We have developed an N-terminated carbon film electrode that allows accurate determination of the diffusion coefficient of electroactive molecules dissolved in a highly concentrated serum protein solution. The carbon film electrode was formed by the unbalanced magnetron sputtering (UBM) method. Then, nitrogen functional groups were introduced by employing NH3 or H2O plasma treatment. Cyclic voltammetry measurements with ferricyanide ion ([Fe(CN)6]3−) showed that the N-terminated carbon film electrode exhibited great anti-fouling property against simulated serum proteins (50 mg/mL human serum albumin and 15 mg/mL γ-globulin dissolved in 1 M KCl solution). In contrast, glassy carbon, H2O plasma-treated, and especially untreated carbon film electrodes were subject to severe electrode fouling, making it difficult to electrochemically determine the diffusion coefficient of the [Fe(CN)6]3− ion. The control experiment using less adsorptive ethylene glycol as a viscosity modifier showed that the increase in viscosity is a main factor of the decrease in diffusion coefficient for nitrogen plasma treated electrode, which is not significantly affected by the possible interaction between [Fe(CN)6]3− ions and serum proteins. Finally, we applied the electrode for the electrochemical analysis of acetaminophen dissolved in phosphate buffer (0.1 M, pH = 7.0), which suggests that NH3 plasma-treated carbon film exhibits the lowest ΔE increase when we compare ΔE with and without proteins and also a more stable peak current in continuous voltametric measurements compared with other carbon electrodes.

Coelenterazine is the most common imidazopyrazinone-based luciferin in marine bioluminescence organisms. The electrochemical main peaks of coelenterazine are observed at approximately around 0.12 and 0.62 Vs in the cyclic voltammetry curve. Theoretical analysis suggests that coelenterazine is converted to the coelenteramide around 0.12 V, and the coelenteramide is deprotonated at around 0.62 V. These approaches can reveal the possible excited states of coelenteramide but do not consistently enable the enzymatic bioluminescence reaction to be traced.

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.

We studied nanocarbon film electrodes with the aim of detecting tryptophan metabolites via the kynurenine pathway. The nanocarbon films were formed by using unbalanced magnetron sputtering, and they exhibited superior electrode properties including a wide potential window and a low background current as a result of the sp(3)-containing structure and ultraflat surface. These properties allowed us to detect certain tryptophan metabolites such as kynurenic acid (KYNA), which has a relatively high oxidation potential. We also investigated the effect of the sp(2)/sp(3) ratio of the nanocarbon film as regards the electrode activity in relation to target molecules. We found that the sp(2)/sp(3) ratio played important roles in both widening the potential window and obtaining superior electrode performance for the metabolites. The nanocarbon film with a high sp(3) content was beneficial as regards the electrode performance with respect to the detection limit and sensitivity. Compared with conventional carbon-based electrodes, the nanocarbon film electrode with a high sp(3) content exhibited higher electrode activity against KYNA while maintaining a low background current. Computational experiments revealed that the theoretical oxidation potential (E-ox) value for some targets coincided with that obtained in electrochemical experiments using our nanocarbon film electrode.

We evaluated the dispersion and diffusion of fluorescent-labeled lipophilic vitamin E (VE) in microemulsions (MEs) including water-in-oil (W/O) type ME, oil-in-water (O/W) type ME, and bicontinuous ME (BME), using fluorescence correlation spectroscopy (FCS). We prepared a fluorescent ATTO 488 or BODIPY group labeled VE (VE-ATTO or VE-BODIPY). VE-ATTO possesses lipophilic and hydrophilic parts, while VE-BODIPY consists solely of the lipophilic part. The VE-ATTO dissolved in heptane solution as an oil phase appeared hot pink in color due to the solvatochromism effect under room light and almost no fluorescent signal, which was unlike the VE-ATTO dissolved in ME solutions and all the VE-BODIPY solutions (typical fluorescent green color). The FCS measurement proved that VE-BODIPY diffuses faster than VE-ATTO. This is presumably because the "surfactant-like" VE-ATTO is localized and trapped at the micro-water/micro-oil interface of the MEs, while the VE-BODIPY exists in the ME phase and macro-oil phase with good dispersion. These results demonstrate that FCS is a powerful tool for the rapid evaluation of the lipophilic probe behavior in heterogeneous ME solutions.

