGA CTG CAT AGT CGC CTG CAT AGA AGC TAC GTC GAC-3 . The recognition sites for T.aqI MTase and HicII endonuclease were marked with italics. The synthesized oligonucleotides were diluted in TE buffer (10 mM Tris–HCl and 1 mM EDTA, pH 8.0) to

Differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) were performed with a CHI 660C electrochemical workstation (Austin, USA). A bare Au electrode or modified Au electrode is used as working electrode. A saturated calomel electrode (SCE) and a platinum wire are used as the reference electrode and auxiliary electrode, respectively. 2.3. Deposition of gold nanoparticles on gold electrode surface The bare Au electrode was carefully polished to a mirror-like with 0.3 and 0.05 ␮m alumina slurry on polishing cloth, and then sonicated with absolute ethanol and double distilled deionized water for 3 min to remove adsorbed particles and dried under the stream of high purity nitrogen for further use. Subsequently, Au electrode was pretreated electrochemically in 0.2 M H2 SO4 aqueous solution by potential cycling in the potential range −0.2 to 1.6 V at a potential scan rate of 100 mV/s. The gold nanoparticles (AuNPs) were electrodeposited on the Au electrode surface in 3 mM HAuCl4 solution containing 0.1 M KNO3 through amperometry i–t technique at −1.4 V for 250 s. After deposition, the electrode surface was thoroughly rinsed with double distilled water. The obtained electrode was named as AuNPs/Au. 2.4. Immobilization of S1 and hybridization For probe immobilization, 5 ␮L probe immobilization buffer containing 5.0 × 10−7 M thiol-capped probe DNA S1 was dripped on

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AuNPs/Au surface and incubated for 2 h at humid condition. Then the modified electrode was rinsed three times with ultrapure water to eliminate the non-specific immobilized probe DNA molecule on the electrode surface. Then, the probe DNA S1 modified electrode was immersed into 10 mM Tris–HCl containing 1.65 × 10−6 M MCH to further eliminate the non-adsorbed DNA molecules and hold a good orientation of probe DNA for its good recognition ability. After that, the modified electrode was rinsed with water to remove the loose DNA S1. Hybridization was conducted at 37 ◦ C by dropping 5 ␮L hybridization buffer containing DNA S2 (5.0 × 10−7 M) for 2 h at humid condition. After hybridization, the electrode was thoroughly rinsed with double distilled water to remove the unhybridized DNA S2 and dried with nitrogen. 2.5. Methylation and assay of T.aqI MTase activity The methylation of S1/S2 hybrid was performed at 37 ◦ C for 2 h by dropping 5 ␮L solution containing 80 ␮M SAM, 100 ␮g/mL BSA and 100 U/mL T.aqI MTase at humid condition. To study the methylation process, we choose some different concentration of T.aqI MTase and different methylation time. 2.6. Cleavage of HincII endonuclease HincII cleavage was performed at 37 ◦ C by dropping 5 ␮L buffer containing 20 U/mL HincII at humid condition. After cleavage, the electrode was rinsed with water and dried with nitrogen. 2.7. Inhibition the activity of T.aqI MTase To study the inhibition effects of fisetin on the T.aqI activity, the methylation of S1/S2 hybrid is performed at 37 ◦ C in a buffer containing 80 ␮M SAM, 100 ␮g/mL BSA and 100 U/mL T.aqI MTase, 40 U/mL COMT and various concentration of the inhibitors. The inhibition efficiency (%) is estimated as follows: Inhibition (%) =

I1 − I2 × 100 I1

where I1 is the current of the hybrid of S1/S2 after treated with T.aqI MTase, and I2 is the inhibited current. 2.8. Methylene blue accumulation MB was accumulated onto the surface of hybrid-modified Au electrode by immersing the electrode into stirred 20 mM Tris–HCl buffer (pH 7.4) containing 20 ␮M MB and 20 mM KCl for 5 min. After accumulation of MB, the electrode was rinsed with 20 mM Tris–HCl buffer (pH 7.4) with stirring for 5 min to remove the nonspecifically bound MB.

