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This report provides a review of stem cells/progenitor cells and their responses to ionising radiation in relation to issues relevant to stochastic effects of radiation that form a major part of the International Commission on Radiological Protection's system of radiological protection. Current information on stem cell characteristics, maintenance and renewal, evolution with age, location in stem cell 'niches', and radiosensitivity to acute and protracted exposures is presented in a series of substantial reviews as annexes concerning haematopoietic tissue, mammary gland, thyroid, digestive tract, lung, skin, and bone. This foundation of knowledge of stem cells is used in the main text of the report to provide a biological insight into issues such as the linear-no-threshold (LNT) model, cancer risk among tissues, dose-rate effects, and changes in the risk of radiation carcinogenesis by age at exposure and attained age. Knowledge of the biology and associated radiation biology of stem cells and progenitor cells is more developed in tissues that renew fairly rapidly, such as haematopoietic tissue, intestinal mucosa, and epidermis, although all the tissues considered here possess stem cell populations. Important features of stem cell maintenance, renewal, and response are the microenvironmental signals operating in the niche residence, for which a well-defined spatial location has been identified in some tissues. The identity of the target cell for carcinogenesis continues to point to the more primitive stem cell population that is mostly quiescent, and hence able to accumulate the protracted sequence of mutations necessary to result in malignancy. In addition, there is some potential for daughter progenitor cells to be target cells in particular cases, such as in haematopoietic tissue and in skin. Several biological processes could contribute to protecting stem cells from mutation accumulation: (a) accurate DNA repair; (b) rapidly induced death of injured stem cells; (c) retention of the DNA parental template strand during divisions in some tissue systems, so that mutations are passed to the daughter differentiating cells and not retained in the parental cell; and (d) stem cell competition, whereby undamaged stem cells outcompete damaged stem cells for residence in the niche. DNA repair mainly occurs within a few days of irradiation, while stem cell competition requires weeks or many months depending on the tissue type. The aforementioned processes may contribute to the differences in carcinogenic radiation risk values between tissues, and may help to explain why a rapidly replicating tissue such as small intestine is less prone to such risk. The processes also provide a mechanistic insight relevant to the LNT model, and the relative and absolute risk models. The radiobiological knowledge also provides a scientific insight into discussions of the dose and dose-rate effectiveness factor currently used in radiological protection guidelines. In addition, the biological information contributes potential reasons for the age-dependent sensitivity to radiation carcinogenesis, including the effects of in-utero exposure.

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.

Currently available methods for stem cell evaluation require both prior knowledge of specific markers and invasive cell lysis or staining, hampering the development of stem cell products with assured safety and quality. Here, we present a strategy using optical cross-reactive sensor arrays for markerless and noninvasive identification of differentiated stem cell lineages with common laboratory equipment. The sensor array consists of a library of polyion complexes (PICs) between anionic enzymes and synthetic poly(ethylene glycol)-modified polyamines, which can recognize "secretomic signatures" in cell culture supernatants. Due to the reversible nature of PIC formation, the incubation of diluted culture supernatants with PICs caused enzyme release through competitive interactions between the secreted molecules and the PICs, generating unique patterns of recovery in enzyme activity for individual cell types or lineages. Linear discriminant analysis of the patterns allowed not only normal/cancer cell discrimination but also lineage identification of osteogenic and adipogenic differentiation of human mesenchymal stem cells, therefore providing an effective way to characterize cultured cells in the fields of regenerative medicine, tissue engineering and cell biology.