The cover image is based on the Research Article Electrochemical Detection of Tryptophan Metabolites via Kynurenine Pathway by Using Nanocarbon Films by Dai Kato et al., DOI: 10.1002/elan.202100241, which is part of the Special Collection on Electroanalysis Based on Carbon Nanomaterials.

General synthesis of a highly oriented metallic heterodimer array based on a selective electrodeposition technique onto a metal nanoparticle-embedded carbon film is proposed, which enables the preparation of heterodimers with a wide variety of metal combinations. This method requires no surfactant, capping agent, organic solvent, or heat treatment. As a representative metal combination, a nickel (Ni)/palladium (Pd) heterodimer array was prepared by selective electrodeposition of Ni nanoparticles (Ni NPs) on top of partially exposed Pd NPs embedded in carbon film electrodes fabricated by a cosputtering technique. Such a selective electrodeposition becomes possible by utilizing the difference in electrodeposition overpotentials between carbon and Pd NP surfaces. X-ray photoelectron spectroscopy revealed a charge transfer from Ni NPs to Pd NPs, implying that the catalytic and optical properties can be expected to be controllable. The formed heterodimer array structure was mechanically stable against ultrasonication in ethanol for over 1 h because most parts of the Pd NPs were tightly embedded in the carbon film. After conversion from Ni to nickel hydroxide (Ni(OH)2), the electrode showed high electrocatalytic activity toward glucose oxidation, with a higher turnover rate and lower overpotential compared to Ni(OH)2 electrodeposited on pure carbon film electrodes.

