The arrangement of differentiated pluripotent embryonic stem cells into three-dimensional aggregates, which are known
as embryonic bodies, is a main step for progressing the embryonic stem cells differentiation. In this work, embryonic
stem cells that were directly produced from the hanging drop step as a three-dimensional structure with no further twodimensional
differentiation were diagnosed with Raman spectroscopy as a non-invasive and label-free technique. Raman
spectroscopy was employed to discriminate between mouse embryonic bodies of different degrees of maturation. EBs
were prepared applying the hanging drop method. The Raman scattering measurements were obtained in vitro with a
Nanophoton RAMAN-11 micro-spectrometer (Japan: URL: www.nanophoton.jp equipped with an Olympus XLUM
Plan FLN 20X/NA= 1.0 objective lens. Spectral data were smoothed, baseline corrected and normalized to the a welldefined
intense 1003 cm-1 band (phenylalanine) which is insensitive to changes in conformation or environment. The
differentiation process of embryonic stem cells is initiated by the removal of LIF from culture medium. 1, 7 and 17-dayold
embryonic stem cells were collected and investigated by Raman spectroscopy. The main differences involve bands
which decreased with maturation such as: 784 cm-1 (U, T, C ring br DNA/RNA, O-P-O str); 1177 cm-1 (cytosine,
guanine) and 1578 cm-1 (G, A). It was found that with the progress of differentiation the protein content was amplified.
The increase of protein to nucleic acid ratio was also previously observed with the progress of the differentiation process.
Raman spectroscopy has the potential to distinguish between the Raman signatures of live embryonic stem cells with
different degrees of maturation.
Nanostructure and molecular interface have currently received the great attractions for highly efficient, simultaneously
analysis of a number of important biomolecules from proteomics to genomics. Outstanding optical property of noble
metal nanostructures, localized surface plasmon resonance (LSPR), is a powerful phenomenon used in many chemical
and biological sensing experiments. This report described two types of gold-capped nanostructures: nanoparticle and
nanopore which reveal the strong excitation of LSPR spectra in the UV-visible region. The optical absorbance properties
of these nanostructures governing its sensitivity to local environment were studied. The flexibility in design of the goldcapped
nanostructures was evidently displayed on the wide-range capacity to develop in many types, from single to
multiple to microfluidic formats. Moreover, chemical modifications on the nanostructure surface were thoroughly
exploited to archive a highly sensitive protein and gene sensors such as using Protein A linker for orientation antibody or
using specific binding of streptavidin and biotinylated PNA or DNA probes... Lastly, we introduced a new form of
optical sensor, involving the coupling between interferometry and LSPR properties on the surface of gold-capped
nanopore structure. Our optical biosensing devices connecting with the gold-capped nanostructures including both
nanoparticle and nanopore are applicable to highly sensitive monitoring the interactions of other biomolecules, such as
proteins, whole cells, or receptors with a massively parallel detection capability in a highly miniaturized package.
High-tech industries are often the cause of groundwater contamination that affects surrounding areas. While steps must be taken to prevent this type of contamination, high-tech industries should be able to procure the required amounts of high-quality groundwater for their manufacturing processes. The objective of the Advanced Environmental Monitoring System (AEMS) project is to develop a new integrated groundwater monitoring system based on innovative technologies
in order to facilitate effective management of groundwater contamination in and around high-tech industrial facilities.
It will be possible to use the biosensors developed in this project not only to monitor ground and other fresh water from various sources for contamination, but also to assess the toxicity and environmental hazards arising from industrial effluents. The AEMS project provides high-tech industries with the means to fulfill their commitments to modern society. Through this project they can pursue sustainable development, compliance with environmental regulations, responsible corporate citizenship, effective life-cycle management, and improved worker safety.