We report the sequence-selective immunochemical discrimination of methylcytosine from genomic DNA that we achieved by utilizing selective antibody binding to a looped-out methylcytosine in a bulge region and without using bisulfite treatment, a methylation-sensitive restriction enzyme, or PCR. First, we investigated the affinity of an anti-methylcytosine antibody for methylcytosine in full match, bulge, mismatch and abasic sites using surface plasmon resonance measurement and a microtiter plate to explore the differences as regards antibody binding to the target methylcytosine. The highest affinity, which was comparable to that in a single strand of DNA, was observed in the bulge region. In particular, no affinity was observed in a full match site. This is because there is no interaction such as hydrogen bond or π-π stacking for the bulged methylcytosine, thus enabling only the target in the bulge to be looped out. Methylated and unmethylated genomic DNA were blended to form a model DNA with which to assess the methylation ratio at a specific site. Fragmented DNA was hybridized with a biotinylated probe DNA, which has a sequence capable of forming a single base bulge at the target. The probe design is simple because it consists solely of the elimination of guanine paired with the target cytosine from a full match sequence. As a result, we successfully obtained a linear relationship (r(2)=0.9962) between the immunoassay signal and the methylation ratio of a specific site within 4 h., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Past nuclear disasters, such as the atomic bombings in 1945 and major accidents at nuclear power plants, have highlighted similarities in potential public health effects of radiation in both circumstances, including health issues unrelated to radiation exposure. Although the rarity of nuclear disasters limits opportunities to undertake rigorous research of evidence-based interventions and strategies, identification of lessons learned and development of an effective plan to protect the public, minimise negative effects, and protect emergency workers from exposure to high-dose radiation is important. Additionally, research is needed to help decision makers to avoid premature deaths among patients already in hospitals and other vulnerable groups during evacuation. Since nuclear disasters can affect hundreds of thousands of people, a substantial number of people are at risk of physical and mental harm in each disaster. During the recovery period after a nuclear disaster, physicians might need to screen for psychological burdens and provide general physical and mental health care for many affected residents who might experience long-term displacement. Reliable communication of personalised risks has emerged as a challenge for health-care professionals beyond the need to explain radiation protection. To overcome difficulties of risk communication and provide decision aids to protect workers, vulnerable people, and residents after a nuclear disaster, physicians should receive training in nuclear disaster response. This training should include evidence-based interventions, support decisions to balance potential harms and benefits, and take account of scientific uncertainty in provision of community health care. An open and joint learning process is essential to prepare for, and minimise the effects of, future nuclear disasters., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Late-onset effects of exposure to ionising radiation on the human body have been identified by long-term, large-scale epidemiological studies. The cohort study of Japanese survivors of the atomic bombings of Hiroshima and Nagasaki (the Life Span Study) is thought to be the most reliable source of information about these health effects because of the size of the cohort, the exposure of a general population of both sexes and all ages, and the wide range of individually assessed doses. For this reason, the Life Span Study has become fundamental to risk assessment in the radiation protection system of the International Commission on Radiological Protection and other authorities. Radiation exposure increases the risk of cancer throughout life, so continued follow-up of survivors is essential. Overall, survivors have a clear radiation-related excess risk of cancer, and people exposed as children have a higher risk of radiation-induced cancer than those exposed at older ages. At high doses, and possibly at low doses, radiation might increase the risk of cardiovascular disease and some other non-cancer diseases. Hereditary effects in the children of atomic bomb survivors have not been detected. The dose-response relation for cancer at low doses is assumed, for purposes of radiological protection, to be linear without a threshold, but has not been shown definitively. This outstanding issue is not only a problem when dealing appropriately with potential health effects of nuclear accidents, such as at Fukushima and Chernobyl, but is of growing concern in occupational and medical exposure. Therefore, the appropriate dose-response relation for effects of low doses of radiation needs to be established., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The RERF International Low-Dose Symposium was held on 5-6 December 2013 at the RERF campus in Hiroshima, Japan, to discuss the issues facing the Life Span Study (LSS) and other low-dose studies. Topics included the current status of low-dose risk detection, strategies for low-dose epidemiological and statistical research, methods to improve communication between epidemiologists and biologists, and the current status of radiological studies and tools. Key points made by the participants included the necessity of pooling materials over multiple studies to gain greater insight where data from single studies are insufficient; generating models that reflect epidemiological, statistical, and biological principles simultaneously; understanding confounders and effect modifiers in the current data; and taking into consideration less studied factors such as the impact of dose rate. It is the hope of all participants that this symposium be used as a trigger for further studies, especially those using pooled data, in order to reach a greater understanding of the health effects of low-dose radiation.

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.

In this study, we developed a micropyramid array electrode that facilitates contact between a nitrocellulose membrane and electrode, which is important to realize a quantitative and sensitive electrochemical detection system for immunochromatography. We evaluated the micropyamid array electrode with our newly developed detection system that can measure contact forces between a membrane and electrode, and also investigated the relationship between redox current and contact forces. By using normal pulse voltammetry, we observed higher reduction currents over a flat surface electrode at lower contact forces with the micropyramid array.

We herein report on the development of an extended-gate type organic field-effect transistor (OFET)-based immunosensor for the detection of human immunoglobulin A (IgA). The titration results of IgA exhibited shifts in the transfer characteristics of the OFET sensor device with increasing IgA concentration. A linear detection range from 0 to 10 μg/mL was realized with a detection limit of 2.1 μg/mL, indicating that the OFET-based immunosensor can be potentially applied to the monitoring of infectious diseases and psychological stress in daily life.

The effect of the sp(2)/sp(3) ratio in an unbalanced magnetron sputtered nanocarbon film electrode was studied for determining Cd(2+) and Pb(2+) by anodic stripping voltammetry (ASV). The signal-to-noise ratio in the ASV measurement improved as the sp(3) concentration in the carbon film increased because the noise current decreased with the increasing sp(3) concentration. The detection limits with a carbon film containing 50% sp(3) were 0.25 and 1.0 μg L(-1) for Cd(2+) and Pb(2+) with high repeatability (Cd: 4.6% and Pb: 6.4%, n = 3). For a real sample measurement, a pretreatment system combining a photooxidation reactor and a cation exchange column was used to eliminate the interference from EDTA and Cu(2+), which forms a stable complex or alloy with Cd(2+) and Pb(2+). More than 99% of the interference was eliminated, and accurate signal currents for Cd(2+) and Pb(2+) were successfully obtained with the pretreatment system.

We studied a nitrogen-doped nanocarbon film electrode with a nitrogen concentration of lower than 10.9 at% formed by the unbalanced magnetron (UBM) sputtering method. The sp(3) content in the nitrogen-doped UBM sputtering nanocarbon film (N-UBM film) slightly increases with increasing nitrogen concentration. The nitrogen-containing graphite-like bonding decreases and pyridine-like bonding increases with increasing nitrogen concentration. The N-UBM film has a very smooth surface with an average roughness of 0.1 to 0.3 nm, which is almost independent of nitrogen concentration. The N-UBM film electrode shows a wider potential window (4.1 V) than a pure-UBM film electrode (3.9 V) due to its slight increase in the sp(3) content. The electrocatalytic activity increased with increasing nitrogen concentration, suggesting that the electroactivity is maximum when the nitrogen concentration is around 10.9 at%, which is confirmed by the peak separation of Fe(CN)6(4-). The hydrogen peroxide (H2O2) reduction potentials at the N-UBM film electrode shifted about 0.1 V, and the peak current of H2O2 increased about 4 times.

