青柳 里果

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is capable of chemically visualizing proteins on insulated samples. Distribution of an immobilized probe protein, fluorescent-labeled protein A-immobilized on a glass plate, and that of a sample protein, immunogloblin G (.IgG) in solution, reacting with protein A on the biosensor surface, were evaluated with TOF-SIMS (TFS-2100, Physical Electronics). TOF-SIMS spectra and images of the protein on the glass plates were obtained, and this "mutual information", as defined by information theory. was employed to analyze the TOF-SIMS spectra of proteins. Fragment ions from protein A and IgG were distinguished by the mutual, reinforcing information and specific fragment ions to each protein were selected to obtain the TOF-SIMS image of the protein. It is evident from the TOF-SIMS images of each protein that protein A was immobilized on the substrate homogeneously and that the reaction between the immobilized protein A and IgG is not localized in this condition. Chemical images of the proteins by TOF-SIMS will contribute to a better understanding of the reaction on the biosensor surface, and thus will help the development of more sophisticated biosensors. In addition, the requisite chemical conditions as well as the interaction between the biosensor surface and the immobilized proteins were investigated by TOF-SIMS by means of sets of reinforcing, mutually supportive information. (C) 2004 Elsevier B.V. All rights reserved.

A reagent-less, regenerable and portable optic immunosensor was developed. A model sample, immunoglobulin G (IgG), was detected with this system based on changes in fluorescent intensity of fluorescent labeled protein A with specific reactivity to IgG depending on a reaction between the proteins. A glass plate immobilized with Qdot(TM)-labeled protein A was placed on the top of optic fibers designed for both excitation and fluorescence emission. The optic fibers with the Qdot(TM)-labeled protein A-immobilized glass plate were inserted into a solution of pH 7.4 phosphate buffered saline. After stabilization of the fluorescence intensity, IgG was added and the time-course of the fluorescence intensity was measured on a fluorometer connected with the optic fibers. Furthermore, the fluorescence response of a transient state was evaluated with the same system. When the Qdot-labeled protein A bound to IgG, fluorescence intensity decreased because of the inhibition by IgG. The degree of fluorescence decrease depends on the IgG concentration at a steady state and also in a transient state. (C) 2004 Elsevier B.V. All rights reserved.

Protein adsorption on dialysis membrane was studied by time-of-flight secondary ion mass spectrometry (TOF-SIMS), which is expected to be capable of chemical imaging of insulated samples, such as hollow-fiber dialysis membranes. Three commercially available hollow-fiber dialysis membranes having different pore sizes and structures, were tested in the present study. Bovine serum albumin (BSA) solutions were used to adsorb the protein on the samples. TOF-SIMS images and spectra of native membranes and membranes treated with BSA were compared in order to identify secondary ions related to BSA and membranes. Mutual information, one of applications of information theory, was employed to select fragment ions related to BSA. TOF-SIMS images show distribution of adsorbed BSA on the dialysis membranes and indicate that BSA permeability and interaction between the membranes and BSA definitely depend on pore size, structure and material. (C) 2004 Elsevier B.V. All rights reserved.

A novel method of deconvolution without using a reference sample was investigated for dopant depth profiling of Si in the AlGaAs/GaAs epilayers. The true Si distribution was numerically analyzed by deconvolution using a depth resolution function (DRF) obtained from an Al composition profile. The composition analysis by thickness fringe-transmission electron microscopy (CAT-TEM) revealed that the Al composition changed rapidly at the AlGaAs/GaAs interface and followed an ideal step function. The DRF of Al was, thus, derived by differentiating the measured Al profile. Deconvolution of the measured Si profile was performed by least-squares fitting of the convolved Si profile with the measured profile, under the assumption that a true Si distribution was in a Gaussian function. The variance sigma of the true distribution was found to be 1.9 nm after deconvolution. Simultaneously, decay length of the Si profile decreased by almost one-third compared to that before deconvolution. The deconvolved Si profile agreed well with the carrier profile of a high electron mobility transistor (HEMT). (C) 2004 Elsevier B.V. All rights reserved.

The applicability of secondary ion mass spectrometry (SIMS) was evaluated for measuring the variation of hydrogen distributions in chemical vapor deposition (CVD) SiO2 films by post-oxidation annealing (POA). No noticeable hydrogen migration induced by electron or ion beam irradiation was observed. An increase of hydrogen ion intensity at the thermal SiO2/Si interface was observed regardless of the presence of a CVD SiO2 cap. Also, it was found that POA caused remarkable reduction of the hydrogen concentration at the SiO2/Si interface. This result agreed well with those by grazing incidence X-ray reflectivity (GIXR) and C-V measurements. These results lead us to the conclusion that the excess hydrogen present at the interfacial region was released during re-oxidation. (C) 2004 Elsevier B.V. All rights reserved.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is capable of chemical imaging of proteins on insulated samples such as hollow-fiber dialysis membranes. Albumin loss and a lowering of diffusive permeability caused by protein adsorption on dialysis membranes should be reduced in order to enhance dialysis adequacy of the patients. Bovine serum albumin (BSA)adsorbed hollow-fiber dialysis membranes were tested in the present study. TOF-SIMS images and spectra of both native membranes and BSA-adsorbed membranes were compared in order to identify secondary ions related to BSA and membranes. Peaks of secondary ions related to BSA and each membrane were selected by means of information theory, and they are characterized by principal component analysis (PCA). Chemical images of BSA adsorption on both native and treated membranes were obtained to find that BSA permeability and interaction between the membranes and BSA definitely depend on the properties of a membrane. TOF-SIMS imaging obtained with information theory is a powerful tool to estimate protein adsorption on the dialysis membranes. (C) 2004 Elsevier B.V. All rights reserved.

