This work reports the use of modified reduced graphene oxide (rGO) as a platform for a label-free DNA-based electrochemical biosensor as a possible diagnostic tool for a DNA methylation assay. The biosensor sensitivity was enhanced by variously modified rGO. The rGO decorated with three nanoparticles (NPs)-gold (AuNPs), silver (AgNPs), and copper (CuNPs)-was implemented to increase the electrode surface area. Subsequently, the thiolated DNA probe (single-stranded DNA, ssDNA-1) was hybridized with the target DNA sequence (ssDNA-2). After the hybridization, the double-stranded DNA (dsDNA) was methylated by M.SssI methyltransferase (MTase) and then digested via a HpaII endonuclease specific site sequence of CpG (5'-CCGG-3') islands. For monitoring the MTase activity, differential pulse voltammetry (DPV) was used, whereas the best results were obtained by rGO-AuNPs. This assay is rapid, cost-effective, sensitive, selective, highly specific, and displays a low limit of detection (LOD) of 0.06 U·mL-1. Lastly, this study was enriched with the real serum sample, where a 0.19 U·mL-1 LOD was achieved. Moreover, the developed biosensor offers excellent potential in future applications in clinical diagnostics, as this approach can be used in the design of other biosensors.

Central European Institute of Technology Brno University of Technology Purkynova 123 612 00 Brno Czech Republic

Centre for Biosensors Bioelectronics and Biodevices and Department of Electronic and Electrical Engineering University of Bath Bath BA2 7AY UK

Department of Chemistry and Biochemistry Mendel University in Brno Zemedelska 1665 1 613 00 Brno Czech Republic

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Krejcova L., Richtera L., Hynek D., Labuda J., Adam V. Current trends in electrochemical sensing and biosensing of DNA methylation. Biosens. Bioelectron. 2017;97:384–399. doi: 10.1016/j.bios.2017.06.004. PubMed DOI

Yin H., Xu Z., Wang M., Zhang X., Ai S. An electrochemical biosensor for assay of DNA methyltransferase activity and screening of inhibitor. Electrochim. Acta. 2013;89:530–536. doi: 10.1016/j.electacta.2012.11.093. DOI

Chen X., Huang J., Zhang S., Mo F., Su S., Li Y., Fang L., Deng J., Huang H., Luo Z., et al. Electrochemical Biosensor for DNA Methylation Detection through Hybridization Chain-Amplified Reaction Coupled with a Tetrahedral DNA Nanostructure. ACS Appl. Mater. Interfaces. 2019;11:3745–3752. doi: 10.1021/acsami.8b20144. PubMed DOI

Zhu B., Booth M.A., Shepherd P., Sheppard A., Travas-Sejdic J. Distinguishing cytosine methylation using electrochemical, label-free detection of DNA hybridization and ds-targets. Biosens. Bioelectron. 2015;64:74–80. doi: 10.1016/j.bios.2014.08.049. PubMed DOI

Yin H., Yin H., Xu Z., Chen L., Zhang D., Ai S. An electrochemical assay for DNA methylation, methyltransferase activity and inhibitor screening based on methyl binding domain protein. Biosens. Bioelectron. 2013;41:492–497. doi: 10.1016/j.bios.2012.09.010. PubMed DOI

Chen S., Su J., Zhao Z., Shao Y., Dou Y., Li F., Deng W., Shi J., Li Q., Zuo X., et al. DNA framework-supported electrochemical analysis of DNA methylation for prostate cancers. Nano Lett. 2020;20:7028–7035. doi: 10.1021/acs.nanolett.0c01898. PubMed DOI

Zhang Q., Wu Y., Xu Q., Ma F., Zhang C.-Y. Recent advances in biosensors for in vitro detection and in vivo imaging of DNA methylation. Biosens. Bioelectron. 2020:112712. doi: 10.1016/j.bios.2020.112712. PubMed DOI