Germ cells are thought to exhibit a unique DNA damage response that differs from that of somatic stem cells, and previous studies suggested that Trp53 is not involved in the survival of spermatogonial stem cells (SSCs) after irradiation. Here, we report a critical role for the Trp53-Trp53inp1-Tnfrsf10b pathway during radiation-induced SSC apoptosis. Spermatogonial transplantation revealed that Trp53 deficiency increased the survival of SSCs after irradiation. Although Bbc3, a member of the intrinsic apoptotic pathway, was implicated in apoptosis of germ and somatic stem cells, Bbc3 depletion inhibited apoptosis in committed spermatogonia, but not in SSCs. In contrast, inhibition of Tnfrsf10b, an extrinsic apoptosis regulator, rescued SSCs. Tnfrsf10b, whose deficiency protected SSCs, was upregulated by Trp53inp1 upon irradiation. These results suggest that the Trp53-Trp53inp1-Tnfrsf10b pathway responds to genotoxic damage in SSCs and that stem and progenitor cells exhibit distinct DNA damage responses in self-renewing tissue., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

A novel biosensor for immunoglobulin G (IgG) detection based on an extended-gate type organic field effect transistor (OFET) has been developed that possesses an anti-IgG antibody on its extended-gate electrode and can be operated below 3 V. The titration results from the target IgG in the presence of a bovine serum albumin interferent, clearly exhibiting a negative shift in the OFET transfer curve with increasing IgG concentration. This is presumed to be due an interaction between target IgG and the immobilized anti-IgG antibody on the extended-gate electrode. As a result, a linear range from 0 to 10 µg/mL was achieved with a relatively low detection limit of 0.62 µg/mL (=4 nM). We believe that these results open up opportunities for applying extended-gate-type OFETs to immunosensing.

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.

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.

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.

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.

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.

We report the electrochemical determination of mite allergen in real house dust by using a thin layer electrochemical flow cell and magnetic beads. Der p1, which is an allergen from Dermatophagoides pteronyssinus, was immunochemically sandwiched between two dispersed monoclonal antibodies; one was modified on the surface of magnetic beads and the other was modified with alkaline phosphatase. After washing the beads, a small volume of p-aminophenol phosphate (p-APP) was added to produce p-aminophenol (p-AP). And then the p-AP concentration was measured electrochemically with a homemade electrochemical cell. The Der p1 assay was completed within 30 min and a low detection limit of 0.3 ng/mL was achieved. This is because the diffusion distance of Der p1 and the detection antibody was reduced to 22.3 μm by using dispersed magnetic beads. Only 10 min was required to complete the entire immunoreaction, and 54% of the Der p1 was confirmed to have immunoreacted in only 1 min of mixing. Furthermore, the p-APP volume could be reduced using the thin-layer electrochemical flow cell. This is advantageous in terms of concentrating p-AP, and provides a high signal-to-noise ratio measurement in a short time. We achieved a high correlation (r=0.967, p<0.001) between our assay and a conventional enzyme-linked immunosorbent assay (ELISA) for real house dust measurements., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Following the Fukushima accident, the International Commission on Radiological Protection (ICRP) convened a task group to compile lessons learned from the nuclear reactor accident at the Fukushima Daiichi nuclear power plant in Japan, with respect to the ICRP system of radiological protection. In this memorandum the members of the task group express their personal views on issues arising during and after the accident, without explicit endorsement of or approval by the ICRP. While the affected people were largely protected against radiation exposure and no one incurred a lethal dose of radiation (or a dose sufficiently large to cause radiation sickness), many radiological protection questions were raised. The following issues were identified: inferring radiation risks (and the misunderstanding of nominal risk coefficients); attributing radiation effects from low dose exposures; quantifying radiation exposure; assessing the importance of internal exposures; managing emergency crises; protecting rescuers and volunteers; responding with medical aid; justifying necessary but disruptive protective actions; transiting from an emergency to an existing situation; rehabilitating evacuated areas; restricting individual doses of members of the public; caring for infants and children; categorising public exposures due to an accident; considering pregnant women and their foetuses and embryos; monitoring public protection; dealing with 'contamination' of territories, rubble and residues and consumer products; recognising the importance of psychological consequences; and fostering the sharing of information. Relevant ICRP Recommendations were scrutinised, lessons were collected and suggestions were compiled. It was concluded that the radiological protection community has an ethical duty to learn from the lessons of Fukushima and resolve any identified challenges. Before another large accident occurs, it should be ensured that inter alia: radiation risk coefficients of potential health effects are properly interpreted; the limitations of epidemiological studies for attributing radiation effects following low exposures are understood; any confusion on protection quantities and units is resolved; the potential hazard from the intake of radionuclides into the body is elucidated; rescuers and volunteers are protected with an ad hoc system; clear recommendations on crisis management and medical care and on recovery and rehabilitation are available; recommendations on public protection levels (including infant, children and pregnant women and their expected offspring) and associated issues are consistent and understandable; updated recommendations on public monitoring policy are available; acceptable (or tolerable) 'contamination' levels are clearly stated and defined; strategies for mitigating the serious psychological consequences arising from radiological accidents are sought; and, last but not least, failures in fostering information sharing on radiological protection policy after an accident need to be addressed with recommendations to minimise such lapses in communication.