Electrocatalytic performance of metal nanoparticles (NPs) has been studied to apply for electrochemical devices such as fuel cells and electrochemical sensors. We have developed metal NPs embedded carbon film electrodes by co-sputtering of metal and carbon [1]. Various kinds of metals including Pt, Pd, Au, Ni, Cu and their alloys can be fabricated in the carbon film by using unbalanced magnetron (UBM) sputtering and applied for detecting hydrogen peroxide, glucose [1], heavy metals [2,3] and sugar markers [4, 5]. The electrocatalytic activity of metal NPs can be modulated by changing electronegativity of substrate such as carbon electrodes. Here, we proposed Ni NPs electrodeposited on nitrogen containing carbon film electrodes. Method The carbon films were prepared by unbalanced magnetron sputtering equipment and then treated by N2 plasma. The surface of the films were characterized by XPS. Then NiNP was electrodeposited onto both plasma treated and untreated carbon films by changing the deposition potentials. The fabricated electrodes were potential cycled to sufficiently form surface Ni(OH)2 on the surface of NiNPs, then applied for measuring sugar oxidation such as glucose and oligosaccharide in alkaline solutions with different pH. Results and Discussion The nitrogen containing carbon film electrode show unique electrochemical performances including reduction of overpotentials for oxygen reduction and oxidation of some biochemicals such as NADH and L-ascorbic acid [6]. The films also show excellent biocompatibility to suppress the fouling of proteins during electrochemical measurements [7]. When we deposited NiNPs onto the nitrogen containing carbon film, the size of NiNPs became smaller by decreasing potential from -1000 to -1300 mV. Since the size of NiNPs at N2 plasma treated surface is larger than that at pure carbon film, we adjusted the potential to obtain similar NiNPs size on both N2 plasma treated and untreated carbon surfaces. After deposition, both electrodes were potential cycled between 0 and 0.70 V (vs Ag/AgCl) to form surface Ni(OH)2 , which is confirmed by HR-TEM, and HAADF-STEM-EDS images. The redox reaction peaks of Ni(OH)2 oxidation and NiOOH reduction is almost identical when the scan rate is slow (1 mV/s). However, the oxidation and reduction peaks shifted positive and negative directions, respectively at Ni(OH)2 modified untreated carbon film with increasing potential scan rate up to 100 mV/s. In contrast, peak separation increase at Ni(OH)2 modified N2 plasma treated carbon film is greatly suppressed suggesting fast redox reaction similar to at Ni(OH)2. We applied both NiNPs deposited plasma treated and untreated carbon film electrodes for electrocatalytic oxidation of glucose and maltopentaose (G5) in alkaline media. Higher electrocatalytic oxidation currents of glucose was observed at NiNPs on nitrogen containing carbon film compared with those at NiNPs on untreated carbon film particularly in higher glucose concentration region (> 1mM). Moreover, the electrocatalytic current was started to increase sharply at +0.28 V at the Ni@Ni(OH)2-NP/N-C, while that at Ni@Ni(OH)2-NP/C was started to increased gradually at +0.34 V. These results could be due to the slightly formed NiOOH at the low potential region which cannot be detected as current change.And 60 mV potential difference could be interpreted as electrostatic interaction between G5 and electrode surface. In conclusion, the electrocatalytic activity of NiNPs can be enhanced by modifying NiNPs on the N2 plasma treated carbon films, which shows enhanced current and lower onset potential for electrocatalytically oxidation of G5. References [1] T. You, O. Niwa, M. Tomita, S. Hirono,” Characterization of platinum nanoparticle- embedded carbon film electrode and its detection of hydrogen peroxide”, Anal. Chem .,75 (2003) 2080. [2] D. Kato, T. Kamata, D. Kato, H. Yanagisawa, O. Niwa, “Au nanoparticle-embeded carbon films for electrochemical As3+ detection with high sensitivity and stability”, Anal. Chem ., 88 (2016) 2944. [3] S. Shiba, S. Takahashi, T. Kamata, H. Hachiya, D. Kato, O. Niwa, “Selective Au Electrodeposition on Au Nanoparticles Embedded in Carbon Film Electrode for Se(IV) Detection” Sensors and Materials ., 31 (2019) 1135. [4] S. Shiba, D. Kato, T. Kamata, O. Niwa,” Co-sputter deposited Nickel-Copper bimetallic nanoalloy embedded carbon films for electrocatalytic biomarker detection”, Nanoscale , 8 (2016) 12887. [5] S. Shiba, R. Maruyama, T. Kamata, D. Kato, O. Niwa, “Chromatographic determination of sugar probes used for gastrointestinal permeability test by employing nickel-copper nanoalloy embedded in carbon film electrodes”, Electroanalysis , 30 (2018) 1407. [6] T. Kamata, D. Kato, O. Niwa,”Electrochemical performance at sputter-deposited nanocarbon film with different surface nitrogencontaining groups”, Nanoscale, 11 (2019) 10239. [7] S. Ohta, S.Shiba, T. Yajima, T. Kamata, D. Kata, O. Niwa, “Gas-phase Treatment Methods for Chemical Termination of Sputtered Nanocarbon Film Electrodes to Suppress Surface Fouling by Proteins”, J. Photopolym. Sci. Tech., 32 (2019) 523

