6 research outputs found
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 sp<sup>2</sup> and sp<sup>3</sup> 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 sp<sup>3</sup> 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 sp<sup>2</sup> and sp<sup>3</sup> 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
sp<sup>3</sup> content. The N-ECR electrode (N = 9.0 at. %) shows
improved electrochemical activity because the lowest peak separation
of FeÂ(CN)<sub>6</sub><sup>3–/4–</sup> was observed at
this nitrogen concentration. The oxygen and hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) reduction potentials at the N-ECR electrode
shifted about 0.3 and 0.15 V, respectively, and the peak height of
H<sub>2</sub>O<sub>2</sub> is greatly increased. As a result, a linear
relationship was obtained from 0.2 to 17 mM for the reductive current
detection of H<sub>2</sub>O<sub>2</sub>. 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 As<sup>3+</sup> 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 As<sup>3+</sup> 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) As<sup>3+</sup> 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 As<sup>3+</sup> and 1000 ppb Cu<sup>2+</sup> 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 sp<sup>2</sup> and sp<sup>3</sup> hybrid
bonds and is fabricated with an unbalanced magnetron (UBM) sputtering
method. First, we studied the effect of the sp<sup>2</sup>/sp<sup>3</sup> 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 sp<sup>2</sup> content in the UBM nanocarbon film electrode increases because of
the π–π interaction between aromatic <i>p</i>-aminophenol and the graphene-like sp<sup>2</sup> 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