Huang J., Zhang S., Mo F., Su S., Chen X., Li Y., Fang L., Huang H., Deng J., Liu H., et al. An electrochemical DNA biosensor analytic technique for identifying DNA methylation specific sites and quantify DNA methylation level. Biosens. Bioelectron. 2019;127:155–160. doi: 10.1016/j.bios.2018.12.022. PubMed DOI

Mascini M., Tombelli S. Biosensors for biomarkers in medical diagnostics. Biomarkers. 2008;13:637–657. doi: 10.1080/13547500802645905. PubMed DOI

Mehrotra P. Biosensors and their applications—A review. J. Oral Biol. Craniofacial Res. 2016;6:153–159. doi: 10.1016/j.jobcr.2015.12.002. PubMed DOI PMC

Gao L., Lian C., Zhou Y., Yan L., Li Q., Zhang C., Chen L., Chen K. Graphene oxide–DNA based sensors. Biosens. Bioelectron. 2014;60:22–29. doi: 10.1016/j.bios.2014.03.039. PubMed DOI

Guex L.G., Sacchi B., Peuvot K.F., Andersson R.L., Pourrahimi A.M., Ström V., Farris S., Olsson R.T. Experimental review: Chemical reduction of graphene oxide (GO) to reduced graphene oxide (rGO) by aqueous chemistry. Nanoscale. 2017;9:9562–9571. doi: 10.1039/C7NR02943H. PubMed DOI

Shin H., Kim K.K., Benayad A., Yoon S., Park H.K., Jung I., Jin M.H., Jeong H., Kim J.M., Choi J., et al. Efficient Reduction of Graphite Oxide by Sodium Borohydride and Its Effect on Electrical Conductance. Adv. Funct. Mater. 2009;19:1987–1992. doi: 10.1002/adfm.200900167. DOI

Zhou M., Zhai Y., Dong S. Electrochemical Sensing and Biosensing Platform Based on Chemically Reduced Graphene Oxide. Anal. Chem. 2009;81:5603–5613. doi: 10.1021/ac900136z. PubMed DOI

Liu P., Wang D., Zhou Y., Wang H., Yin H., Ai S. DNA methyltransferase detection based on digestion triggering the combination of poly adenine DNA with gold nanoparticles. Biosens. Bioelectron. 2016;80:74–78. doi: 10.1016/j.bios.2015.12.100. PubMed DOI

Kokkinos C. Electrochemical DNA Biosensors Based on Labeling with Nanoparticles. Nanomaterials. 2019;9:1361. doi: 10.3390/nano9101361. PubMed DOI PMC

Qing Z., Bai A., Xing S., Zou Z., He X., Wang K., Yang R. Progress in biosensor based on DNA-templated copper nanoparticles. Biosens. Bioelectron. 2019;137:96–109. doi: 10.1016/j.bios.2019.05.014. PubMed DOI

Zhang X., Guo Q., Cui D.-X. Recent Advances in Nanotechnology Applied to Biosensors. Sensors. 2009;9:1033–1053. doi: 10.3390/s90201033. PubMed DOI PMC

Bao J., Geng X., Hou C., Zhao Y., Huo D., Wang Y., Wang Z., Zeng Y., Yang M., Fa H.-B. A simple and universal electrochemical assay for sensitive detection of DNA methylation, methyltransferase activity and screening of inhibitors. J. Electroanal. Chem. 2018;814:144–152. doi: 10.1016/j.jelechem.2018.02.060. DOI

Jamróz E., Kopel P., Tkaczewska J., Dordević D., Jančíková S., Kulawik P., Milosavljevic V., Dolezelikova K., Smerkova K., Svec P., et al. Nanocomposite Furcellaran Films—The Influence of Nanofillers on Functional Properties of Furcellaran Films and Effect on Linseed Oil Preservation. Polymers. 2019;11:2046. doi: 10.3390/polym11122046. PubMed DOI PMC

Besenhard J.O. Inorganic Reactions and Methods. Wiley-VCH Publishers; Weinheim, Germany: 1990. Preparation of Graphite Oxides; pp. 261–262.