Biomolecules such as extracellular matrix and adhesive proteins generated by adhered cells on metallic specimens were characterized by means of time-of-flight secondary ion mass spectrometry (TOF-SIMS) in order to clarify the interaction between cells and metal surfaces. Since composition and structure of the extracellular matrix depends on conditions of cells, characterization of the interaction between cells and metallic specimens is important in order to evaluate the biocompatibility and the degradation behavior of metallic biomaterials and artificial organs. Moreover, the obtained data can contribute to the development of new metallic biomaterials. TOF-SIMS spectra were analyzed by means of mutual information described by information theory and principal components analysis (PCA). The results show that cells have great influence on adsorption of biomolecules on metallic materials because they change surface conditions of the materials. Thus TOF-SIMS is a useful technique to investigate the interaction between metallic biomaterials and cells. (C) 2004 Elsevier B.V. All rights reserved.

Control of the biosensor surface is required for developing high performance sensing systems. The optic immunosensor based on a solid-liquid and antigen-antibody reaction, which requires homogeneous immobilization of a probe protein, is used in this study. Distribution of fluorochrome-labeled protein A-immobilized on the biosensor surface and immunoglobulin G (IgG) reacted with protein A were estimated by means of time-of-flight secondary ion mass spectrometry (TOF-SIMS) spectra. Specific chemical distribution including amino acid fragments related to proteins will provide useful information on immobilization of proteins and their reactions. Peak selection of proteins for chemical imaging is difficult because proteins generate similar fragment ions. In this study, specific peaks of fragment ions from different proteins were determined by means of mutual information, one of useful applications of information theory. Aammosilanized glass plates immobilized with protein A and immunoglobulin G were measured with TOF-SIMS, respectively. TOF-SIMS spectra of the protein immobilized glass plates were analyzed with information theory and principal component analysis (PCA). Both methods suggest differences between proteins and mutual information clearly indicates specific peaks from each protein. Thus the analysis of protein immobilization on the biosensor surface with TOF-SIMS suggests that this technique will contribute to the improvement and the development of a sophisticated biosensor. (C) 2004 Elsevier B.V. All rights reserved.

Enhanced hydroxyl radical production in the Fenton reaction with ATP or ADP based on luminol chemiluminescence was clarified. Luminol chemiluminescence intensity increases with the concentration of ATP or ADP due to their influence on the Fenton reagents which produce hydroxyl radicals. Fenton reagents containing hydrogen peroxide and ferrous ion produce hydroxyl radicals which attack luminol and this emits light. The effect of ATP and ADP on the Fenton reaction is detected as luminol CL intensity with a photon counting system. This method allows measurement of ATP or ADP concentration ranging from 0 to 15 mM, which covers the physiological concentration range.

A measurement without the influence of protein adsorption on the sensor surface has been developed using transient current of an enzyme-immobilized electrode. Response current decreases due to protein adsorption on the sensor surface and the enzyme-immobilized membrane causing glucose concentration gradient. By measuring glucose concentration in the transient state while controlling GOD activity, it is possible to prevent the influence of protein adsorption. A needle-type electrode glucose sensor on which enzyme is immobilized with a polyurethane membrane, with electrochemical on-off control of enzyme activity, is capable of accurate measurement of glucose concentration in bovine serum at least for 24 hrs.

This report describes a method of continuously, stably, and inexpensively measuring endotoxin (ET) concentrations in dialysate fluid using an ET sensor with intermittent injection of limulus reagent. An ET solution simulating dialysate fluid was sampled in a single tube at a flow rate of 260 mu l/min and mixed with 30 mu l of limulus reagent intermittently injected into the tube. The absorbance of the solution was measured after the limulus reaction at 313 or 318 degrees K at 26 min. A good linear relationship (r = 0.98) between peak area of absorbance and ET concentration at ET concentrations ranging from 0 to 0.12 endotoxin unit (EU)/ml was obtained, using a spectrophotometer with a cell volume of 8 mu l. The baseline rose after the measurements were taken because the cell volume was so small that the cell was stuffed with gel. A good linear relationship (r = 1.00) at ET concentrations of 0.1-0.25 EU/ml was also obtained, and the baseline was unchanged after measurements, using a metal free spectrophotometer with a cell volume of 420 mu l. In conclusion, to measure ET concentrations below 0.1 EU/ml, the cell volume of a metal free spectrophotometer should be minimal.