Analytical and Bioanalytical Chemistry

, Volume 410, Issue 20, pp 4943–4952 | Cite as

A colorimetric assay of DNA methyltransferase activity based on peroxidase mimicking of DNA template Ag/Pt bimetallic nanoclusters

  • Hanie Ahmadzade Kermani
  • Morteza Hosseini
  • Andrea Miti
  • Mehdi Dadmehr
  • Giampaolo Zuccheri
  • Saman Hosseinkhani
  • Mohammad Reza Ganjali
Research Paper


DNA methylation catalyzed by DNA methyl transferase (MTase) is a significant epigenetic process for modulating gene expression. Abnormal levels of DNA MTase enzyme have been regarded as a cancer biomarker or a sign of bacterial diseases. We developed a novel colorimetric method to assay M.SssI MTase activity employing peroxidase-like activity of DNA template Ag/Pt NCs without using restriction enzymes. Based on inhibiting the peroxidase reaction that occurred in the TMB-H2O2 system, in the presence of MTase, a highly sensitive and selective colorimetric biosensor was fabricated with a detection limit (LOD) of 0.05 U/mL and a linear range from 0.5 to 10 U/mL. The changes in absorption intensity were monitored to quantify the M.SssI activity. This strategy had a high selectivity over other proteins. Furthermore, it is also demonstrated that this method can be used for the evaluation and screening of inhibitors for DNA MTase.


Ag/Pt nanoclusters DNA methyltransferase Colorimetric detection Enzyme mimic 



The authors thank the research Council of University of Tehran (Grant 28645/01/02) for financial support of this work.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2018_1143_MOESM1_ESM.pdf (512 kb)
ESM 1 (PDF 512 kb)