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.

Background and Purpose: Anticonvulsants have been developed according to the traditional neurotransmission imbalance hypothesis. However, the anticonvulsive pharmacotherapy currently available remains unsatisfactory. To develop new antiepileptic drugs with novel antiepileptic mechanisms, we have tested the antiepileptic actions of ONO-2506, a glial modulating agent, and its effects on tripartite synaptic transmission., Experimental Approach: Dose-dependent effects of ONO-2506 on maximal-electroshock seizure (MES), pentylenetetrazol-induced seizure (PTZ) and epileptic discharge were determined in a genetic model of absence epilepsy in mice (Cacna1a(tm2Nobs/tm2Nobs) strain). Antiepileptic mechanisms of ONO-2506 were analysed by examining the interaction between ONO-2506 and transmission-modulating toxins (tetanus toxin, fluorocitrate, tetrodotoxin) on release of l-glutamate, d-serine, GABA and kynurenic acid in the medial-prefrontal cortex (mPFC) of freely moving rats using microdialysis and primary cultured rat astrocytes., Key Results: ONO-2506 inhibited spontaneous epileptic discharges in Cacna1a(tm2Nobs/tm2Nobs) mice without affecting MES or PTZ. Given systemically, ONO-2506 increased basal release of GABA and kynurenic acid in the mPFC through activation of both neuronal and glial exocytosis, but inhibited depolarization-induced releases of all transmitters. ONO-2506 increased basal glial release of kynurenic acid without affecting those of l-glutamate, d-serine or GABA. However, ONO-2506 inhibited AMPA-induced releases of l-glutamate, d-serine, GABA and kynurenic acid., Conclusions and Implications: ONO-2506 did not affect traditional convulsive tests but markedly inhibited epileptic phenomena in the genetic epilepsy mouse model. ONO-2506 enhanced release of inhibitory neuro- and gliotransmitters during the resting stage and inhibited tripartite transmission during the hyperactive stage. The results suggest that ONO-2506 is a novel potential glial-targeting antiepileptic drug., Linked Article: This article is commented on by Onat, pp. 1086-1087 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12050., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

During meiosis, telomeres cluster and promote homologous chromosome pairing. Telomere clustering requires the interaction of telomeres with the nuclear membrane proteins SUN (Sad1/UNC-84) and KASH (Klarsicht/ANC-1/Syne homology). The mechanism by which telomeres gather remains elusive. In this paper, we show that telomere clustering in fission yeast depends on microtubules and the microtubule motors, cytoplasmic dynein, and kinesins. Furthermore, the γ-tubulin complex (γ-TuC) is recruited to SUN- and KASH-localized telomeres to form a novel microtubule-organizing center that we termed the "telocentrosome." Telocentrosome formation depends on the γ-TuC regulator Mto1 and on the KASH protein Kms1, and depletion of either Mto1 or Kms1 caused severe telomere clustering defects. In addition, the dynein light chain (DLC) contributes to telocentrosome formation, and simultaneous depletion of DLC and dynein also caused severe clustering defects. Thus, the telocentrosome is essential for telomere clustering. We propose that telomere-localized SUN and KASH induce telocentrosome formation and that subsequent microtubule motor-dependent aggregation of telocentrosomes via the telocentrosome-nucleated microtubules causes telomere clustering.

We studied the direct electron transfer (DET) of proteins on a carbon nanofiber (CNF) modified carbon film electrode by employing the one-step UV/ozone treatment of CNF. This treatment changed the CNF surface from hydrophobic to hydrophilic because a sufficient quantity of oxygen functional groups was introduced onto the CNF surface. Furthermore, this simple approach increased both the effective surface area and the number of edge-plane defect sites. As a result, the reversibility of redox species, such as ferrocyanide and dopamine, was greatly improved on the treated electrode surface. We obtained on efficient DET of bilirubin oxidase (BOD) and cytochrome c (cyt c) at the treated CNF electrode, which exhibited 38 (for BOD) and 6 (for cyt c) times higher than that at untreated CNF modified electrode. These results indicate that the combination of nanostructured carbon and this UV/ozone treatment process can efficiently create a functionalized surface for the electron transfer of proteins.

This review addresses recent studies of newly developed carbon-based electrode materials and their use for DNA electroanalysis. Recently, new carbon materials including carbon nanotubes (CNT), graphene and diamond-based nanocarbon electrodes have been actively developed as sensing platforms for biomolecules, such as DNA and proteins. Electrochemical techniques using these new material-based electrodes can provide very simple and inexpensive sensing platforms, and so are expected to be used as one of the "post-light" DNA analysis methods, which include coulometric detection, amperometric detection with electroactive tags or intercalators, and potentiometric detection. DNA electroanalysis using these new carbon materials is summarized in view of recent advances on electrodes.