Fig. 1. Electrochemical impedance spectra of different electrodes in 5 mM [Fe(CN)6 ]3−/4− (1:1) solution containing 0.1 M KCl. (a) The bare Au electrode, (b) AuNPs/Au, (c) probe DNA S1 assembled on the modified electrode, and (d) probe DNA 1 hybridized with target DNA S2.

electrode showed an electron transfer resistance (Ret ) of about 236.5  (curve a). After modifying with electrodeposited AuNPs, only an almost straight line was observed (curve b). However, the Ret increased to 1112  after probe DNA S1 immobilization (curve c), causing by the electrostatic repulsion between the negatively charged deoxyribose–phosphate backbone of probe oligonucleotides and Fe(CN)6 3−/4− . It proved that the probe was successfully assembled on the electrode surface. The Ret value was further increased to about 3000  after being hybridized with complementary DNA S2, which could be also well ascribed to the electrostatic repulsion of the negatively charged Fe(CN)6 3−/4− from the approaching electrode surface by the negatively charged phosphate skeletons of DNA The result demonstrated that DNA S2 was successfully immobilized on the electrode surface. 3.2. Feasibility investigation of the electrochemical biosensor As shown in Fig. 2, after DNA S1 hybridized with S2, methylene blue was accumulated as the electrochemical indicator and DPV showed a well-defined anodic peak after the backgroundsubtraction at −0.264 V (curve a). After the S1/S2 hybridization (unmethylated) was treated with HincII endonuclease, the electrochemical reduction signal decreased significantly (curve b) because HincII can specifically recognize the site of 5 -GTCG/AC-3 and less MB was adsorbed on dsDNA. However, if the S1/S2 hybridization were first methylated by T.aqI, then digested by HincII, the response of MB was almost unchangeable (curve c) because the digestion

2.9. Electrochemical determination EIS was carried out in 5 mM Fe(CN)6 3−/4− (1:1, molar ratio) solution containing 0.1 M KCl. The frequency was ranged from 10−1 to 105 Hz. The applied potential is the formal potential. DPV was performed in 10 mL 0.1 M PBS (pH 7.0) from −0.6 to 0.1 V. The parameters are as follows: increment potential, 0.004 V; pulse amplitude, 0.05 V; pulse width, 0.05 s; sample width, 0.0167 s; pulse period, 0.2 s; quiet time, 2 s. 3. Results and discussion 3.1. Characterization of the biosensor EIS could provide important information on the impedance changes of the electrode surface. As shown in Fig. 1, the bare Au

Fig. 2. Background-subtracted DPV response of MB–S1/S2 (a), S1/S2 hybrid treated with HincII (b), MB–S1/S2 (c) and MB–S1/S3 (d) hybrids treated with T.aqI and then HincII in 0.1 M PBS (pH 7.0), respectively.

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effect by the HincII is blocked. This result also demonstrated that the hybrids of DNA hybrid have been successively methylated by T.aqI MTase. We also evaluate the methylation specificity by using one-base mismatched synthetic DNA S3. DNA S1 first hybridized with S3, and then treated with T.aqI and HincII. It is obvious that the DPV signal of MB can still be observed when S1/S3 was cleaved (curve d) and peak currents were higher than that obtained at curves b and c. As we known, MB could interact with DNA. Although the formation of dsDNA improved the electrostatic interaction between MB and the deoxyribose–phosphate backbone, and the intercalation of MB to dsDNA, the specific interaction between MB and guanine residues was greatly obstructed because guanine residues were enwrapped in dsDNA. Therefore, less MB was adsorbed on dsDNA than that on single-stranded DNA (ssDNA) [13]. There were some single S3 which did not hybridize with the DNA probe (S1) because of one-base mismatched synthetic DNA S3. It suggested that HincII had no effect on the S1/S3 hybrid because the hybrid did not contain a specific recognition sequence (5 -GTCG/AC-3 ) of the endonuclease. These results demonstrated that the developed method can distinguish even one-base mismatched DNA. Furthermore, this result proved that the developed method had good selectivity to detect DNA methylation.

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(S/N = 3), which is lower than those reported previously 2.5 U/mL [32], 0.07 U/mL [29] and 0.1 U/mL [31]. 3.4. Dependence of DPV signals of MB on the methylation time The effect of the methylation time on the peak current was investigated. The anodic peak currents of MB was linearly proportional to the methylation time with the equations I (␮A) = 0.084t (min) + 0.43. However, the growth trend decreased when the methylation more than 120 min, which indicated the methylation has reached maximum. 3.5. Precision and repeatability As an analytical method, the precision and repeatability are important factors. Therefore, the two factors of the method developed in this work were evaluated. The precision was investigated according to the slope of the regression equation of 100 U/mL T.aqI obtained from three independent assay process. The RSD (relative standard deviation) of the three slopes is 5.33%, which indicated that the developed method has good precision. For repeatability, the RSD for five independent treatment hybrids is 6.52%, indicating an acceptable repeatability of the developed method. 3.6. The inhibition of fisetin on the activity of T.aqI