  1. 1.
    Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science. 2001;293:1089–93.CrossRefPubMedGoogle Scholar
  2. 2.
    Wu SC, Zhang Y. Active DNA demethylation: many roads lead to Rome. Nat Rev Mol Cell Biol. 2010;11:607–20.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Lopez-Serra P, Esteller M. DNA methylation-associated silencing of tumor-suppressor microRNAs in cancer. Oncogene. 2012;31:1609–22.CrossRefPubMedGoogle Scholar
  4. 4.
    Jeltsch A. Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases. Chembiochem. 2002;3:274–93.CrossRefPubMedGoogle Scholar
  5. 5.
    Turek-Plewa J, Jagodzinski PP. The role of mammalian DNA methyltransferases in the regulation of gene expression. Cell Mol Biol Lett. 2005;10:631–47.PubMedGoogle Scholar
  6. 6.
    Mutze K, Langer R, Schumacher F, Becker K, Ott K, Novotny A, et al. DNA methyltransferase 1 as a predictive biomarker and potential therapeutic target for chemotherapy in gastric cancer. Eur J Cancer. 2011, 47:1817–25.Google Scholar
  7. 7.
    Belinsky SA, Nikula KJ, Baylin SB, Issa JP. Increased cytosine DNA-methyltransferase activity is target-cell-specific and an early event in lung cancer. Proc Natl Acad Sci. 1996;93:4045–50.CrossRefPubMedGoogle Scholar
  8. 8.
    Kobayashi Y, Absher DM, Gulzar ZG, Young SR, Mckenney JK, Peehl DM, et al. DNA methylation profiling reveals novel biomarkers and important roles for DNA methyltransferases in prostate cancer. Genome Res. 2011;21:1017–27.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Baylin SB, Herman JG. DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet. 2000;16:168–74.CrossRefPubMedGoogle Scholar
  10. 10.
    Das PM, Singal R. DNA methylation and cancer. J Clin Oncol. 2004;22:4632–42.CrossRefPubMedGoogle Scholar
  11. 11.
    Lyko F, Brown R. DNA methyltransferase inhibitors and the development of epigenetic cancer therapies. JNCI. 2005;97:1498–506.CrossRefPubMedGoogle Scholar
  12. 12.
    Myrnes B, Norstrand K, Giercksky KE, Sjunneskog C, Krokan H. A simplified assay for O6 -methylguanine-DNA methyltransferase activity and its application to human neoplastic and non-neoplastic tissues. Carcinogenesis. 1984:1061–1064Google Scholar
  13. 13.
    Zou D, Zhang D, Liu S, Zhao B, Wang H. Interplay of binding stoichiometry and recognition specificity for the interaction of MBD2b protein and methylated DNA revealed by affinity capillary electrophoresis coupled with laser-induced fluorescence analysis. Anal Chem. 2014;86:1775–82.CrossRefPubMedGoogle Scholar
  14. 14.
    Li X, Franke AA. High-throughput and cost-effective global DNA methylation assay by liquid chromatography-mass spectrometry. Anal Chim Acta. 2011;703:58–63.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Lyko F, Ramsahoye BH, Jaenisch R. Development: DNA methylation in Drosophila melanogaster. Nature. 2000;408:538–40.CrossRefPubMedGoogle Scholar
  16. 16.
    Hu J, Zhang CY. Single base extension reaction-based surface enhanced Raman spectroscopy for DNA methylation assay. Biosens Bioelectron. 2012;31:451–7.CrossRefPubMedGoogle Scholar
  17. 17.
    Boye E, Marinus MG, Løbner-Olesen A. Quantitation of Dam methyltransferase in Escherichia coli. J Bacteriol. 1992;174:1682–5.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Wan Y, Wang Y, Luo J, Lu Z. Bisulfite modification of immobilized DNAs for methylation detection. Biosens Bioelectron. 2007;22:2415–21.CrossRefPubMedGoogle Scholar
  19. 19.
    Yin H, Zhou Y, Xu Z, Wang M, Ai S. Ultrasensitive electrochemical immunoassay for DNA methyltransferase activity and inhibitor screening based on methyl binding domain protein of MeCP2 and enzymatic signal amplification. Biosens Bioelectron. 2013;49:39–45.CrossRefPubMedGoogle Scholar
  20. 20.
    Yang Z, Wang F, Wang M, Yin H, Ai S. A novel signal-on strategy for M.SssI methyltransferase activity analysis and inhibitor screening based on photoelectrochemical immunosensor. Biosens Bioelectron. 2015;66:109–14.CrossRefPubMedGoogle Scholar
  21. 21.
    Deng H, Yang X, Yeo SPX, Gao Z. Highly sensitive electrochemical methyltransferase activity assay. Anal Chem. 2014;86:2117–23.CrossRefPubMedGoogle Scholar
  22. 22.
    Zhang L, Liu Y, Li Y, Zhao Y, Wei W, Liu S. Sensitive electrochemical assaying of DNA methyltransferase activity based on mimic-hybridization chain reaction amplified strategy. Anal Chim Acta. 2016;933:75–81.CrossRefPubMedGoogle Scholar
  23. 23.