The electrokinetic parameters of all the DNA bases were evaluated using a sputter-deposited nanocarbon film electrode. It is very difficult to evaluate the electrokinetic parameters of DNA bases with conventional electrodes, and particularly those of pyrimidine bases, owing to their high oxidation potentials. Nanocarbon film formed by employing an electron cyclotron resonance sputtering method consists of a nanocrystalline sp(2) and sp(3) mixed bond structure that exhibits a sufficient potential window, very low adsorption of DNA molecules, and sufficient electrochemical activity to oxidize all DNA bases. A precise evaluation of rate constants (k) between all the bases and the electrodes is achieved for the first time by obtaining rotating disc electrode measurements with our nanocarbon film electrode. We found that the k value of each DNA base was dominantly dependent on the surface oxygen-containing group of the nanocarbon film electrode, which was controlled by electrochemical pretreatment. In fact, the treated electrode exhibited optimum k values for all the mononucleotides, namely, 2.0 × 10(-2), 2.5 × 10(-1), 2.6 × 10(-3), and 5.6 × 10(-3) cm s(-1) for GMP, AMP, TMP, and CMP, respectively. The k value of AMP was sufficiently enhanced by up to 33 times with electrochemical pretreatment. We also found the k values for pyrimidine bases to be much lower than those of purine bases although there was no large difference between their diffusion coefficient constants. Moreover, the theoretical oxidation potential values for all the bases coincided with those obtained in electrochemical experiments using our nanocarbon film electrode.

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.

Microarrays of RNA aptamers are fabricated in a one-step, multiplexed enzymatic synthesis on gold thin films in a microfluidic format and then employed in the detection of protein biomarkers with surface plasmon resonance imaging (SPRI) measurements. Single-stranded RNA (ssRNA) oligonucleotides are transcribed on-chip from double-stranded DNA (dsDNA) templates attached to microarray elements (denoted as generator elements) by the surface transcription reaction of T7 RNA polymerase. As they are synthesized, the ssRNA oligonucleotides diffuse in the microfluidic channel and are quickly captured by hybridization adsorption onto adjacent single-stranded DNA (ssDNA) microarray elements (denoted as detector elements) that contain a sequence complementary to 5'-end of the ssRNA. The RNA aptamers attached to these detector elements are subsequently used in SPRI measurements for the bioaffinity detection of protein biomarkers. The microfluidic generator-detector element format permits the simultaneous fabrication of multiple ssRNA oligonucleotides with different capture sequences that can hybridize simultaneously to distinct detector elements and thus create a multiplexed aptamer microarray. In an initial set of demonstration experiments, SPRI measurements are used to monitor the bioaffinity adsorption of human thrombin (hTh) and vascular endothelial growth factor (VEGF) proteins onto RNA aptamer microarrays fabricated in situ with this on-chip RNA polymerase synthesis methodology. Additional SPRI measurements of the hydrolysis and desorption of the surface-bound ssRNA aptamers with a surface RNase H are used to verify the capture of ssRNA with RNA-DNA surface hybridization onto the detector elements. The on-chip RNA synthesis described here is an elegant, one-step multiplexed methodology for the rapid and contamination-free fabrication of RNA aptamer microarrays for protein biosensing with SPRI.

In this paper, we describe our development of an electrochemical surface plasmon resonance (EC-SPR) measurement device based on a bottom-filled gold nanohole array. The polymer based gold nanohole array was fabricated with a UV nanoimprint technique and electron beam gold deposition. Direct reflection mode measurement was used to monitor the SPR dip in the reflection spectra. A cyclic voltammogram was also operated by using the standard three electrodes containing working electrode having a gold nanohole array and counter and reference electrodes. The gold nanohole array was modified with an osmium-poly(vinylpyridine)-wired horseradish peroxidase (Os-gel-HRP) film, and its redox state induced by the change in potential was monitored simultaneously. The redox state of the local film was obtained simply by scanning the sample substrate stage. The substrate modified with Os-gel-HRP film was incorporated in a microfluidic chip, and then the hydrogen peroxide was determined in terms of the redox change in the Os complex mediator from the slope of the SPR dip shift. The linear relation of hydrogen peroxide from 10 to 250 μM was successfully monitored, and a high conversion efficiency was realized.

Cytosine methylation in DNA was determined by an enzyme linked immunosorbent assay (ELISA) with electrochemiluminescence (ECL) detection and employed for the DNA methylation assay of a long and real genomic sample for the first time. The developed method employed an antimethyl cytosine antibody labeled with acetylcholinesterase, which was added to recognize single methylated cytosine in a DNA oligomer. The acetylcholinesterase converted acetylthiocholine (substrate) to thiocholine (product), which was accumulated on a gold electrode surface via gold-thiol binding. This surface accumulated preconcentration made it possible to observe bright and distinctive ECL by applying a potential to the gold electrode in the presence of a tris(2,2-bipyridyl)ruthenium complex luminophore when the analyte DNA contained a methylation region. Methyl-cytosine was measured quantitatively in the 1-100 pmol range, which exhibits sufficiently high sensitivity to achieve real DNA measurements without amplification by a polymerase chain reaction (PCR). The proposed ECL method also exhibited high selectivity for methyl-cytosine against nonmethylated cytosine, guanine, thymine, and adenine nucleotides. Finally, original and methylated DNA samples were clearly distinguished with our method using a real DNA bacteriophage sample (48,502 base pairs).