Nanocarbon films fabricated by using unbalanced magnetron (UBM) or electron cyclotron resonance (ECR) sputtering show a wide potential window and high stability. We fabricated nitrogen-doped nanocarbon film electrodes by using sputtering in the presence of N-2 gas, a heat treatment in the presence of ammonia (NH3), and a plasma treatment using NH3 vapor. The nitrogen concentration, sp(2)/sp(3) ratio and the structure of nitrogen-containing surface functional groups of the carbon films were dependent on the fabrication methods and plasma treatment conditions, such as the nitrogen or ammonia gas concentrations, which caused a positive shift of the oxygen reduction reaction (ORR) potential. For detecting biomolecules, the oxidation peaks of nicotinamide adenine dinucleotide (NADH) and L-ascorbic acid shifted to more negative potentials. Furthermore, the UBM carbon film showed improved biocompatibility after a NH3 plasma treatment since the Delta E of ferrocyanide ([Fe(CN)(6)](3-)) showed excellent redox properties with very small Delta E increase, even in a solution containing a large concentration of bovine serum albumin (BSA). The magnitude of the current for serotonin oxidation (neurotransmitter) was almost unchanged by continuous CV measurements, although the oxidation product of serotonin is adsorptive to the electrode surface. The UBM sputtered nanocarbon film introduced nitrogen-containing functional groups onto the surface, which suggests an anti-fouling property by the adsorption of serotonin oxidized products and proteins.

We developed carbon film-based electrodes by using an unbalanced magnetron (UBM) and an electron cyclotron resonance (ECR) sputtering equipment, and applied for the electroanalysis of four kinds of heavy metal ions: cadmium (Cd2+), lead (Pb2+), arsenic (As2+) and selenium (se). Au nanoparticle embedded carbon (AuNP-C) film was formed by UBM co-sputtering. Regarding the detection of cadmium (Cd2+) and lead (Pb2+) ions, anodic stripping voltammetry measurement was performed with our carbon thin film electrode. The detection limits of Cd2+ and Pb2+ were 0.25 ppb and 1.0 ppb on our electrode. These values were better than those on the conventional GC electrode, also sufficient for the environmental analysis. On the other hand, the AuNP-C electrode was used for the detection of arsenic ions (As3+), which cannot be preconcentrated on the pure carbon electrode. A detection limit of 0.55 ppb was achieved, which is a sufficient performance for the environmental analysis. Reproducible results could be obtained for an As measurement after Au was further electrodeposited on AuNPs because AuNPs are embedded in the carbon film, which prevented the AuNPs detaching from the electrode surface. In the case of selenium ion (se) detection, an electrode in which Au was electrodeposited only on AuNPs at AuNP-C (Au/AuNP-C) electrodes was used because of the slow deposition rate of Se4+. Improved sensitivity and low detection limit were achieved compared with the electrode before the electrodeposition.

We studied the diffusion properties of lipophilic vitamin E (VE) through bicontinuous microemulsions (BME) using both electrochemical and fluorescence correlation spectroscopy (FCS) measurements. We investigated the effect of different composition ratios of micro-water and micro-oil phases in BMEs (W/OBME). When we employed the BME with a lower W/OBME value of 40/60 (oil-rich BME) as an electrolyte solution, we obtained a larger current response from VE at a fluorinated nanocarbon film electrode. Further voltammetric studies revealed that a higher VE diffusion coefficient was observed in the oil-rich BME. The FCS results also exhibited faster diffusion through the oil-rich BME, which played a significant role in accelerating the VE diffusion probably due to the widening of the micro-oil phase pathway in the BME. Moreover, the effect of increasing the VE diffusion was pronounced at the interface between the electrode surface and the BME solution. These results indicate that controlling the conditions of the BME as the measurement electrolyte is very effective for achieving superior electrochemical measurements in a BME.

We developed and used the nanocarbon film electrode to successfully detect epigenetic mark 5-hydro-xymethylcytosine (5hmC) through direct oxidation after digesting genomic DNA (gDNA) with nuclease P1. The film showed superior key film properties including a very chemically stable structure and an ultraflat surface (R-a = 0.21 nm) that resulted in better electrode properties such as a wider potential window and lower background current than the widely used carbon electrode. Computational experiments revealed that the theoretical oxidation potential (E-ox) value for 5hmC coincided with that obtained in electrochemical experiments using our nanocarbon film electrode. We successfully detected 5hmC by using this film electrode in combination with HPLC. Due to the difference of each oxidation potential, we achieved the oxidation potential-dependent selective detection of 5hmC in gDNA from excess amounts of unmethylated cytosine with a similar polarity to 5hmC.