3.3. Effect of MTase concentration on methylation process The catalytic activity and availability of T.aqI is investigated by changing the T.aqI concentration from 0.1 to 200 U/mL. The DNA hybrid on the electrode surface was first methylated with different concentration of T.aqI MTase for 2 h and treated with HincII endonuclease 2 h, respectively. After that, MB was accumulated onto the surface of hybrid-modified Au electrode. Finally, the response of MB is recorded in 0.1 M PBS by DPV. As seen in Fig. 3A, the DPV response after the background-subtraction increased with the increasing of T.aqI concentration. Fig. 3B showed the calibration curve of the peak current with different MTase concentrations. The peak current increased with MTase from 0.1 to 200 U/mL and then slowed down at higher concentration. The reason was that almost all of the DNA hybrids were methylated. The current increases linearly with the T.aqI concentration from 0.1 to 10 U/mL and follows the regression equation of I (␮A) = 0.003c (U/mL) + 0.5856 (R = 0.999); and by the equation I (␮A) = 0.007c (U/mL) + 0.5356 (R = 0.999), at the 10–100 U/mL range. The limit of detection was 0.03 U/mL

Since DNA methylation plays an important role in both prokaryotes and eukaryotes, the pharmacological inhibitors of DNA MTase have a broad spectrum of application in cancer therapy and antimicrobial. To study the inhibition effects on the T.aqI, the experiments have been performed in the presence of MTase inhibitor [32,33]. It is also well known that COMT can catalyze the O-methylation reaction of bioflavonoids. In order to confirm the validity of this developed method on inhibitor screening, we selected fisetin as model complex to investigate the inhibition effect on T.aqI MTase activity in the presence of COMT. As shown in Fig. 4, the activity of the T.aqI decreased with the increasing concentration of fisetin. It showed that the biosensor also has the potential ability to screen the inhibitors to the DNA MTase. The IC50 value, the inhibitor concentration required to reduce the enzyme activity by 50%, was found to be 280 ␮M for fisetin. The maximum inhibition was about 53.61% for fisetin with the inhibitor concentration of 600 ␮M. The results proved that the novel method was available to screen the inhibitors to the DNA MTase.

Fig. 3. (A) The effects of the concentration of T.aqI on background-subtracted the DPV response of the MB in the modified electrodes for different concentrations of MTase. Curves (a) to (o) are 0.1, 0.5, 1.0, 2.5, 5.0, 10, 20, 30, 50, 60, 70, 90, 100, 120 and 200 U/mL, respectively. (B) Calibration curve in the range 0.1–10 U/mL and 10–100 U/mL.

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DNA MTase activity. This method was carried out on the T.aqI methylase–HincII endonuclease interaction system. The method used MB as electrochemical indicator. It is convenient to detect DNA methylation without PCR amplification or bisulfite processes compared with the traditional method. Moreover, this work could detect the DNA methylation site based on the current change of the MB. The results showed the DNA biosensor could be applied for the determination of DNA MTase, which provided a new platform for screening DNA MTase inhibitors. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 21075078, 21105056) and the Natural Science Foundation of Shandong Province, China (No. ZR2010BM005, ZR2011BQ001). Fig. 4. The inhibition of the activity of T.aqI by fisetin. The inhibitor concentration: 0, 0.5, 1.0, 5.0, 10, 50, 100, 200, 300, 400, 500, and 600 ␮M.

Fig. 5. Background-subtracted DPV responses of MB with different treatment process in 0.1 M PBS (pH 7.0). (a) S4/S5 (b) and S1/S2 hybrid after digestion with HincII for 2 h at 37 ◦ C, (c) MB–S1/S2, and (d) MB–S4/S5.

3.7. The study of methylation site Electrochemical method has many advantages such as high sensitivity, rapidity and easy controllability, and has proved to be very useful in the detection of DNA methylation. However, electrochemical methods for the determination methylation site have not been reported. The present work developed a sensitive electrochemical method for the detection of DNA methylation site based on the current change of the redox indicator. As shown in Fig. 5, when S1 hybridized with S2, an anodic peak current after the background-subtraction of about 1.233 ␮A was achieved (curve c). After the S1/S2 hybridization was treated with HincII endonuclease for 2 h at 37 ◦ C, the DPV response decreased to 0.5757 ␮A (curve b), that is to say the decrease of current was 53.30%. The result was clear that the methylation site of the hybrid S1/S2 was at the middle of the DNA chain. The nearly current was also obtained at S4/S5 (1.229 ␮A, curve d), however, the current was 0.238 ␮A (curve a) after the S4/S5 treated with of HincII endonuclease, which was less than that of S1/S2. The decrease of current about S4/S5 was 80.63%, which indicated the methylation site of S4/S5 was nearer to electrode surface than S1/S2. It was agree with the DNA sequences. The results were clear that the methylation site could be found based on this method. 4. Conclusion In this article, we developed a sensitive electrochemical approach for detection of DNA methylation level and assay of

