On-Chip Evaluation of DNA Methylation with Electrochemical Combined Bisulfite Restriction Analysis Utilizing a Carbon Film Containing a Nanocrystalline Structure

This paper reports an on-chip electrochemical assessment of the DNA methylation status in genomic DNA on a conductive nanocarbon film electrode realized with combined bisulfite restriction analysis (COBRA). The film electrode consists of sp² and sp³ hybrid bonds and is fabricated with an unbalanced magnetron (UBM) sputtering method. First, we studied the effect of the sp²/sp³ ratio of the UBM nanocarbon film electrode with <i>p</i>-aminophenol, which is a major electro-active product of the labeling enzyme from <i>p</i>-aminophenol phosphate. The signal current for <i>p</i>-aminophenol increases as the sp² content in the UBM nanocarbon film electrode increases because of the π–π interaction between aromatic <i>p</i>-aminophenol and the graphene-like sp² structure. Furthermore, the capacitative current at the UBM nanocarbon film electrode was successfully reduced by about 1 order of magnitude thanks to the angstrom-level surface flatness. Therefore, a high signal-to-noise ratio was achieved compared with that of conventional electrodes. Then, after performing an ELISA-like hybridization assay with a restriction enzyme, we undertook an electrochemical evaluation of the cytosine methylation status in DNA by measuring the oxidation current derived from <i>p</i>-aminophenol. When the target cytosine in the analyte sequence is methylated (unmethylated), the restriction enzyme of HpyCH4IV is able (unable) to cleave the sequence, that is, the detection probe cannot (can) hybridize. We succeeded in estimating the methylation ratio at a site-specific CpG site from the peak current of a cyclic voltammogram obtained from a PCR product solution ranging from 0.01 to 1 nM

Au Nanoparticle-Embedded Carbon Films for Electrochemical As³⁺ Detection with High Sensitivity and Stability

Au nanoparticle (AuNP)-embedded carbon films were formed with a one-step reproducible process by using unbalanced magnetron (UBM) cosputtering to make it possible to detect As³⁺ in water. The sputtered Au components formed NPs (typically 5 nm in diameter) spontaneously in the carbon films, owing to the poor intermiscibility of Au with carbon. The surface contents of embedded AuNPs in the carbon film were widely controllable (Au = 13–21 at %) by regulating the target powers of Au and carbon individually. The obtained film had a flat surface (Ra = 0.1 nm) despite the fact the AuNPs were partially exposed at the surface. By anodic stripping voltammetry (ASV) As³⁺ detection, a limit of detection of 0.55 ppb and linear dynamic range of 1–100 ppb were obtained with our electrode. These values meet the requirements imposed by international regulation. Moreover, our electrode structure realized good electrode stability for repetitive ASV measurements (relative standard deviation (RSD) = 11.7%, <i>n</i> = 15) because the partially embedded AuNP structures prevented the AuNPs from detaching from the surface. This result was achieved by the electrode recovery only by a potential scan from 0.1 to 1.5 V. Our electrodes can be stocked for a long time (2 years) with maintaining the electrode performance, which is very attractive for practical electrode. Selectivity test by using Tsukuba tap water added 10 ppb As³⁺ and 1000 ppb Cu²⁺ was successfully achieved with existence of 0.1 M EDTA (RSD = 2.6%, <i>n</i> = 3). The ASV results with tap water samples agreed well with those by the conventional ICPMS method