DNA methylation plays a significant role in various biological events, and its precise determination is vital for the prognosis and treatment of cancer. Here, we proposed an ultrasensitive electrochemical biosensor for the quantitative analysis of multiple methylation-locus in DNA sequence via DNA anchoring the gold nanoparticles (DNA-AuNPs) and bienzyme dual signal amplifications. After the target DNA captured by the DNA-AuNPs of the biosensor, the methyl-CpG binding protein MeCP2 could specifically conjugate to the methylation-loci in the double-stranded DNA. Successively, the glucose oxidase (GOD) and horseradish (HRP) co-labeled antibody captured the His tagged MeCP2, which leads to a cascade enzymatic catalysis of the substrates to yield a detectable electrochemical signal. Both the two strategies, including the high content of DNA-AuNPs and the associated catalysis of bienzyme, dramatically enhanced the sensitivity of the biosensor. The response current elevated with the increasing numbers of methylation-locus, thus the multiple methylated DNA was identified by detecting the corresponding current signals. This method could detect the methylated target as low as 0.1 fM, and showed a wide linear range from 10 - 15 M to 10 - 7 M. Besides, the long-term stability and repeatability of the biosensor were also validated. The prepared electrochemical immunosensor exhibits ultrasensitivity through the bienzyme labeling process, which can be applied for the detection of DNA methylation with low concentration.
References
[1]
Suzuki, M.M. and Bird, A. (2008) DNA Methylation Landscapes: Provocative Insights from Epigenomics. Nature Reviews Genetics, 9, 465-476. https://doi.org/10.1038/nrg2341
[2]
Dirk, S. (2015) Function and Information Content of DNA Methylation. Nature, 517, 321-326. https://doi.org/10.1038/nature14192
[3]
Smith, Z.D. and Meissner, A. (2013) DNA Methylation: Roles in Mammalian Development. Nature Reviews Genetics, 14, 204-220. https://doi.org/10.1038/nrg3354
[4]
Ehrlich, M., Gama-Sosa, M.A., Huang, L.H., et al. (1982) Amount and Distribution of 5-Methylcytosine in Human DNA from Different Types of Tissues of Cells. Nucleic Acids Research, 10, 2709-2721. https://doi.org/10.1093/nar/10.8.2709
[5]
Kader, F. and Ghai, M. (2015) DNA Methylation and Application in Forensic Sciences. Forensic Science International, 249, 255-265. https://doi.org/10.1016/j.forsciint.2015.01.037
[6]
Chen, M., Voeller, D., Marquez, V.E., et al. (2010) Enhanced Growth Inhibition by Combined DNA Methylation/HDAC Inhibitors in Lung Tumor Cells with Silenced CDKN2A. International Journal of Oncology, 37, 963-971. https://doi.org/10.3892/ijo_00000747
[7]
Scesnaite, A., Jarmalaite, S., Mutanen, P., et al. (2012) Similar DNA Methylation Pattern in Lung Tumours from Smokers and Never-Smokers with Second-Hand Tobacco Smoke Exposure. Mutagenesis, 27, 423-429. https://doi.org/10.1093/mutage/ger092
[8]
Baek, S.J., Yang, S., Kang, T.W., et al. (2013) MENT: Methylation and Expression Database of Normal and Tumor Tissues. Gene, 518, 194-200. https://doi.org/10.1016/j.gene.2012.11.032
[9]
Von Kanel, T., Gerber, D., Schaller, A., et al. (2010) Quantitative 1-Step DNA Methylation Analysis with Native Genomic DNA as Template. Clinical Chemistry, 56, 1098-1106. https://doi.org/10.1373/clinchem.2009.142828
[10]
Bakshi, A., Ekram, M.B, and Kim, J. (2015) Locus-Specific DNA Methylation Analysis of Retrotransposons in ES, Somatic and Cancer Cells Using High-Throughput Targeted Repeat Element Bisulfite Sequencing. Genomics Data, 3, 87-89. https://doi.org/10.1016/j.gdata.2014.11.013