    Zhao HF, Liang RP, Wang JW, Qiu JD. One-pot synthesis of GO/AgNPs/luminol composites with electrochemiluminescence activity for sensitive detection of DNA methyltransferase activity. Biosens Bioelectron. 2015;63:458–64.CrossRefPubMedGoogle Scholar
  24. 24.
    Zeng YP, Hu J, Long Y, Zhang CY. Sensitive detection of DNA methyltransferase using hairpin probe-based primer generation rolling circle amplification-induced chemiluminescence. Anal Chem. 2013;85:6143–50.CrossRefPubMedGoogle Scholar
  25. 25.
    Kermani HA, Hosseini M, Dadmehr M, Hosseinkhani S, Ganjali MRDNA. methyltransferase activity detection based on graphene quantum dots using fluorescence and fluorescence anisotropy. Sens Actuat B. 2017;241:217–23.CrossRefGoogle Scholar
  26. 26.
    Ji L, Cai Z, Qian Y, Wu P, Zhang H, Cai C. Highly sensitive methyltransferase activity assay and inhibitor screening based on fluorescence quenching of graphene oxide integrated with the site-specific cleavage of restriction endonuclease. Chem Commun. 2014;50:10691–4.CrossRefGoogle Scholar
  27. 27.
    Ouyang X, Liu J, Li J, Yang R. A carbon nanoparticle-based low-background biosensing platform for sensitive and label-free fluorescent assay of DNA methylation. Chem Commun (Camb). 2012;48:88–90.CrossRefGoogle Scholar
  28. 28.
    Gao C, Li H, Liu Y, Wei W, Zhang Y, Liu S. Label-free fluorescence detection of DNA methylation and methyltransferase activity based on restriction endonuclease HpaII and exonuclease III. Analyst. 2014;139:6387–92.CrossRefPubMedGoogle Scholar
  29. 29.
    Wang H, Liu P, Jiang W, Li X, Yin H, Ai S. Photoelectrochemical immunosensing platform for M.SssI methyltransferase activity analysis and inhibitor screening based on g-C3N4 and CdS quantum dots. Sens Actuat B. 2017;244:458–65.CrossRefGoogle Scholar
  30. 30.
    Liu P, Zhang K, Zhang R, Yin H, Zhou Y, Ai S. A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4. Anal Chim Acta. 2016;920:80–5.CrossRefPubMedGoogle Scholar
  31. 31.
    Li ZM, Zhong ZH, Liang RP, Qiu JD. The colorimetric assay of DNA methyltransferase activity based on strand displacement amplification. Sens Actuat B. 2017;238:626–32.CrossRefGoogle Scholar
  32. 32.
    Cui W, Wang L, Xu X, Wang Y, Jiang W. A loop-mediated cascade amplification strategy for highly sensitive detection of DNA methyltransferase activity. Sens Actuat B. 2017;244:599–605.CrossRefGoogle Scholar
  33. 33.
    Su F, Wang L, Sun Y, Liu C, Duan X, Li Z. Highly sensitive detection of CpG methylation in genomic DNA by AuNP-based colorimetric assay with ligase chain reaction. Chem Commun (Camb). 2015;51:3371–4.CrossRefGoogle Scholar
  34. 34.
    Song G, Chen C, Ren J, Qu X. A simple universal colorimetric assay for endonuclease/methyltransferase activity and inhibition based on an enzyme-responsive nanoparticle system. ACS Nano. 2009;3:1183–9.CrossRefPubMedGoogle Scholar
  35. 35.
    Liu T, Zhao J, Zhang D, Li G (2010) Novel method to detect DNA methylation using gold nanoparticles coupled with enzyme-linkage reactions. Anal Chem 82:229–233Google Scholar
  36. 36.
    Wu Z, Wu ZK, Tang H, Tang LJ, Jiang JH. Activity-based DNA-gold nanoparticle probe as colorimetric biosensor for DNA methyltransferase/glycosylase assay. Anal Chem. 2013;85:4376–83.CrossRefPubMedGoogle Scholar
  37. 37.
    Li W, Liu Z, Lin H, Nie Z, Chen J, Xu X, et al. Label-free colorimetric assay for methyltransferase activity based on a novel methylation-responsive DNAzyme strategy. Anal Chem. 2010;82:1935–41.CrossRefPubMedGoogle Scholar
  38. 38.
    Zhao Y, Chen F, Lin M, Fan C. A methylation-blocked cascade amplification strategy for label-free colorimetric detection of DNA methyltransferase activity. Biosens Bioelectron. 2014);54:565–70.CrossRefPubMedGoogle Scholar
  39. 39.
    Kermani HA, Hosseini M, Dadmehr M, Ganjali MR. Rapid restriction enzyme free detection of DNA methyltransferase activity based on DNA-templated silver nanoclusters. Anal Bioanal Chem. 2016;408:4311–8.CrossRefPubMedGoogle Scholar
  40. 40.
    Dadmehr M, Hosseini M, Hosseinkhani S, Ganjali MR, Sheikhnejad R. Label-free colorimetric and fluorimetric direct detection of methylated DNA based on silver nanoclusters for cancer early diagnosis. Biosens Bioelectron. 2015;73:108–13.CrossRefPubMedGoogle Scholar
  41. 41.
    Qiu X, Wang P, Cao Z. Hybridization chain reaction modulated DNA-hosted silver nanoclusters for fluorescent identification of single nucleotide polymorphisms in the let-7 miRNA family. Biosens Bioelectron. 2014;60:351–7.CrossRefPubMedGoogle Scholar
  42. 42.
