Diverse Hierarchical Meso/Nanoporous Pt Film Electrocatalysts Prepared via Hydrogen Adsorption-Assisted Dynamic Soft Templating.

In this study, we present an innovative approach for creating hierarchical meso/nanoporous Pt films using dynamic soft templating. The fabrication process, called dynamic soft templating, involves Pt electrodeposition within a specialized bicontinuous microemulsion (BME) system characterized by a sophisticated three-dimensional network comprising water and oil phases, surfactants, and cosurfactants. Pt electrodeposition exclusively occurs in the water phase of the BME. This results in a porous Pt film exhibiting a nanostructure mirroring the oil solution/water solution nanostructure (solution/solution structure) of the BME, the size of which can be tailored by adjusting the BME composition. Through a simultaneous interplay of Pt electrodeposition and overpotential deposition of hydrogen (H-OPD, dissociative adsorption of water), potential-dependent Pt mesostructures are dynamically shaped. As a result, we achieve diverse morphologies in the form of hierarchical meso/nanoporous Pt films. The potential applications of the films are evaluated as electrocatalysts for the methanol oxidation reaction (MOR), and it was found that the electrocatalytic performances seem to be sensitive to nanoporosity and not relevant to mesoporosity.

The influence mechanism of the molecular structure on the peak current and peak potential in electrochemical detection of typical quinolone antibiotics.

Antibiotic pollution in water has become an increasingly serious problem, posing a potentially huge threat to human health. Ofloxacin (OFL), norfloxacin (NOR), and enoxacin (ENX) are typical broad-spectrum quinolone antibiotics, which are frequently detected in various water environments. An electrochemical sensor is a rapid and effective tool to detect antibiotics in the aquatic environment. The molecular structure of target pollutants is an important factor affecting the detection performance of electrochemical sensors. Based on the electrochemical detection results of antibiotics (OFL, NOR, and ENX), we first used the molecular structure analysis method based on quantum chemistry to accurately identify the electronegativity and the electrocatalytic degree of the oxidizable (and non-oxidizable) functional groups of pollutants. We also clarified the influence mechanism of the molecular structure on the peak current and peak potential. These results can provide theoretical support for rapidly selecting electrodes with a suitable electrochemical window to efficiently detect trace organic pollutants (such as antibiotics) in water based on the molecular structure of the target pollutant.

Stand-Alone Semi-Solid-State Electrochemical Systems Based on Bicontinuous Microemulsion Gel Films.

Bicontinuous microemulsion (BME)-based hydrogel films were integrated with screen-printed electrodes (SPEs) comprising working, counter, and reference electrodes to form stand-alone, semi-solid-state electrochemical systems that do not require an outer electrolyte solution. The gel network of the BME hydrogel only exists in the microaqueous phase and retains the structure of the entire BME gel. Following gelation, a microaqueous phase with sufficient ionic strength ensured effective ionic conductivity, even in thin gel films. This enabled the electrochemical reaction to proceed using a thin gel film as an electrolyte solution. However, an intact micro-oil phase with no gel network enabled efficient extraction from an external oil solution and exhibited rapid electrochemistry that was comparable to that of a BME solution. Cyclic voltammograms of lipophilic redox species in oil using the gel-integrated SPE system demonstrated successfully in the oil itself and in the air with dropped oil onto the system.

Study of the More Suitable Drugs and Dosage Forms for Administration to Newborn Infants via Feeding Tube Using the Collection Rate as an Indicator.