[11]
Head, J.A., Mittal, K. and Basu, N. (2015) Application of the LUminometric Methylation Assay to Ecological Species: Tissue Quality Requirements and a Survey of DNA Methylation Levels in Animals. Molecular Ecology Resources, 14, 943-952. https://doi.org/10.1111/1755-0998.12244
[12]
Huang, W., Qi, C.B., Lv, S.W., et al. (2015) Correction to Determination of DNA and RNA Methylation in Circulating Tumor Cells by Mass Spectrometry. Analytical Chemistry, 88, 1378-1384. https://doi.org/10.1021/acs.analchem.5b03962
[13]
Bibikova, M., Barnes, B., Tsan, C., et al. (2011) High Density DNA Methylation Array with Single CpG Site Resolution. Genomics, 98, 288-295. https://doi.org/10.1016/j.ygeno.2011.07.007
[14]
Su, J., He, X., Wang, Y., Wang, K., Chen, Z.F. and Yan, G. (2012) A Sensitive Signal-on Assay for MTase Activity Based on Methylation-Responsive Hairpin-Capture DNA Probe. Biosensors and Bioelectronics, 36, 123-128. https://doi.org/10.1016/j.bios.2012.04.012
[15]
Shen, Q., Han, L., Fan, G., et al. (2015) Highly Sensitive Photoelectrochemical Assay for DNA Methyltransferase Activity and Inhibitor Screening by Exciton Energy Transfer Coupled with Enzyme Cleavage Biosensing Strategy. Biosensors and Bioelectronics, 64, 449-455. https://doi.org/10.1016/j.bios.2014.09.044
[16]
Peng, X., Hu, T., Bao, T., Zhao, L., et al. (2017) A Label-Free Electrochemical Biosensor for Methytransferase Activity Detection and Inhibitor Screening Based on Graphene Quantum Dot and Enzyme-Catalyzed Reaction. Journal of Electroanalytical Chemistry, 799, 327-332. https://doi.org/10.1016/j.jelechem.2017.06.030
[17]
Bao, J., Geng, X., Hou, C., et al. (2018) A Simple and Universal Electrochemical Assay for Sensitive Detection of DNA Methylation, Methyltransferase Activity and Screening of Inhibitors. Journal of Electroanalytical Chemistry, 814, 144-152. https://doi.org/10.1016/j.jelechem.2018.02.060
[18]
Wang, M., Xu, Z., Chen, L., Yin, H. and Ai, S. (2012) Electrochemical Immunosensing Platform for DNA Methyltransferase Activity Analysis and Inhibitor Screening. Analytical Chemistry, 84, 9072-9078. https://doi.org/10.1021/ac301620m
[19]
Xu, Z., Yin, H., Huo, L., Zhou, Y. and Ai, S. (2014) Electrochemical Immunosensor for DNA Methyltransferase Activity Assay Based on Methyl CpG-Binding Protein and Dual Gold Nanoparticle Conjugate-Based Signal Amplification. Sensors and Actuators B: Chemical, 192, 143-149. https://doi.org/10.1016/j.snb.2013.10.099
[20]
Yu, Y., Blair, S., Gillespie, D., et al. (2010) Direct DNA Methylation Profiling Using Methyl Binding Domain Proteins. Analytical Chemistry, 82, 5012-5019. https://doi.org/10.1021/ac1010316
[21]
Du, Q., Lu, P.L., Stirzaker, C. and Clark, S.J. (2015) Methyl-CpG-Binding Domain Proteins: Readers of the Epigenome. Epigenomics, 7, 1051-1073. https://doi.org/10.2217/epi.15.39
[22]
Zhuo, Y., Yi, W.J., Lian, W.B., et al. (2011) Ultrasensitive Electrochemical Strategy for NT-proBNP Detection with Gold Nanochains and Horseradish Peroxidase Complex Amplification. Biosensors and Bioelectronics, 26, 2188-2193. https://doi.org/10.1016/j.bios.2010.09.033
[23]
Lu, D.Q., Xu, Q.D., Pang, G.C. and Lu, F. (2018) A Novel Electrochemical Immunosensor Based on Au Nanoparticles and Horseradish Peroxidase Signal Amplification for Ultrasensitive Detection of α-Fetoprotein. Biomedical Microdevices, 20, 46-58. https://doi.org/10.1007/s10544-018-0291-7
[24]