Carbon materials have been widely used for electrochemical analysis and include carbon nanotubes, graphene, and boron-doped diamond electrodes in addition to conventional carbon electrodes, such as those made of glassy carbon and graphite. Of the carbon-based electrodes, carbon film has advantages because it can be fabricated reproducibly and micro- or nanofabricated into electrodes with a wide range of shapes and sizes. Here, we report two categories of hybrid-type carbon film electrodes for mainly electroanalytical applications. The first category consists of carbon films doped or surface terminated with other atoms such as nitrogen, oxygen and fluorine, which can control surface hydrophilicity and lipophilicity or electrocatalytic performance, and are used to detect various electroactive biochemicals. The second category comprises metal nanoparticles embedded in carbon film electrodes fabricated by co-sputtering, which exhibits high electrocatalytic activity for environmental and biological samples including toxic heavy metal ions and clinical sugar markers, which are difficult to detect at pure carbon-based electrodes.

Identification of biomolecules expressed on a cell surface is important, because their expressions usually imply condition of the cell, and fluorescence-based labeling and detection is one popular method for the identification of such molecules. In the method, specific antibodies are conjugated with fluorescent dyes, target molecules are reacted with the fluorescent antibodies, and expressions of the target molecules are identified with fluorescence observation. This method is target-specific and highly sensitive, however, detection sensitivity of the method is sometimes still insufficient. For example, one problem for the identification of circulating tumor cells (CTCs) is false-negative results caused on the missing of cancer cells on which target marker molecules are few expressed. Using of electrochemiluminescence (ECL)-based detection is one useful way to improve the sensitivity [1], however, ECL can be only irradiated from very close position of the electrode, and detection of cell surface molecules by ECL-based method is difficult because of large size of cells. We propose a new method to detect cell surface biomolecules with ECL-based method by using cup-shaped microelectrodes. The hemispherical electrodes were composed of thin double layer [2], and in detail, the inner concave was low noise nano-carbon layer [3] and the outer was nickel. Diameter of the cup was almost the same as that of general animal cells [4], therefore, large area of the cell can be closely approached to the electrode surface with capture of the cell to inner concave of the cup. Epithelial cell adhesion molecule (EpCAM), which is a famous marker molecule for the identification of CTCs, was used for a model target in this study, and a human breast cancer cell line on which EpCAM was low expressed (MDA-MB-231) was used for the evaluation of EpCAM detection by ECL-based measurement. ECL probe (ruthenium(II) tris(bipyridine))-conjugated anti-EpCAM antibody was reacted with the cancer cells, the labeled cells were captured to inner concave of the cup-shaped microelectrodes, and ECL observation was performed. When electric voltage was applied, ECL was observed from cups in which cancer cells were captured. These results indicate that ECL-based detection of cell surface molecules can be achieved by using cup-shaped microelectrodes with quite high detection sensitivity. References [1] M. Liang et al., Assay Drug Dev. Technol., 5, 655 (2007) [2] H. Kim et al., Jpn. J. Appl. Phys., 50, 06GJ03 (2011) [3] O. Niwa et al., J. Am. Chem. Soc., 128, 7144 (2006) [4] H. Kim et al., Sci. Rep., 4, 6362 (2014)

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.

