Cytochrome P450 Modified Polycrystalline Indium Tin Oxide Film as a Drug Metabolizing Electrochemical Biosensor with a Simple Configuration

The development of a biocatalytic electrode consisting of cytochrome P450 (CYP) proteins would be a key technology with which to establish simple drug metabolizing biosensors or screening devices for drug inhibitors. We have successfully detected the direct electron transfer (DET) from a human CYP layer or a CYP microsome adsorbed on a bare indium tin oxide (ITO) film electrode without any modification layers and applied it to drug metabolism evaluation. We compared the electrocatalytic properties of the two ITO films with different surface nanostructures (polycrystalline or amorphous). CYP on polycrystalline ITO film enhanced the electron transfer rate of oxygen reduction about fifteen times more than with amorphous film. The polycrystalline ITO film was a suitable electrode for the adsorption of CYP proteins while maintaining efficient DET and enzymatic activity, probably because of its larger surface area and negatively charged surface. The oxygen reduction current at the polycrystalline ITO film electrodes had increased 3- to 4-fold, specifically coupled with the oxidation of drugs (testosterone and quinidine) by the monooxygenase activity of CYP. In contrast, the oxygen reduction current completely disappeared in the presence of the CYP inhibitor (ketoconazole). Similar results could be obtained from the CYP microsome with sufficiently clear responses. These results indicate that the CYP modified polycrystalline ITO electrode offers the potential for electrochemically evaluating CYP activity for drug metabolism with a simple configuration

Structure and Electrochemical Performance of Nitrogen-Doped Carbon Film Formed by Electron Cyclotron Resonance Sputtering

A nitrogen-doped nanocarbon film electrode with mixed sp² and sp³ bonds formed using the electron cyclotron resonance (ECR) sputtering method was studied with respect to the relationship between nitrogen concentration and electrochemical performance. The film (N-ECR) has a nanocrystalline structure, and the sp³ content increases with increasing nitrogen concentration unlike the recently reported nitrogen-containing tetrahedral amorphous carbon film. The film has a very smooth surface with an average roughness of 0.1 to 0.2 nm, which is almost independent of nitrogen concentration. In contrast, the ratio of nitrogen-containing graphite-like bonding is high at low nitrogen concentrations, and then pyridine-like bonding increases as the nitrogen concentration increases. These variations in the chemical structures and the sp² and sp³ content greatly change the electrochemical performance. The N-ECR electrode shows a wider potential window (∼3.8 V) than a pure nanocarbon electrode (∼3.1 V) due to its higher sp³ content. The N-ECR electrode (N = 9.0 at. %) shows improved electrochemical activity because the lowest peak separation of Fe­(CN)₆^3–/4– was observed at this nitrogen concentration. The oxygen and hydrogen peroxide (H₂O₂) reduction potentials at the N-ECR electrode shifted about 0.3 and 0.15 V, respectively, and the peak height of H₂O₂ is greatly increased. As a result, a linear relationship was obtained from 0.2 to 17 mM for the reductive current detection of H₂O₂. The N-ECR electrode also shows better activity for oxidizing certain biomolecules. The oxidation potentials of guanosine and adenosine decreased about 0.1 V, suggesting that the N-ECR electrode is suitable for use as a biosensing platform

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

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

Direct Analysis of Lipophilic Antioxidants of Olive Oils Using Bicontinuous Microemulsions

Quantitative analyses of olive oil for lipophilic antioxidants, such as α-tocopherol and phenolics, by simple electrochemical measurements were conducted in a bicontinuous microemulsion (BME), which was bicontinuously composed of saline and toluene microphases with a surfactant system. Lipophilic antioxidants in oils were directly monitored in BME solutions using a lipophilic, fluorinated nanocarbon-film electrode (F–ECR). The combination of a well-balanced BME and extremely biased electrodes, such as strongly hydrophilic indium/tin oxide and strongly lipophilic (hydrophobic) F–ECR, allowed individual monitoring of hydrophilic and lipophilic antioxidants in the same BME solution without any required extraction. Furthermore, values for the charge <i>Q</i>, integrated from observed currents, showed good linear relationships with the results of conventional assays for antioxidant activity, namely, total phenolics and oxygen radical absorbance capacity assays, even with practical food samples. This proposed methodology provided a very simple, rapid, easily serviceable, and highly reproducible analysis that possesses great potential for applications to a wide range of chemical mixtures, in terms of analyte and media, beyond food oils

Simultaneous Electrochemical Analysis of Hydrophilic and Lipophilic Antioxidants in Bicontinuous Microemulsion

Qualitative and quantitative analyses of hydrophilic and lipophilic antioxidants, such as polyphenols, by simple electrochemical measurements were conducted in a bicontinuous microemulsion (BME), in which water and oil phases coexisted bicontinuously on a microscopic scale. Hydrophilic and lipophilic antioxidants were individually monitored in the same BME solution using a hydrophilic indium tin oxide (ITO) electrode and a lipophilic fluorinated nanocarbon film electrode (F-ECR), respectively. The combination of well-balanced BME and extremely biased electrodes, such as ITO and F-ECR, in terms of hydrophilic–lipophilic balance allowed us to achieve individual monitoring of hydrophilic and lipophilic antioxidants in the same BME solution without extraction. Furthermore, the antioxidant activities of functional liquid foods, such as coffee and olive oil, were also evaluated by means of electrochemical measurements in BME solutions containing analytes in concentrations of several percent. The technique we propose provides a very simple, rapid, easily serviceable, and highly reproducible analysis and can be extended to a wide range of analytes and media