Pelossof, G., Tel-Vered, R., Elbaz, J. and Willner, I. (2010) Amplified Biosensing Using the Horseradish Peroxidase-Mimicking DNAzyme as an Electrocatalyst. Analytical Chemistry, 82, 4396-4402. https://doi.org/10.1021/ac100095u
[25]
Bankar, S.B., Bule, M.V., Singhal, R.S. and Ananthanarayan, L. (2009) Glucose Oxidase—An Overview. Biotechnology Advances, 27, 489-501. https://doi.org/10.1016/j.biotechadv.2009.04.003
[26]
Feng, Q., Liu, K., Fu, J., et al. (2012) Direct Electrochemistry of Hemoglobin Based on Nano-Composite Film of Gold Nanopaticles and Poly (Diallyldimethylammonium Chloride) Functionalized Graphene. Electrochimica Acta, 60, 304-308. https://doi.org/10.1016/j.electacta.2011.11.048
[27]
Baron, R., Willner, B. and Willner, I. (2007) Biomolecule-Nanoparticle Hybrids as Functional Units for Nanobiotechnology. Chemical Communications, 38, 323-332. https://doi.org/10.1039/B610721B
[28]
Cao, Y., Yuan, R., Chai, Y., et al. (2012) Ultrasensitive Luminol Electrochemiluminescence for Protein Detection Based on in Situ Generated Hydrogen Peroxide as Coreactant with Glucose Oxidase Anchored AuNPs@MWCNTs labeling. Biosensors and Bioelectronics, 31, 305-309. https://doi.org/10.1016/j.bios.2011.10.036
[29]
Jing, X., Cao, X., Wang, L., et al. (2014) DNA-AuNPs Based Signal Amplification for Highly Sensitive Detection of DNA Methylation, Methyltransferase Activity and Inhibitor Screening. Biosensors and Bioelectronics, 58, 40-47. https://doi.org/10.1016/j.bios.2014.02.035
[30]
Avrameas, S. (1969) Coupling of Enzymes to Proteins with Glutaraldehyde: Use of the Conjugates for the Detection of Antigens and Antibodies. Immunochemistry, 6, 49-52. https://doi.org/10.1016/0019-2791(69)90177-3
[31]
Avrameas, S. and Ternynck, T. (1969) The Cross-Linking of Proteins with Glutaraldehyde and Its Use for the Preparation of Immunoadsorbents. Immunochemistry, 6, 53-66. https://doi.org/10.1016/0019-2791(69)90178-5
[32]
Regidi, S., Ravindran, S., Vijayan, A.L., et al. (2018) Effect of Lyophilization on HRP—Antibody Conjugation: An Enhanced Antibody Labeling Technology. BMC Research Notes, 11, 596-601. https://doi.org/10.1186/s13104-018-3688-8
[33]
Yang, H., Gong, C.C., Miao, L.F. and Xu, F. (2017) A Glucose Biosensor Based on Horseradish Peroxidase and Gkusoce Oxidase Co-Entrapped in Carbon Nanotubes Modified Electrode. International Journal of Electrochemical Science, 12, 4958-4969. https://doi.org/10.20964/2017.06.05
[34]
Zhang, Q., Wu, S.Y. and Zhang, L. (2011) Fabrication of Polymeric Ionic Liquid/Graphene Nanocomposite for Glucose Oxidase Immobilization and Direct Electrochemistry. Biosenors and Bioelectronics, 26, 2632-2637. https://doi.org/10.1016/j.bios.2010.11.024
[35]
Yin, H., Zhou, Y., Xu, Z., Wang, M. and Ai, S. (2013) Ultrasensitive Electrochemical Immunoassay for DNA Methyltransferase Activity and Inhibitor Screening Based on Methyl Binding Domain Protein of MeCP2 and Enzymatic Signal Amplification. Biosensors and Bioelectronics, 49, 39-45. https://doi.org/10.1016/j.bios.2013.04.040
[36]
Liu, P., Liu, M., Yin, H., Zhou, Y. and Ai, S.Y. (2015) Electrochemical Biosensor for DNA Methyltransferase Detection Based on DpnI Digestion Triggering the Formation of G-Quadruplex DNAzymes. Sensors and Actuators B: Chemical, 220, 101-106. https://doi.org/10.1016/j.snb.2015.05.058
[37]
Koo, K.M., Wee, E.J.H., Rauf, S., et al. (2014) Microdevices for Detecting Locus-Specific DNA Methylation at CpG Resolution. Biosensors and Bioelectronics, 56, 278-285. https://doi.org/10.1016/j.bios.2014.01.029