A hybrid functional biomolecular interface designed at a molecular size level is very effective at capturing an analyte with high sensitivity even if the interaction is very weak, as when detecting proteins with carbohydrate. We designed and processed a protein (lectin) recognition molecular interface taking the following points into consideration: (1) the height (molecular length) difference between the capturing and spacer molecules; (2) the ratio of capturing molecules in the recognition interface. When the height difference between the maltoside part (Concanavalin A (Con A) recognition group) and the OH group terminated spacer molecules exceeded (>(CH(2))(6)), the association rate constant (k(a)) became larger (k(a)(1/Ms): ∼2.6 times) and the dissociation constant (K(D)) became much smaller (K(D)(M): 1.0 × 10(-6): ∼0.17 times) compared with the similar heights (lengths) of both molecular interfaces. With regard to maltoside density, a 100% maltoside monolayer was unsuitable for detecting Con A. We constructed a nanostructured recognition site with a maltoside part of 10%, which was the most suitable ratio for Con A detection. The binding interaction between Con A and the maltoside group was changed from monovalent binding to bivalent binding when the maltoside part was diluted in the recognition interface. From electrochemical measurements, even though there was a small amount of maltoside component on the suitable recognition monolayer, quality similar to that of 100% maltoside was observed.

To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability., Competing Interests: The authors have declared that no competing interests exist.

A hybridized nanocrystalline carbon film electrode consisting of sp(2) and sp(3) bonds was investigated to reveal the reduction properties of Cd(2+) and for application as a highly sensitive and reliable electrochemical immunoassay. Conductive nanocrystalline carbon film consisting of about 60% sp(2) and 40% sp(3) bonds was fabricated using electron cyclotron resonance (ECR) sputtering equipment, and then the Cd(2+) concentrations were measured with an ECR sputtered carbon (ECR nano-carbon) electrode by employing an anodic stripping voltammetry (ASV) technique. The preconcentrated Cd was analyzed with Kelvin probe force microscopy and energy dispersive X-ray spectroscopy while observing the morphology change with an atomic force microscope and a scanning electron microscope. The preconcentrated Cd on the ECR nano-carbon electrode was revealed to be a thin sheet structure, which was significantly different from the Cd on a conventional carbon material that grows with a coralloid structure. The background current during an ASV measurement maintains a low level equivalent to that found with boron-doped diamond because the surface of the ECR nano-carbon is robust and angstrom-level flat. The carbon-electrode performance for ASV was improved by controlling its structure at a nanometer scale without any metal doping or coating. Finally, the ECR nano-carbon was used for biomolecular determination by electrochemical immunoassay with a CdSe nanoparticle label. Electrochemical immunoassay results were successfully obtained with the ECR nano-carbon, and they correlated well with fluorescence results obtained for CdSe nanoparticles.

We have developed a polymer film based plasmonic device whose optical properties are tuned for measuring biological samples. The device has a circular nanohole array structure fabricated with a nanoimprint technique using a UV curable polymer, and then gold thin film is deposited by electron beam deposition. Therefore, the device is mass-producible, which is also very important for bioaffinity sensors. First the gold film thickness and hole depth were optimized to obtain the maximum dip shift for the reflection spectra. The dip shift is equivalent to the sensitivity to refractive index changes at the plasmonic device surface. We also calculated the variation in reflection spectra by changing the above conditions using the finite-difference time domain method, and we obtained agreement between the theoretical and experimental curves. The nanohole periodicity was adjusted from 400 to 900 nm to make it possible to perform measurements in the visible wavelength region to measure the aqueous samples with less optical absorption. The tuned bottom filled gold nanohole array was incorporated in a microfluidic device covered with a PDMS based microchannel that was 2 mm wide and 20 μm deep. As a proof of concept, the device was used to detect TNF-α by employing a direct immunochemical reaction on the plasmonic array, and a detection limit of 21 ng mL(-1) was obtained by amplification with colloidal gold labeling instead of enzymatic amplification.

We describe the electrochemical detection of DNA methylation through the direct oxidation of both 5-methylcytosine (mC) and cytosine (C) in 5'-CG-3' sequence (CpG) oligonucleotides using a sputtered nanocarbon film electrode after digesting a longer CpG oligonucleotide with endonuclease P1. Direct electrochemistry of the longer CpG oligonucleotides was insufficient for obtaining the oxidation currents of these bases because the CG rich sequence inhibited the direct oxidation of each base in the longer CpG oligonucleotides, owing to the conformational structure and its very low diffusion coefficient. To detect C methylation with better quantitativity and sensitivity in the relatively long CpG oligonucleotides, we successfully used an endonuclease P1 to digest the target CpG oligonucleotide and yield an identical mononucleotide 2'-deoxyribonucleoside 5'-monophosphate (5'-dNMP). Compared with results obtained without P1 treatment, we achieved 4.4 times higher sensitivity and a wider concentration range for mC detection with a resolution capable of detecting a subtle methylated cytosine difference in the CpG oligonucleotides (60mer)., (© 2011 American Chemical Society)

Aneuploidy decreases cellular fitness, yet it is also associated with cancer, a disease of enhanced proliferative capacity. To investigate one mechanism by which aneuploidy could contribute to tumorigenesis, we examined the effects of aneuploidy on genomic stability. We analyzed 13 budding yeast strains that carry extra copies of single chromosomes and found that all aneuploid strains exhibited one or more forms of genomic instability. Most strains displayed increased chromosome loss and mitotic recombination, as well as defective DNA damage repair. Aneuploid fission yeast strains also exhibited defects in mitotic recombination. Aneuploidy-induced genomic instability could facilitate the development of genetic alterations that drive malignant growth in cancer.