In recent years, we have developed an advanced environmental monitoring system (AEMS) containing the eco-sensor, which means a sensor for the measurement of environmental pollutants, based on lipid membranes for continuous monitoring of ground water in industry areas such as semiconductor factories. The AEMS project is composed of three work packages as follows, 1) Eco -sensor, 2) Prediction of plume propagation using a computer simulation technique, and 3) Environmental protection method. In this paper, we would like to focus on the study of the eco-sensor. We considered that modified lipid membranes serve as good models for cell membranes because they would be ideal hosts for receptor molecules of biological origin or disruptive environmental pollutants. Thus, we selected lipid membrane as a sensing element for environmental pollutants. We have already confirmed that the eco-sensor could detect a 10 ppb level of volatile organic chlorinated compounds (VOCs) such as trichloroethylene in ground water. Here, we tried to apply the eco-sensor to measure other environmental pollutants containing pesticides and endocrine disrupting chemicals. We made a novel automatic bilayer lipid membrane preparation device and a new system for the continuous
measurement of environmental pollutants in ground water.
Biosensors in connection with enzyme linked immunosorbent assay (ELISA) can be applied in many fields of research. In this paper, the reduction in the size of ELISA utilizing micro-chemical reaction is described in a microchamber array chip, and also a micro-flow antibody chip. The chips were fabricated by micro electromechanical system (MEMS) technology. The quantitative determination of dioxins was performed by using the chips. Glass or polystyrene beads were used for immobilization of an antibody at these chips. The antibody-immobilized beads were introduced into micro-flow channel or microchamber. As a competitive ELISA, sample solution mixed with horseradish peroxidase (HRP)-conjugated antigen, and non-HRP conjugated antigen was allowed to react in the microchamber or flow channel. As a sandwich assay, sample solution and HRP-conjugated antibody were sequentially added to the chamber. After the antigen-antibody reaction, addition of PBS buffer, hydrogen peroxide, and fluorogenic substrate produced the fluorescent dye. The resulting change in the fluorescence intensity was monitored by a fluorescence microscope.
In recent years, we have developed an advanced environmental monitoring system (AEMS) containing the eco-sensor, which means a sensor for the measurement of environmental pollutants, based on lipid membranes for continuous monitoring of underground water in industry areas such as semiconductor factories. The AEMS project is composed of three work packages as follows, 1) Eco-sensor, 2) Prediction of plume propagation using a computer simulation technique, and 3) Environmental protection method. In this presentation, we would like to focus on the study of the eco-sensor. The reason why lipid membranes were selected as a sensing element for environmental pollutants is that the pollutants should be interacted with cell membranes because cells are surrounded by cell membranes containing lipid components. Improving the applicability and the responsibility of bilayer lipid membranes (BLMs) in the eco-sensor, we have investigated automatic BLMs preparation devices. An automatic BLMs preparation device was made by use of an inkjet mechanism. The reproducibility of the BLMs preparation was remarkably improved. The sensitivity to volatile organic chlorinated compounds such as cis-1,2-dichloroethylene was in the order of 10 ppb using monoolein BLMs even in real underground water. We have been also developing a smaller sized eco-sensor for the practical use.
In recent years, we have developed an advanced environmental monitoring system (AEMS) containing the eco-sensor, which means a sensor for the measurement of environmental pollutants, based on lipid membranes for continuous monitoring of underground water in industry areas such as semiconductor factories. The AEMS project is composed of three work packages followed by 1)Eco-sensor, 2)Prediction of plume propagation using a computer simulation technique, and 3)Environmental protection method. In this presentation, we would like to focus on the study of the eco-sensor. The reason why lipid membranes selected as a sensing element for environmental pollutants is that the pollutants should be interacted with cell membranes because cells are surrounded by cell membranes containing lipid components. Improving the applicability and the responsibility of bilayer lipid membranes (BLMs) in the eco-sensor, we have investigated the automatic BLMs preparation device. An automatic BLMs preparation was remarkably improved. The sensitivity to volatile organic chlorinated compounds such as cis-1,2-dichloroethylene was in the order of 10ppb using the monoolein BLMs even in real underground water. We also have been developing a smaller sized eco-sensor for the practical use.
