Sedlackova Eliska, Bytesnikova Zuzana, Birgusova Eliska, Svec Pavel, Ashrafi Amir M, Estrela Pedro, Richtera Lukas
Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1665/1, 613 00 Brno, Czech Republic.
Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00 Brno, Czech Republic.
Materials (Basel). 2020 Nov 3;13(21):4936. doi: 10.3390/ma13214936.
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. Lastly, this study was enriched with the real serum sample, where a 0.19 U·mL 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.
本研究报道了使用改性还原氧化石墨烯（rGO）作为基于DNA的无标记电化学生物传感器的平台，作为DNA甲基化检测的一种可能的诊断工具。通过对rGO进行各种修饰提高了生物传感器的灵敏度。用三种纳米颗粒（NPs）——金（AuNPs）、银（AgNPs）和铜（CuNPs）修饰的rGO被用于增加电极表面积。随后，硫醇化DNA探针（单链DNA，ssDNA - 1）与目标DNA序列（ssDNA - 2）杂交。杂交后，双链DNA（dsDNA）由M.SssI甲基转移酶（MTase）进行甲基化，然后通过CpG（5'-CCGG-3'）岛的HpaII内切酶特异性位点序列进行消化。为监测MTase活性，使用了差分脉冲伏安法（DPV），而rGO - AuNPs获得了最佳结果。该检测方法快速、经济高效、灵敏、具有选择性、高度特异性，检测限低至0.06 U·mL。最后，本研究使用真实血清样本进行了验证，检测限达到了0.19 U·mL。此外，所开发的生物传感器在未来临床诊断应用中具有巨大潜力，因为这种方法可用于设计其他生物传感器。