We have developed a new carbon film electrode material with thornlike surface nanostructures to realize efficient direct electron transfer (DET) with enzymes, which is very important for various enzyme biosensors and for anodes or cathodes used in biofuel cells. The nanostructures were fabricated using UV/ozone treatment without a mask, and the obtained nanostructures were typically 2-3.5 nm high as confirmed by atomic force microscopy measurements. X-ray photoelectron spectroscopy and transmission electron microscopy revealed that these nanostructures could be formed by employing significantly different etching rates depending on nanometer-order differences in the local sp(3) content of the nanocarbon film, which we fabricated with the electron cyclotron resonance sputtering method. These structures could not be realized using other carbon films such as boron-doped diamond, glassy carbon, pyrolyzed polymers based on spin-coated polyimide or vacuum-deposited phthalocyanine films, or diamond-like carbon films because those carbon films have relatively homogeneous structures or micrometer-order crystalline structures. With physically adsorbed bilirubin oxidase on the nanostructured carbon surface, the DET catalytic current amplification was 30 times greater than that obtained with the original carbon film with a flat surface. This efficient DET of an enzyme could not be achieved by changing the hydrophilicity of the flat carbon surface, suggesting that DET was accelerated by the formation of nanostructures with a hydrophilic surface. Efficient DET was also observed using cytochrome c., (© 2011 American Chemical Society)

Mercaptododecyl glycosides containing a terminal β-galactosyl group were prepared from D-galactose or from D-lactose via hexa-O-acetyl-lactal (10) as a key intermediate. Interactions of these glycolipids (5 kinds) and galectins (β-galactoside binding lectins, 6 species) were evaluated by surface plasmon resonance (SPR) method. High binding responses were observed for the lactoside, 2-deoxy-lactoside, and lactosaminide with some galectins (Gal-3, -4, -8), whereas the galactoside and 2,3-dideoxy-lactoside showed low binding activities., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Rituximab (chimeric anti-CD20 mAb) is currently used in the treatment of B-NHL and B cell malignancies, alone or in combination with chemotherapy. However, subsets of patients do not initially respond and/or develop resistance to additional treatments. Hence, there is a need to develop more effective anti-CD20 mAbs that may improve clinical response. BM-ca is a novel humanized anti-CD20 mAb that was tested against several B-NHL cell lines and was compared to several anti-CD20 mAbs (Rituximab, ofatumumab, 2H7, B1 and B-Ly1). BM-ca was shown to strongly induce both homotypic cell aggregation and redistribution of CD20 to membrane lipid rafts. BM-ca was also able to induce programmed cell death (apoptosis) without the need for cross-linking and demonstrated potent complement-dependent cytotoxicity (CDC). BM-ca was more cytotoxic than rituximab even in malignant B cells expressing low amounts of membrane CD20. Type I anti-CD20 mAbs typically induce minimal levels of homotypic cell aggregation and apoptosis but strong localization of CD20 to lipid rafts and potent CDC. Type II anti-CD20 mAbs typically exert the reverse activities. Noteworthy, BM-ca exhibits properties that are shared by both type I and type II anti-CD20 mAbs, which may reflect the recognition of a new CD20 epitope and/or exhibit different molecular signaling. Overall, the present data show that BM-ca is a novel anti-CD20 mAb that may be classified as a type I/II. The therapeutics efficacy of BM-ca awaits its use in clinical trials.

Ferrocene-attached polymyxin B (PMB-Fc) was prepared by the reaction of polymyxin B with ferrocenoyl chloride in a toluene/pyridine mixture. An electrochemical detection of lipopolysaccharide (LPS) was carried out using a combination of PMB-Fc and an enzyme-modified electrode constructed from a glassy carbon electrode modified with a bovine serum albumin membrane containing glucose oxidase. The ferrocene units of the PMB-Fc molecules were oxidized on the electrode, and then reduced to the original neutral form by a glucose oxidase-catalyzed reaction in the presence of D-glucose. The consumption/regeneration cycle for PMB-Fc resulted in a chemically amplified current response. The current response for PMB-Fc decreased in association with its complexation with LPS, and the magnitude of this current decrease caused by LPS was also amplified by the recycling process. The enzyme-modified electrode exhibited a rapid response of 5 min for LPS with the detection limit as low as 50 ng ml(-1). Further, the addition of D-solbitol or poly(vinyl alcohol) of high concentration over 1 mg ml(-1) substantially induced no response, and three kinds of LPS from different strains exhibited similar magnitudes of current response for the same concentrations; these results suggest the advantages of this detection system for practical applications. Ferrocene-attached colistin, an analogue of PMB-Fc, was also effective for the LPS detection using the glucose oxidase-modified electrode., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Surface modification of glassy carbon (GC) and highly oriented pyrolytic graphite (HOPG) was carried out with diazonium, amine, azide, and olefin derivatives bearing ferrocene as an electroactive moiety. Features of the modified surfaces were evaluated by surface concentrations of immobilized molecule, blocking effect of the modified surface against redox reaction, and surface observation using cyclic voltammetry and electrochemical scanning tunneling microscope (EC-STM). The measurement of surface concentrations of immobilized molecule revealed the following three aspects: (i) Diazonium and olefin derivatives could modify substrates with the dense-monolayer concentration. (ii) The surface concentration of immobilized amine derivative did not reach to the dense-monolayer concentration reflecting their low reactivity. (iii) The surface modification with the dense-monolayer concentration was also possible with azide derivative, but the modified surface contained some oligomers produced by the photoreaction of azides. Besides, the blocking effect against redox reaction was observed for GC modified with diazonium derivative and for HOPG modified with diazonium and azide derivatives, suggesting fabrication of a densely modified surface. Finally, the surface observation for HOPG modified with diazonium derivative by EC-STM showed a typical monolayer structure, in which the ferrocene moieties were packed densely at random. On the basis of those results, it was demonstrated that surface modification of carbon substrates with diazonium could afford a dense monolayer similar to the self-assembled monolayer (SAM) formation.