In recent years, we have developed an advanced environmental monitoring system (AEMS) containing the eco-sensor, which means a sensor for the measurement of environmental pollutants, based on lipid membranes for continuous monitoring of underground water in industry areas such as semiconductor factories. The AEMS project is composed of three work packages followed by 1) Eco-sensor, 2) Prediction of plume propagation using a computer simulation technique, and 3) Environmental protection method. In this presentation, we would like to focus on the study of the eco-sensor. The reason why lipid membranes were selected as a sensing element for environmental pollutants is that the pollutants should be interacted with cell membranes because cells are surrounded by cell membranes containing lipid components. Improving the applicability and the responsibility of bilayer lipid membranes (BLMs) in the eco-sensor, we have investigated the automatic BLMs preparation device. An automatic BLMs preparation device was made by use of an inkjet mechanism. The reproducibility of the BLMs preparation was remarkably improved. The sensitivity to volatile organic chloride compounds such as cis-1,2-dichloroethylene was in the order of 10 ppb using the monoolein BLMs even in real underground water. We also have been developing a smaller sized eco-sensor for the practical use.
12 In recent years, we have developed the eco-sensor, which means the sensor for the measurement of environmental pollutants, based on lipid membranes for continuous monitoring of underground water in industry areas such as semiconductor factories. In order to improve the stability and the responsibility of bilayer lipid membranes (BLMs) in the eco-sensor, we have investigated both the stabilization of BLMs based on the supporting structure and the test making of the automatic BLMs preparation device. The application of the supporting structure using gelatin, etc showed the some times' extension of the BLMs life. But the decrease in the sensitivity of the sensor was observed in the storage of the sensor. An automatic BLMs preparation device was made by use of an inkjet mechanism. The reproducibility of the BLMs preparation was remarkably improved. The sensitivity to organochlorine compounds was in the order of 10 ppb using the monoolein BLMs.
KEYWORDS: Neurons, Luminescence, Near field scanning optical microscopy, Aluminum, Near field optics, Optical fibers, Microscopes, Atomic force microscopy, Neurotransmitter release, Near field
A SNOAM system is capable of obtaining simultaneous topographic and optical images with a resolution beyond than the diffraction limit of far field optical imaging. Fluorescence tagging combined with optical resolutions of better than 100nm allow us to detect structures not possible with conventional microscopes. Also in contrast with electron microscopy SNOAM has the ability to look at biological structures in the liquid medium. Presently there is much interest in understanding the processes that lead to LTP in neuron synapses. LTP is widely associated with memory function in neurons. Hence, better understanding will lead to advances in medicine, as well as neuron-based memory and processing devices. Better understanding is also crucial to the development of neuron-electronic interfaces. In this research, neuron networks are grown on a patterned polylysine substrate. Polylysine is patterned using micro lithographic techniques. Neurons are extracted from the hippocampus of chick embryos, and are then grown on this pattern under standard sterile incubating conditions. The neurons are stimulated to release the neurotransmitter glutamate. The glutamate is then fluorescently imaged with Amplex-red SNOAM.
Conformation change of DNA under binding a DNA binding protein was studied using an AFM. An originally designed AFM mounted on an inverse microscope was used for imaging DNA. High mobility group (HMG) protein and pUC118 DNA were used as DNA binding protein and DNA for investigation in this study. The pUC118 DNA and its mixture with HMG2BJ were imaged by the AFM. In the AFM image of pUC118, DNA strands showed open structure with a few helix sites. The height of the helix part is 1.5 times higher than the ordinary DNA strand. Centrally AFM images of the mixture showed HMG-like particles on DNA strands. The height of the particle is 2-3 times higher than the DNA strands which showed the particles were not aggregating parts but HMG. The HMG bound on a crossing part of DNA which make a loop.
KEYWORDS: Near field scanning optical microscopy, Luminescence, Particles, Optical spheres, Near field optics, Microscopy, Optical fibers, Near field, Genetics, Target detection
Scanning near-field optical/atomic-force microscopy was first applied to detect fluorescence hybridization of DNA immobilized on nano-particle media. Hybridization can also be used to determine the sequence of unknown DNA. 100-nm in diameter of polystyrene sphere carboxylate was used as the nano-particle media. Template DNA including target sequence was chemically modified with animo group at the 5'-end of single-stranded DNA. Amine-coupling reaction made covalent bond between template DNA and carboxyl group on the surface of the media. Single-stranded DNA of specific base sequence labeled either fluorescent dye, that is used to detect the complementary base sequence by hybridization. Simultaneous imaging the colloidal particles showed us topography and near-field fluorescence images of them. All particles were observed in the topographic image, however, some particles were realized in the fluorescence image. This result indicated that fluorescent hybridized DNAs on the surface of the media were visualized specifically. High density arrays or integration of media is a fast and effective means of accessing the gene variation. However, in this study, detection of hybridized fluorescent DNA conjugated with particle is a major purpose rather than arrangement technique.