Gao W., Alemany L.B., Ci L., Ajayan P.M. New insights into the structure and reduction of graphite oxide. Nat. Chem. 2009;1:403–408. doi: 10.1038/nchem.281. PubMed DOI

Goncalves G., Marques P.A.A.P., Granadeiro C.M., Nogueira H.I.S., Singh M.K., Graácio J. Surface Modification of Graphene Nanosheets with Gold Nanoparticles: The Role of Oxygen Moieties at Graphene Surface on Gold Nucleation and Growth. Chem. Mater. 2009;21:4796–4802. doi: 10.1021/cm901052s. DOI

Keighley S.D., Li P., Estrela P., Migliorato P. Optimization of DNA immobilization on gold electrodes for label-free detection by electrochemical impedance spectroscopy. Biosens. Bioelectron. 2008;23:1291–1297. doi: 10.1016/j.bios.2007.11.012. PubMed DOI

Bizzotto D., Burgess I.J., Doneux T., Sagara T., Yu H.-Z. Beyond Simple Cartoons: Challenges in Characterizing Electrochemical Biosensor Interfaces. ACS Sensors. 2018;3:5–12. doi: 10.1021/acssensors.7b00840. PubMed DOI

Macdonald J.R., Johnson W.B. Fundamentals of Impedance Spectroscopy. Impedance Spectroscopy. 2005;1:1–26. doi: 10.1002/0471716243.ch1. DOI

Jing X., Cao X., Wang L., Lan T., Li Y., Xie G. DNA-AuNPs based signal amplification for highly sensitive detection of DNA methylation, methyltransferase activity and inhibitor screening. Biosens. Bioelectron. 2014;58:40–47. doi: 10.1016/j.bios.2014.02.035. PubMed DOI

Liu S., Wu P., Li W., Zhang H., Cai C. An electrochemical approach for detection of DNA methylation and assay of the methyltransferase activity. Chem. Commun. 2011;47:2844–2846. doi: 10.1039/c0cc05153e. PubMed DOI

Xu Z., Wang M., Zhou T., Yin H., Ai S. Electrochemical biosensing method for the detection of DNA methylation and assay of the methyltransferase activity. Sensors Actuators B Chem. 2013;178:412–417. doi: 10.1016/j.snb.2012.12.124. DOI

Su J., He X., Wang Y., Zou Z., Chen Z., Yan G. A sensitive signal-on assay for MTase activity based on methylation-responsive hairpin-capture DNA probe. Biosens. Bioelectron. 2012;36:123–128. doi: 10.1016/j.bios.2012.04.012. PubMed DOI

Wang M., Xu Z., Chen L., Yin H., Ai S. Electrochemical Immunosensing Platform for DNA Methyltransferase Activity Analysis and Inhibitor Screening. Anal. Chem. 2012;84:9072–9078. doi: 10.1021/ac301620m. PubMed DOI

Wang G.L., Zhou L.Y., Luo H.Q., Li N.B. Electrochemical strategy for sensing DNA methylation and DNA methyltransferase activity. Anal. Chim. Acta. 2013;768:76–81. doi: 10.1016/j.aca.2013.01.026. PubMed DOI

Li W., Wu P., Zhang H., Cai C. Signal Amplification of Graphene Oxide Combining with Restriction Endonuclease for Site-Specific Determination of DNA Methylation and Assay of Methyltransferase Activity. Anal. Chem. 2012;84:7583–7590. doi: 10.1021/ac301990f. PubMed DOI

Huang B., Ji L., Liang B., Cao Q., Tu T., Ye X. A simple and low-cost screen printed electrode for hepatocellular carcinoma methylation detection. Analyst. 2019;144:3282–3288. doi: 10.1039/C9AN00191C. PubMed DOI

DNA Methylation in Solid Tumors: Functions and Methods of Detection