We describe the electrochemical determination of oxidative damaged DNA by using a nanocarbon film electrode combined with a high performance liquid chromatography (HPLC) system. The nanocarbon film was formed by employing the electron cyclotron resonance sputtering method, and has a nano-crystalline sp(2) and sp(3) mixed bond structure with an atomically flat surface. This film electrode provided the high electrode activity and stability needed to quantitatively detect oxidative damaged DNA, 8-hydroxy-2'-deoxyguanosine (8-OHdG), by direct electrochemical oxidation. The coefficient of variation (C.V.) value of 1 µM 8-OHdG at our film electrode was 0.75% (n = 12), which constitutes superior reproducibility to that of a conventional glassy carbon (GC) electrode (9.28%, n = 12) in flow-injection analysis. This was because the nanocarbon film suppressed fouling for the oxidized product of 8-OHdG owing to its hydrophilically ultraflat and chemically stable surface. We also investigated the performance of HPLC with an electrochemical detection (HPLC-ECD) system using our nanocarbon film electrode. The detection limit for 8-OHdG at the nanocarbon film electrode was 3 nM, which was superior to the detection limit of the GC electrode (7.2 nM). Furthermore, this electrode was more suitable for use in a urinary 8-OHdG experiment than the GC electrode. The concentration of urinary 8-OHdG in the urinary sample was 8.30 nM. These results indicate that this HPLC-ECD system with our nanocarbon film electrode enables us to realize an accurate, sensitive, reproducible and easy to use analysis technique.

An on-chip enzyme-linked immunosorbent assay combined with an electrochemical detection method (EC-ELISA) was employed to detect a leptin, one of the most important adipose derived hormones, using gold electrodes modified with a tri(ethylene glycol) terminated short alkanethiol (TEGCnSH, Cn = (CH(2))n, n = 2, 4, 6, and 8) monolayer. These TEGCnSH monolayers on gold electrodes can suppress non-specific protein adsorption without affecting the electrochemical activity required for detecting p-aminophenol (PAP), which is an alkaline phosphatase (ALP) product. We measured leptin with a highly sensitive detection range (100 pg mL(-1) to 10 ng mL(-1) level) and with the desired detection limit (13.6 pg mL(-1)) by using electrochemical detection. For detecting leptin, the EC-ELISA method using TEGC4SH modified gold electrode with a poly(dimethylsiloxane) based microchannel was superior to the conventional ELISA method. With the EC-ELISA method, we were able to measure leptin with a satisfactory detection range and a pg level detection limit within 30 min, which is a much lower detection level than that obtained with conventional plate based ELISA.

We have developed a miniaturized electrochemical surface plasmon resonance biosensor for measuring two biomolecules that have very different molecular sizes, one is transferrin (MW=75 kDa) as a disease marker protein, the other is creatinine (MW=113) as a calibration marker for the accurate measurement of human urinary samples. The sensor has a PDMS based microchannel that is 2 mm wide and 20 μm deep. Two gold films were integrated in the microchannel; one was modified with anti-transferrin antibody for immuno-reaction, and the other was modified with osmium-poly-vinylpyridine wired horseradish peroxidase (Os-gel-HRP). We further immobilized a tri-enzyme layer of creatininase, creatinase and sarcosine oxidase in order to measure creatinine by converting it to hydrogen peroxide in the upstream channel. We measured the transferrin concentration from the refractive index change involved in an immuno-complex formation, and we were simultaneously able to measure creatinine by employing the refractive index change in the Os-gel-HRP caused by oxidation with the hydrogen peroxide produced from creatinine by the tri-enzyme. The effects of ascorbic acid and uric acid in urine samples were sufficiently eliminated by adding ascorbate oxidase and uricase to the urine samples during sampling. We were able to measure two analyte concentrations within 15 min by one simple injection of 50 μL of diluted human urine into our sensor. The detectable transferrin and creatinine ranges were 20 ng/mL to 10 μg/mL, and 10 μM to 10 mM, respectively, which are sufficient levels for clinical tests. Finally, we compared the results obtained using our sensor with those obtained with a conventional immunoassay and the Jaffe method. We obtained a similar trend that can reduce the fluctuation in the urinary transferrin concentration from three different samples by calibrating the creatinine concentration., (Copyright © 2010 Elsevier B.V. All rights reserved.)