KEYWORDS: Luminescence, Near field scanning optical microscopy, Green fluorescent protein, Near field optics, Biomedical optics, Liquids, Optical fibers, Microscopes, Glasses, Spatial resolution
A scanning near-field optical/atomic-force microscope (SNOAM) system was applied to simultaneous topographic and fluorescence imaging of biological samples in air and liquid. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic-force microscopy cantilever and a scanning near-field optical microscopy probe. The SNOAM system used 458 or 488 nm from Ar ion laser multiline of excitation of green fluorescent protein (GFP), since a native GFP has been known to give a maximum at 395 nm and a broad absorption spectrum until 500 nm. Topographic and fluorescence images of recombinant E.coli were obtained simultaneously with a high spatial resolution which was apparently better than that of a conventional confocal microscope.
We propose a novel method for individual immobilization. Biomaterials were first immobilized enlarges the size of biomaterials to the size of sensor element. Then the support is arranged on the sensor element by self-assembling. The element was microfabricated to have a microstructure for self-assembling. When both size of biomaterial and the element are the same, self-assemble is expected to give one- to-one correspondence, and then individual response. Mixed glass beads immobilized each of various enzyme were put near the pits fabricated one chip and the chip was slanted to roll the beads into the pits. When the beads immobilized only with peroxidase were arranged, the addition of luminol and hydrogen peroxide gave chemiluminescence at almost every site. Next, the beads immobilized only with glucose oxidase as dummy enzyme were mixed with HRP-beads and arranged to the sites. Addition of substrate gave limited number of luminescence-giving sites, though every site had an enzyme- immobilized bead. These results show that two kinds of enzymes were separately arranged in the site in one-to-one correspondence.
KEYWORDS: Near field scanning optical microscopy, Luminescence, Green fluorescent protein, Near field optics, Optical fibers, Biomedical optics, Atomic force microscopy, Microscopy, Spectrographs, Chromium
We have developed scanning near-field optical/atomic force microscopy (SNOM/AFM). The SNOM/AFM uses a bent optical fiber simultaneously as a dynamic force AFM cantilever and a SNOM probe. Resonant frequency of the optical fiber cantilever is 15 - 40 kHz. Optical resolution of the SNOM/AFM images shows less than 50 nm. The SNOM/AFM system contains photon counting system and polychrometer/ICCD system to observe fluorescence image and spectrograph of micro areas, respectively. A SNOM-AFM system was newly applied to analyses of biological samples. In this system a feedback signal from AFM in the noncontact mode was used to scan the probe tip along the surface contour of the sample. An optical fiber with a sharp tip on one end was bent for use as cantilever, and ac amplitude of the cantilever deflection was held constant during scanning by moving the stage. Green fluorescent protein (GFP) absorbs blue light and emits green light. GFP should be a convenient indicator of transformation and one that could allow cells to be separated with fluorescence-activated cell sorting. The gene coding to GFP was cloned in recombinant E.coli and plant cells. Spatial distribution of GFP gene expression was clarified using a SNOM-AFM system. Fluorescent spectroscopic analyses supported GFP was surely produced in E.coli and plant cells. Applications to gene identification in human genomes were also discussed.
Environmental assessment is important to evaluate the overall health and ecological impact of domestic and industrial wastes. Biosensors are kinds of analytical devices which consist of biomaterials and transducers. Photoluminescence of recombinant E.coli containing lux related genes were used as indicators of environmental pollutions. This paper deals with sensitive and rapid optical sensing systems for monitoring BOD (Biochemical Oxygen Demand), toxic compounds and mutagens.
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