The dosages of drugs in newborn infants are small. Small dose necessitate consideration of the loss of drug when administered via feeding tube. In this study, we conducted a tube administration test for seven kinds of antiepileptic drugs and two kinds of potassium supplements using a neonatal feeding tube and investigated the drug loss using the collection rate. We also studied the differences in collection rates among different dosage forms and drugs to determine the more suitable dosage forms and drugs. We investigated three dosage forms: powder, fine granules or dry syrup (powdery form) drugs, powdery form drugs that have been pulverized (pulverized powdery forms), and pulverized tablets. Additionally, we investigated two potassium supplements to determine which was more suitable: potassium L-aspartate and potassium gluconate. For topiramate, only the powdery form caused tube obstructions; the collection rates of the pulverized powdery form and pulverized tablets were > 90%. All antiepileptic drugs other than topiramate that were tested had collection rates of about > 90%. Considering stability and pharmacokinetics, the more suitable dosage form for topiramate is pulverized tablets, whereas the more suitable dosage form for other antiepileptic drugs is powdery form. Collection rate of potassium gluconate was higher than that of potassium L-aspartate. The current study, which indicates that potassium gluconate powdery form is the more suitable drug, presents the more suitable dosage form and drug for administration via feeding tube to newborn infants. These results show that it is essential to evaluate passage through the tube using the collection rate.

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 p-aminophenol, which is a major electro-active product of the labeling enzyme from p-aminophenol phosphate. The signal current for p-aminophenol increases as the sp2 content in the UBM nanocarbon film electrode increases because of the π-π interaction between aromatic p-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 p-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.

Artificial Modification of an Enzyme for Construction of Cross-Reactive Polyion Complexes To Fingerprint Signatures of Proteins and Mammalian Cells.

A novel strategy for construction of cross-reactive enzyme-based sensors have been developed based on chemical modification of lysine ε-NH3(+) groups in ß-galactosidase (ß-Gal) from Aspergillus oryzae with various acid anhydrides. Modification of lysine side chains markedly altered the kinetic parameters of ß-Gal (KM and kcat), whereas catalytic activity remained and the tertiary structure was maintained for all modified ß-Gals. The addition of cationic PEGylated polymers to reactive solutions caused the formation of polyion complexes (PICs) with marked inhibition of the modified ß-Gal activity. Therefore, the obtained PICs can be used to construct cross-reactive enzyme-based sensors without any purification. With addition of each analyte protein or mammalian cell to the PIC library, the modified ß-Gals were partially released from PICs, and therefore the catalytic activities showed characteristic recovery fingerprints. Linear discriminant analysis (LDA) of fingerprints generated by the combination of only three PICs enabled successful discrimination of 0.5 µg/mL human plasma proteins (albumin, immunoglobulin G, transferrin, fibrinogen, α1-antitrypsin, C-reactive protein), and semiquantification of inflammatory biomarker proteins in buffer (0.3-8.1 µg/mL) and human serum (2-100 µg/mL) with comparable sensitivity for diagnosis in human blood samples. Moreover, we identified five mammalian (human, bovine, fetal bovine, horse, and rabbit) sera containing 2.5 µg/mL serum proteins, and three human cancer cell lines [A549 (lung), MG63 (bone), HuH7 (liver)] and a human mesenchymal stem cell line (UE7T-13) (1500 cells/mL) with 100% accuracy.

Au Nanoparticle-Embedded Carbon Films for Electrochemical As(3+) 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(3+) 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(3+) 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%, n = 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(3+) and 1000 ppb Cu(2+) was successfully achieved with existence of 0.1 M EDTA (RSD = 2.6%, n = 3). The ASV results with tap water samples agreed well with those by the conventional ICPMS method.

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 Q, 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.

Label-Free Detection of Human Glycoprotein (CgA) Using an Extended-Gated Organic Transistor-Based Immunosensor.

Herein, we report on the fabrication of an extended-gated organic field-effect transistor (OFET)-based immunosensor and its application in the detection of human chromogranin A (hCgA). The fabricated OFET device possesses an extended-gate electrode immobilized with an anti-CgA antibody. The titration results of hCgA showed that the electrical changes in the OFET characteristics corresponded to the glycoprotein recognition ability of the monoclonal antibody (anti-CgA). The observed sensitivity (detection limit: 0.11 µg/mL) and selectivity indicate that the OFET-based immunosensor can be potentially applied to the rapid detection of the glycoprotein concentration without any labeling.