We investigated sputtered nanocarbon films with respect to the effect of suppressing surface oxygen on their electrochemical properties. The nanocarbon film consisted of nanocrystallites with mixed sp^2 and sp^3 bonds formed by unbalanced magnetron sputtering. Ultraviolet/ozone (UV/O^3) irradiation and electrochemical pretreatment (ECP) were conducted to change the surface oxygen concentration of nanocarbon film. X-ray photoelectron spectroscopy (XPS) measurements revealed that nanocarbon films with different amounts of surface oxygen could be prepared. In addition, we observed no significant increase of the surface roughness ( R _a) at the angstrom level after treatments, owing to a stable structure containing 40% of sp^3 bonds. The electrode characteristics, including the potential window and electrochemical properties for some redox species, such as Ru(NH_3)_6^3+/2+, were investigated. Some electrochemical measurements of zinc ions (Zn^2+) and hydrogen peroxide (H_2O_2) showed that the electrochemical reaction was improved by suppressing the surface oxygen. These results clearly indicated that the low surface oxygen concentration plays an important role in these electrochemical reactions.

Electrodes that suppress protein adsorption are particularly important for the development of electrochemical biosensors and electroanalysis of biological samples. We studied the electrochemical performances of carbon film electrodes before and after water vapor (H 2 O) and ammonia gas (NH 3 ) plasma treatments. The H 2 O plasma treatment substantially increased the surface oxygen concentration and decreased the contact angle. The NH 3 plasma treatment increased the surface nitrogen content to about 5 at %, but a similar amount of oxygen remained on the surface. The sp 2 bond amounts decreased and the sp 3 bond amounts increased after the H 2 O plasma treatment, whereas both amounts changed little after the NH 3 plasma treatment. Cyclic voltammetry with the plasma-treated electrodes showed an increase in the peak separation (Δ E ) of less than 20 mV for 1 mM Fe(CN) 63-/4- containing 100 mg/mL bovine serum albumin (BSA), whereas Δ E of the untreated carbon film increased by about 600 mV. Thus, both plasma-treated electrodes strongly suppressed the protein adsorption. The NH 3 plasma-treated film showed the highest electrochemical activity and lowest redox peak separation with and without BSA despite its higher contact angle value than of the H 2 O plasma-treated film. For both plasma-treated films, Δ E did not depend on BSA concentration.

Carbon materials containing nitrogen have been extensively studied as electrode materials for use in non-platinum cathodes of fuel cells due to their high electrocatalytic activity for oxygen reduction. The activity is strongly dependent on the structure of surface nitrogen-containing functional groups. Carbon film containing nitrogen is also suitable for analytical applications because of its low background noise and its electrocatalytic activity, which is superior to that of pure carbon film. Here, we fabricated sputter-deposited nanocarbon film with a nitrogen-containing group and estimated the efficacy of a surface nitrogen-containing group for detecting biomolecules. Two types of carbon films, one rich in graphite-like nitrogen-containing bonds and the other rich in pyridine-like bonds, were successfully fabricated without changing their nitrogen concentration, sp2/sp3 ratio or surface flatness. The carbon film rich in pyridine-like bonds shows a positive oxygen reduction peak of about 250 mV compared with pure carbon film and is also 200 mV more positive compared with film with graphite-like nitrogen-containing bonds. This indicates that pyridine-like bonds contribute more effectively to electrocatalytic activity than graphite-like nitrogen-containing bonds. For detecting biomolecules, carbon film rich in pyridine-like bonds also exhibits more negative peak potentials for the oxidation of NADH and l-ascorbic acid, suggesting that carbon film rich in pyridine-like bonds will show improved performance for detecting electroactive biomolecules.

We report the ppb level detection of selenium by anodic stripping voltammetry (ASV) using finely dispersed gold nanoparticles (grown Au NPs) electrodeposited on sputter-deposited Au NPs embedded in carbon film electrodes. The Au NP-embedded carbon (AuNP-C) films were fabricated by the unbalanced magnetron (UBM) cosputtering of Au and carbon targets. The difference in overpotential between gold and carbon surfaces allows the selective deposition of gold ions only on the exposed Au parts of a AuNP-C film. As a result, we could successfully realize the highly dense deposition of Au NPs at the electrode surface without forming a Au film. By ASV with the developed electrode, we successfully increased the stripping current of selenium while maintaining the stable response derived from selectively grown Au NP structures.