References [1] C. Frauer, H. Leonhardt, Twists and turns of DNA methylation, Proceedings of the National Academy of Science 108 (2011) 8919–8920. [2] J.S. Choy, S. Wei, J.Y. Lee, S. Tan, S. Chu, T.H. Lee, DNA methylation increases nucleosome compaction and rigidity, Journal of the American Chemical Society 132 (2010) 1782–1783. [3] A. Jeltsch, R.Z. Jurkowska, T.P. Jurkowski, K. Liebert, P. Rathert, M. Schlickenrieder, Application of DNA methyltransferases in targeted DNA methylation, Applied Microbiology and Biotechnology 75 (2007) 1233–1240. [4] J. Lewandowska, A. Bartoszek, DNA methylation in cancer development, diagnosis and therapy—multiple opportunities for genotoxic agents to act as methylome disruptors or remediators, Mutagenesis 26 (2011) 475–487. [5] P. Wang, Z. Mai, Z. Dai, X. Zou, Investigation of DNA methylation by direct electrocatalytic oxidation, Chemical Communications 46 (2010) 7781–7783. [6] M. Ehrlich, DNA methylation in cancer: too much, but also too little, Oncogene 21 (2002) 5400–5413. [7] P.A. Jones, P.W. Laird, Cancer-epigenetics comes of age, Nature Genetics 21 (1999) 163–167. [8] J.G. Herman, S.B. Baylin, Gene silencing in cancer in association with promoter hypermethylation, New England Journal of Medicine 349 (2003) 2042–2054. [9] M. Esteller, CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future, Oncogene 21 (2002) 5427–5440. [10] J.R. Horton, K. Liebert, M. Bekes, A. Jeltsch, X. Cheng, Structure and substrate recognition of the Escherichia coli DNA adenine methyltransferase, Journal of Molecular Biology 358 (2006) 559–570. [11] X. Ouyang, J. Liu, J. Li, R. Yang, A carbon nanoparticle-based low-background biosensing platform for sensitive and label-free fluorescent assay of DNA methylation, Chemical Communications 48 (2012) 88–90. [12] J.G. Herman, J.R. Graff, S. Myöhänen, B.D. Nelkin, S.B. Baylin, Methylationspecific PCR: a novel PCR assay for methylation status of CpG islands, Proceedings of the National Academy of Science 93 (1996) 9821–9826. [13] Z. Dai, X. Hu, H. Wu, X. Zou, A label-free electrochemical assay for quantification of gene-specific methylation in a nucleic acid sequence, Chemical Communications 48 (2012) 1769–1771. [14] J. Hu, C. Zhang, Single base extension reaction-based surface enhanced Raman spectroscopy for DNA methylation assay, Biosensors and Bioelectronics 31 (2011) 451–457. [15] J.D. Suter, D.J. Howard, H. Shi, C.W. Caldwell, X. Fan, Label-free DNA methylation analysis using opto-fluidic ring resonators, Biosensors and Bioelectronics 26 (2010) 1016–1020. [16] S. Pan, J. Xu, Y. Shu, F. Wang, W. Xia, Q. Ding, T. Xu, C. Zhao, M. Zhang, P. Huang, Double recognition of oligonucleotide and protein in the detection of DNA methylation with surface plasmon resonance biosensors, Biosensors and Bioelectronics 26 (2010) 850–853. [17] Z. Wu, J. Luo, Q. Ge, Z. Lu, Microarray-based Ms-SNuPE: near-quantitative analysis for a high-throughput DNA methylation, Biosensors and Bioelectronics 23 (2008) 1333–1339. [18] Y. Wan, Y. Wang, J.F. Luo, Z.H. Lu, Bisulfite modification of immobilized DNAs for methylation detection, Biosensors and Bioelectronics 22 (2007) 2415–2421. [19] F. Feng, H. Wang, L. Han, S. Wang, Fluorescent conjugated polyelectrolyte as an indicator for convenient detection of DNA methylation, Journal of the American Chemical Society 130 (2008) 11338–11343. [20] X. Wang, Y. Song, M. Song, Z. Wang, T. Li, H. Wang, Fluorescence polarization combined capillary electrophoresis immunoassay for the sensitive detection of genomic DNA methylation, Analytical Chemistry 81 (2009) 7885–7891. [21] J. Li, H. Yan, K. Wang, W. Tan, X. Zhou, Hairpin fluorescence DNA probe for real-time monitoring of DNA methylation, Analytical Chemistry 79 (2007) 1050–1056. [22] T. Liu, J. Zhao, D. Zhang, G. Li, Novel method to detect DNA methylation using gold nanoparticles coupled with enzyme-linkage reactions, Analytical Chemistry 82 (2009) 229–233.