On-Chip Sequence-Specific Immunochemical Epigenomic Analysis Utilizing Outward-Turned Cytosine in a DNA Bulge with Handheld Surface Plasmon Resonance Equipment.

This paper reports a sequence-specific immunoassay chip for DNA methylation assessment by microfluidic-based surface plasmon resonance (SPR) detection. This was achieved by utilizing an affinity measurement involving the target, (methyl)cytosine, in a single-base bulge region and an anti-methylcytosine antibody in a microchannel, following hybridization with a biotinylated bulge-inducing DNA probe. The probe alters the target cytosine in a looped-out state because of the π-π stacking between flanking bases of the target. The probe design is simple and consists of the elimination of guanine paired with the target cytosine from a fragmented full-match sequence. We obtained the single methylation status in 6 amol (48 fg) of synthesized oligo DNA in 45 min, which is the fastest DNA methylation assessment yet reported, without employing a conventional bisulfite reaction, PCR, or sequencing. We also succeeded in discrimination of the methylation status of single cytosine in genomic λ DNA and HCT116 human colon cancer cells. The advantages of the proposed method are its small equipment, simple microfluidics design, ease of handling (two injections of DNA and antibody), lack of need for a methylation-sensitive enzyme, and neutral buffer conditions.

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.

Pd-Ni alloy nanoparticle/carbon nanofiber composites: preparation, structure, and superior electrocatalytic properties for sugar analysis.

Novel Pd-Ni alloy nanoparticle/carbon nanofiber (Pd-Ni/CNF) composites were successfully prepared by a simple method involving electrospinning of precursor polyacrylonitrile/Pd(acac)2/Ni(acac)2 nanofibers, followed by a thermal process to reduce metals and carbonize polyacrylonitrile. The nanostructures of the resulting Pd-Ni/CNF nanocomposites were carefully examined by a combination of scanning electron microscopy (SEM), transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM), high-angle annular dark field (HAADF)-scanning transmission electron microscopy (STEM), energy dispersive X-ray (EDX), thermogravimetric analysis (TGA), X-ray diffraction (XRD), and X-ray photoelectron spectra (XPS). For all the nanocomposites, the Pd-Ni alloy nanoparticles (NPs) were dispersed uniformly and embedded firmly within the framework or on the surface of CNF. The size, composition, and alloy homogeneity of the Pd-Ni alloy NPs could be readily tailored by controlling the feed ratio of metal precursors and the thermal treatment process. Cyclic voltammetric studies showed enhanced redox properties for Pd-Ni/CNF-based electrodes relative to the Ni-metal electrode and significantly improved electrocatalytic activity for sugar (e.g., glucose, fructose, sucrose, and maltose) oxidation. The application potential of Pd-Ni/CNF-based electrodes in flow systems for sugars detection was explored. A very low limit of detection for sugars (e.g., 7-20 nM), high resistance to surface fouling, excellent signal stability and reproducibility, and a very wide detection linear range (e.g., 0.03-800 µM) were revealed for this new type of Pd-Ni/CNF nanocomposite as the detecting electrode. Such detection performances of Pd-Ni/CNF-based electrodes are superior to those of state-of-the-art nonenzymatic sugar detectors that are commercially available or known in the literature.

Structure and electrochemical performance of nitrogen-doped carbon film formed by electron cyclotron resonance sputtering.

A nitrogen-doped nanocarbon film electrode with mixed sp(2) and sp(3) 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(3) content increases with increasing nitrogen concentration unlike the recently reported nitrogen-containing tetrahedral amorphous carbon film.1 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(2) and sp(3) 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(3) 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.

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.

Surface modification of silicon oxide with trialkoxysilanes toward close-packed monolayer formation.