    Zhang S, Wang K, Li K. B, Shi W, Jia WP, Chen X, Sun T, Han DM (2017) A DNA-stabilized silver nanoclusters/graphene oxide-based platform for the sensitive detection of DNA through hybridization chain reaction. Biosens Bioelectron 91:374–379Google Scholar
  43. 43.
    Shokri E, Hosseini M, Faridbod F, Rahaie M. Synthesis and assessment of DNA/silver nanoclusters probes for optimal and selective detection of Tristeza virus mild strains. J Fluorescence. 2016;26:1795–803.CrossRefGoogle Scholar
  44. 44.
    Shokri E, Hosseini M, Faridbod F, Rahaie M. Rapid presymptomatic recognition of Tristeza viral RNA by a novel fluorescent self-dimerized DNA-silver nanocluster probe. RSC Adv. 2016;6:99437–43.CrossRefGoogle Scholar
  45. 45.
    Liu W, Lai H, Huang R, Zhao C, Wang Y, Weng X, Zhou X (2015) DNA methyltransferase activity detection based on fluorescent silver nanocluster hairpin-shaped DNA probe with 5’-C-rich/G-rich-3’ tails. Biosens Bioelectron 68:736–7740Google Scholar
  46. 46.
    Zheng C, Zheng AX, Liu B, Zhang XL, He Y, Li J, et al. One-pot synthesized DNA-templated Ag/Pt bimetallic nanoclusters as peroxidase mimics for colorimetric detection of thrombin. Chem Commun. 2014;50:13103–6.CrossRefGoogle Scholar
  47. 47.
    You H, Peng Z, Wu J, Yang H. Lattice contracted AgPt nanoparticles. Chem Commun. 2011;47:12595–7.CrossRefGoogle Scholar
  48. 48.
    Gao Z, Xu M, Hou L, Chen G, Tang D. Irregular-shaped platinum nanoparticles as peroxidase mimics for highly efficient colorimetric immunoassay. Anal Chim Acta. 2013;776:79–86.CrossRefPubMedGoogle Scholar
  49. 49.
    Higuchi A, Siao YD, Yang ST, Hsieh PV, Fukushima H, Chang Y, et al. Preparation of a DNA aptamer–Pt complex and its use in the colorimetric sensing of thrombin and anti-thrombin antibodies. Anal Chem. 2008;80:6580–6.CrossRefPubMedGoogle Scholar
  50. 50.
    Wu LL, Wang LY, Xie ZJ, Xue F, Peng CF. Colorimetric detection of Hg2+ based on inhibiting the peroxidase-like activity of DNA-Ag/Pt nanoclusters. RSC Adv. 2016;6:75384–9.CrossRefGoogle Scholar
  51. 51.
    Wu LL, Wang LY, Xie ZJ, Pan N, Peng CF. Colorimetric assay of l-cysteine based on peroxidase-mimicking DNA-Ag/Pt nanoclusters. Sens Actuat B. 2016;235:110–6.CrossRefGoogle Scholar
  52. 52.
    Fu XM, Liu ZJ, Cai SX, Zhao YP, Wu DZ, Li CY, et al. Electrochemical aptasensor for the detection of vascular endothelial growth factor (VEGF) based on DNA-templated Ag/Pt bimetallic nanoclusters. Chinese Chem Lett. 2016;27:920–6.CrossRefGoogle Scholar
  53. 53.
    Stier I, Kiss A. Cytosine-to-uracil deamination by SssI DNA methyltransferase. PloS One. 2013;8(10):e79003.CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    New SY, Lee ST, Su XD. DNA-templated silver nanoclusters: structural correlation and fluorescence modulation. Nanoscale. 2016;8:17729–46.CrossRefPubMedGoogle Scholar
  55. 55.
    An H, Jin B. Prospects of nanoparticle–DNA binding and its implications in medical biotechnology. Biotechnol Adv. 2012;30:1721–32.CrossRefPubMedGoogle Scholar
  56. 56.
    Zhang Y, Xu WJ, Zeng YP, Zhang CY. Sensitive detection of DNA methyltransferase activity by transcription-mediated duplex-specific nuclease-assisted cyclic signal amplification. Chem Commun. 2015;51:13968–71.CrossRefGoogle Scholar
  57. 57.
    Xing XW, Tang F, Wu J, Chu JM, Feng YQ, Zhou X, Yuan BF (2014) Sensitive detection of DNA methyltransferase activity based on exonuclease-mediated target recycling. Anal Chem 86:11269-11274.Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Hanie Ahmadzade Kermani
    • 1
  • Morteza Hosseini
    • 1
  • Andrea Miti
    • 2
  • Mehdi Dadmehr
    • 3
  • Giampaolo Zuccheri
    • 2
  • Saman Hosseinkhani
    • 4
  • Mohammad Reza Ganjali
    • 5
    • 6
  1. 1.Department of Life Science Engineering, Faculty of New Sciences and TechnologiesUniversity of TehranTehranIran
  2. 2.Department of Pharmacy and Biotechnology and Interdepartmental Center for Industrial Research on Health Sciences and Technologies at the University of BolognaThe Nanoscience Institute of CNR, the National Interuniversity Consortium of Materials Science and TechnologyBolognaItaly
  3. 3.Department of BiotechnologyPayame Noor UniversityTehranIran
  4. 4.Department of BiochemistryTarbiat Modares University, Jalal AleAhmad NasrTehranIran
  5. 5.Center of Excellence in Electrochemistry, School of Chemistry, College of ScienceUniversity of TehranTehranIran
  6. 6.Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences InstituteTehran University of Medical SciencesTehranIran

Personalised recommendations