Z. Xu et al. / Sensors and Actuators B 178 (2013) 412–417 [23] C. Ge, Z. Fang, J. Chen, J. Liu, X. Lu, L. Zeng, A simple colorimetric detection of DNA methylation, Analyst 137 (2012) 2032–2035. [24] Y. Li, C. Huang, J. Zheng, H. Qi, Electrogenerated chemiluminescence biosensing method for the detection of DNA methylation and assay of the methyltransferase activity, Biosensors and Bioelectronics 38 (2012) 407–410. [25] R. Kurita, K. Arai, K. Nakamoto, D. Kato, O. Niwa, Determination of DNA methylation using electrochemiluminescence with surface accumulable coreactant, Analytical Chemistry 84 (2012) 1799–1803. [26] K. Goto, D. Kato, N. Sekioka, A. Ueda, S. Hirono, O. Niwa, Direct electrochemical detection of DNA methylation for retinoblastoma and CpG fragments using a nanocarbon film, Analytical Biochemistry 405 (2010) 59–66. [27] S. Liu, P. Wu, W. Li, H. Zhang, C. Cai, An electrochemical approach for detection of DNA methylation and assay of the methyltransferase activity, Chemical Communications 47 (2011) 2844–2846. [28] H. Wu, S. Liu, J.H. Jiang, G.L. Shen, R. Yu, A sensitive electrochemical biosensor for DNA methyltransferase activity by combining DNA methylation-sensitive cleavage and terminal transferase-mediated extension, Chemical Communications 48 (2012) 6280–6282. [29] J. Su, X. He, Y. Wang, K. Wang, Z. Chen, G. Yan, A sensitive signal-on assay for MTase activity based on methylation-responsive hairpin-capture DNA probe, Biosensors and Bioelectronics 36 (2012) 123–128. [30] X. He, J. Su, Y. Wang, K. Wang, X. Ni, Z. Chen, A sensitive signal-on electrochemical assay for MTase activity using AuNPs amplification, Biosensors and Bioelectronics 28 (2011) 298–303. [31] M. Wang, Z. Xu, L. Chen, H. Yin, S. Ai, An electrochemical immunosensing platform for DNA methyltransferase activity analysis and inhibitor screening, Analytical Chemistry 84 (2012) 9072–9078. [32] G. Song, C. Chen, J. Ren, X. Qu, A simple, universal colorimetric assay for endonuclease/methyltransferase activity and inhibition based on an enzymeresponsive nanoparticle system, ACS Nano 3 (2009) 1183–1189. [33] B. Brueckner, R. Garcia Boy, P. Siedlecki, T. Musch, H.C. Kliem, P. Zielenkiewicz, S. Suhai, M. Wiessler, F. Lyko, Epigenetic reactivation of tumor suppressor genes

417

by a novel small-molecule inhibitor of human DNA methyltransferases, Cancer Research 65 (2005) 6305–6311.

Biographies Zhenning Xu received her bachelor’s degree in applied chemistry from College of Chemistry and Material Science, Shandong Agricultural University in 2011. She is currently a master candidate in College of Chemistry and Material Science, Shandong Agricultural University and her research interest is bioelectroanalysis. Mo Wang received her bachelor’s degree in applied chemistry from College of Chemistry and Material Science, Shandong Agricultural University in 2012. She is currently a master candidate in College of Chemistry and Material Science, Shandong Agricultural University and her research interest is bioelectroanalysis. Tingting Zhou received her bachelor’s degree in applied chemistry from College of Chemistry and Material Science, Shandong Agricultural University in 2012. Huanshun Yin received master degree in applied chemistry from College of Chemistry, Xiangtan University in 2004 and doctor degree in Soil Science in College of Resources and Environment, Shandong Agricultural University in 2012. Now he is an associate Professor in College of Chemistry and Material Science, Shandong Agriculture University. His current interests are bioelectroanalysis. Shiyun Ai is a professor in College of Chemistry and Material Science, Shandong Agricultural University. He received his master degree (physics chemistry) and doctor degree (analytical chemistry) from Department of Chemistry, East China Normal University in 1995 and in 2004, respectively. His current research interests include chemically modified electrode, bioelectroanalysis, preparation and application of nano functional material, new method for environmental pollutant analysis.