In order to scrutinize potential of trialkoxysilanes to form close-packed monolayer, surface modification of silicon oxide was carried out with the trialkoxysilanes bearing a ferrocene moiety for analysis by electrochemical methods. As it was found that hydrogen-terminated silicon reacts with trialkoxysilane through natural oxidation in organic solvents, where the silicon oxide layer is thin enough to afford conductivity for electrochemical analysis, hydrogen-terminated silicon wafer was immersed in trialkoxysilane solution for surface modification without oxidation treatment. Cyclic voltammetry measurements to determine surface concentrations of the immobilized ferrocene-silane on silicon surface were carried out with various temperature, concentration, solvent, and molecular structure, while the blocking effect in the cyclic voltammogram was investigated to obtain insight into density leading to the close-packed layer. The results suggested that a monolayer modification tended to occur under milder conditions when the ferrocene-silane had a longer alkyl chain, and formation of a close-packed layer to show significant blocking effect was observed. However, the surface modification proceeded even when surface concentration of the immobilized ferrocene-silane was greater than that expected for the monolayer. On the basis of these tendencies, the surface of silicon oxide modified with trialkoxysilane is considered to be a partial multilayer rather than monolayer although a close-packed layer is formed. This result is supported by the comparison with carbon surface modified with ferrocene-diazonium, in which a significant blocking effect was observed when surface concentrations of the immobilized ferrocene moiety are lower than that for silicon oxide modified with ferrocene-silane.

Design and fabrication of biosensing interface for waveguide-mode sensor.

In order to develop a biosensing system with waveguide-mode sensor, fabrication of a biosensing interface on the silica surface of the sensing chip was carried out using triethoxysilane derivatives with anti-leptin antibody. Triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties were synthesized to immobilize the antibody and to suppress nonspecific adsorption of proteins, respectively. The chip modified with triethoxysilane derivatives bearing oligoethylene glycol moiety suppressed nonspecific adsorption of proteins derived from human serum effectively by rinse with PBS containing surfactant (0.05% Tween 20). On the other hand, it was confirmed that antibody was immobilized on the chip by immersion into antibody solution to show response of antigen-antibody reaction, where the chip was modified with triethoxysilane derivatives bearing succinimide ester moiety. When the interface was fabricated with antibody and a mixture of triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties, the response of antigen-antibody reaction depended on composition of the mixture and enhanced with the increase of ratio for triethoxysilane derivatives bearing succinimide ester moiety reflecting the antibody concentration immobilized on the chip. While introduction of excess triethoxysilane derivatives bearing succinimide ester moiety induced nonspecific adsorption of proteins derived from human serum, the immobilized antibody on the chip kept its activity after 1-month storage in a refrigerator. Taking into consideration those factors, the biosensing interface was fabricated using triethoxysilane derivatives with anti-leptin antibody to examine performance of the waveguide-mode sensor. It was found that the detection limits for human leptin were 50 ng/mL in PBS and 100 ng/mL in human serum. The results demonstrate that the waveguide-mode sensor powered by the biosensing interface fabricated with those triethoxysilane derivatives and antibody has potential to detect several tens of nanograms per milliliter of biomarkers in human serum with an unlabeled detection method.

Human cytochrome P450 3A4 and a carbon nanofiber modified film electrode as a platform for the simple evaluation of drug metabolism and inhibition reactions.

Electrochemical biosensors consisting of cytochrome P450 enzyme modified electrodes have been developed to provide a simple method for screening the metabolism of a drug and its inhibitor. Here, we report a very simple electrochemically driven biosensor for detecting drug metabolism and its inhibition based on cytochrome P450 3A4 (CYP3A4) and a carbon nanofiber (CNF) modified film electrode without any other modified layers such as mediator films. Direct electron transfer (DET) between CYP3A4 and CNFs was observed at a formal potential of -0.302 V. The electrocatalytic reduction current increased with the addition of drugs including testosterone and quinidine. In contrast, the reduction current was greatly suppressed in the presence of ketoconazole, which is a CYP3A4 inhibitor. CNFs with high conductivity, a large surface area and sufficient edge planes provide a suitable microenvironment for achieving excellent DET and biocatalysis properties, which could not be observed when we used other carbon materials such as carbon nanotube (CNT) and carbon black (CB) modified electrodes, indicating that our system is promising as a new bioelectronic platform for electrochemical biosensing.

A Novel Polyurethane-Based Polyion Complex Material with Tunable Selectivity against Interferents for Selective Dopamine Determination.

Polyion complex (PIC) materials have been widely used in biosensors due to their molecular selectivity. However, achieving both widely controllable molecular selectivity and long-term solution stability with traditional PIC materials has been challenging due to the different molecular structures of polycations (poly-C) and polyanions (poly-A). To address this issue, we propose a novel polyurethane (PU)-based PIC material in which the main chains of both poly-A and poly-C are composed of PU structures. In this study, we electrochemically detect dopamine (DA) as the analyte and L-ascorbic acid (AA) and uric acid (UA) as the interferents to evaluate the selective property of our material. The results show that AA and UA are significantly eliminated, while DA can be detected with a high sensitivity and selectivity. Moreover, we successfully tune the sensitivity and selectivity by changing the poly-A and poly-C ratios and adding nonionic polyurethane. These excellent results were employed in the development of a highly selective DA biosensor with a detection range from 500 nM to 100 µM and a 3.4 µM detection limit. Overall, our novel PIC-modified electrode has the potential to advance biosensing technologies for molecular detection.

Electrochemistry in bicontinuous microemulsions derived from two immiscible electrolyte solutions for a membrane-free redox flow battery.

HYPOTHESES: Bicontinuous microemulsions (BMEs) have attracted attention as unique heterogeneous mixture for electrochemistry. An interface between two immiscible electrolyte solutions (ITIES) is an electrochemical system that straddles the interface between a saline and an organic solvent with a lipophilic electrolyte. Although most BMEs have been reported with nonpolar oils, such as toluene and fatty acids, it should be possible to construct a sponge-like three-dimensionally expanded ITIES comprising a BME phase. EXPERIMENTS: Dichloromethane (DCM)-water microemulsions stabilized by a surfactant were investigated in terms of the concentrations of co-surfactants and hydrophilic/lipophilic salts. A Winsor III microemulsion three-layer system, consisting of an upper saline phase, a middle BME phase, and a lower DCM phase, was prepared, and electrochemistry was conducted in each phase. FINDINGS: We found the conditions for ITIES-BME phases. Regardless of where the three electrodes were placed in the macroscopically heterogeneous three-layer system, electrochemistry was possible, as in a homogeneous electrolyte solution. This indicates that the anodic and cathodic reactions can be divided into two immiscible solution phases. A redox flow battery comprising a three-layer system with a BME as the middle phase was demonstrated, paving the way for applications such as electrolysis synthesis and secondary batteries.

DNA methylation analysis triggered by bulge specific immuno-recognition.

We report the sequence-selective discrimination of the cytosine methylation status in DNA with anti methylcytosine antibody for the first time. This was realized by employing an affinity measurement involving the target methylcytosine in a bulge region and anti methylcytosine antibody, following hybridization with a bulge-inducing DNA to ensure that only the target methylcytosine is located in the bulge. The affinity of the antibody for methylcytosine in the bulge was 79% of that in a single strand of DNA; however, the affinity for nontarget methylcytosine in a double strand of DNA decreased greatly. This is because the antibody cannot bind with an inwardly turned methylcytosine in the duplex region owing to the large antibody size. In contrast, the methylcytosine in the bulge is recognized by the antibody because it is available to rotate freely owing to the single bond between deoxyribose and phosphate in a DNA chain. By employing the difference between the affinity in the bulge and that in the duplex, we could determine selectively whether or not the target cytosine was methylated in an O(6)-methylguanine DNA methyltransferase (MGMT) promoter sequence with a single base level. The proposed bulge-specific assay technique can be combined with a widely used absorbance measurement method that employs the color change in tetramethyl benzidine induced by horseradish peroxidase-labeled secondary antibody. The sequence-selective discrimination of the methylation status could also be obtained with various types of interfering genomic DNA contamination without any conventional bisulfite treatment, polymerase chain reaction, (PCR) or electrophoresis.