Satoka Aoyagi

Oriented immobilization of antibodies on a silicon wafer using Si-tagged protein A.

We previously reported a silica-binding protein, designated Si-tag, which can be used to immobilize proteins on silica surfaces. Here, we constructed a fusion protein of Si-tag and immunoglobulin-binding staphylococcal protein A for oriented immobilization of antibodies on a silicon wafer whose surface is oxidized to silicon dioxide (silica). The fusion protein, Si-tagged protein A, strongly bound to the silica surface with a dissociation constant of 0.31 nM. Time-of-flight secondary ion mass spectrometry analysis of the silicon wafer coated with Si-tagged protein A, combined with principal component analysis and mutual information, demonstrated that protein A is localized on the outermost surface of the bound protein layer. Immunoglobulin G (IgG) was immobilized both on the silicon wafer coated with Si-tagged protein A and, as a control, directly on the intact silicon wafer via physical adsorption. The silicon wafer coated with Si-tagged protein A bound 30-70% more IgG than the uncoated silicon wafer, whereas the antigen-binding activity was 4- to 5-fold higher for the former, indicating that IgG was functionally immobilized on the silicon wafer via Si-tagged protein A in an oriented manner.

Reagentless and regenerable immunosensor for monitoring of immunoglobulin G based on non-separation immunoassay.

Based on the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by reactions between proteins, we developed a reagentless, regenerable and rapid immunosensing system to determine immunoglobulin G (IgG). Fluorescence intensity of the immobilized FITC depends on IgG concentration, ranging from 10 to 50 microg/ml, specifically, even with co-existing proteins. The response time is 30 min during steady-state measurement and is less than a minute during transient measurement. When the FITC-labeled protein A binds to IgG, the surrounding atmosphere of FITC becomes hydrophobic. Since the fluorescence intensity of fluorescent substances generally increases at a hydrophobic environment, FITC fluorescence intensity increases with the concentration of protein A bonding to IgG. This system is regenerable because the fluorescence enhancement repeatedly occurs every time the immobilized fluorescent reagent is immersed in sample solutions.

Peptide Fragmentation and Surface Structural Analysis by Means of ToF-SIMS Using Large Cluster Ion Sources

Peptide or protein structural analysis is crucial for the evaluation of biochips and biodevices, therefore an analytical technique with the ability to detect and identify protein and peptide species directly from surfaces with high lateral resolution is required. In this report, the efficacy of ToF-SIMS to analyze and identify proteins directly from surfaces is evaluated. Although the physics governing the SIMS bombardment process precludes the ability for researchers to detect intact protein or larger peptides of greater than a few thousand mass unit directly, it is possible to obtain information on the partial structures of peptides or proteins using low energy per atom argon cluster ion beams. Large cluster ion beams, such as Ar clusters and C60 ion beams, produce spectra similar to those generated by tandem MS. The SIMS bombardment process also produces peptide fragment ions not detected by conventional MS/MS techniques. In order to clarify appropriate measurement conditions for peptide structural analysis, peptide fragmentation dependency on the energy of a primary ion beam and ToF-SIMS specific fragment ions are evaluated. It was found that the energy range approximately 6 ≤ E/n ≤ 10 eV/atom is most effective for peptide analysis based on peptide fragments and [M + H] ions. We also observed the cleaving of side chain moieties at extremely low-energy E/n ≤ 4 eV/atom.

ToF-SIMS observation for evaluating the interaction between amyloid β and lipid membranes

The adsorption behaviour of amyloid beta (Aβ), thought to be a key peptide for understanding Alzheimer’s disease, was investigated by means of time-of-flight secondary ion mass spectrometry (ToF-SIMS). Aβ aggregates depending on the lipid membrane condition though it has not been fully understood yet. In this study, Aβ samples on different lipid membranes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), were observed with ToF-SIMS and the complex ToF-SIMS data of the Aβ samples was interpreted using data analysis techniques such as principal component analysis (PCA), gentle-SIMS (G-SIMS) and g-ogram. DOPC and DMPC are liquid crystal at room temperature, while DPPC is gel at room temperature. As primary ion beams, Bi3+ and Ar cluster ion beams were used and the effect of an Ar cluster ion for evaluating biomolecules was also studied. The secondary ion images of the peptide fragment ions indicated by G-SIMS and g-ogram were consistent with the PCA results. It is suggested that Aβ is adsorbed homogeneously on the liquid-crystalline-phase lipid membranes, while it aggregates along the lipid on the gel-phase lipid membrane. Moreover, in the results using the Ar cluster, the influence of contamination was reduced.

Fluorescence Enhancement of Fluorescein Isothiocyanate-Labeled Protein A Caused by Affinity Binding with Immunoglobulin G in Bovine Plasma

Fluorescence enhancement of fluorescein isothiocyanate-labeled protein A (FITC-protein A) caused by the binding with immunoglobulin G (IgG) in bovine plasma was studied. FITC-protein A was immobilized onto a glass surface by covalent bonds. An increase in fluorescence intensity was dependent on IgG concentration ranging from 20 to 78 μg/mL in both phosphate buffer saline and bovine plasma. This method requires no separation procedure, and the reaction time is less than 15 min. A fluorescence enhancement assay by the affinity binding of fluorescence-labeled reagent is thus available for the rapid determination of biomolecules in plasma.

SIMS characterization of hydrogen transport through SiO2 by low-temperature hydrogen annealing

The variation of hydrogen distribution at the SiO 2 /Si interface by low-temperature hydrogen annealing was investigated using secondary ion mass spectrometry (SIMS). As the amount of hydrogen atoms incorporated at SiO 2 /Si is considered to be comparable to the silicon dangling bond density (1 x 10 10 to 1 x 10 12 atoms/cm 2 ), an analytical method with a high sensitivity is necessary for the detection of hydrogen at SiO 2 /Si. In this study, the experimental conditions of SIMS were optimized in order to obtain a sufficient reproducibility of interfacial hydrogen ion intensity. There are two main causes that influence the measurement reproducibility: (1) misalignment of the relative irradiation areas of the electron beam and ion beam and (2) the contribution of background hydrogen from surface contaminants and residual gas. We obtained a high measurement reproducibility within a 5.5% relative standard deviation (2σ). This enabled us to observe an increase of hydrogen at SiO 2 /Si by hydrogen annealing at 400 °C, which resulted from the incorporation of hydrogen from the ambient. From the results of nuclear reaction analysis (NRA) and thermal desorption spectroscopy (TDS), it was also found that the incorporated hydrogen had two chemical states.

A new reagentless immunosensor for measuring IgG concentration in human plasma based on fluorescence-enhancement immunoassay

Abstract Based on the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by binding to proteins, we developed a reagentless immunosensor to directly determine immunoglobulin G (IgG) in human plasma. The fluorescence-enhancement immunoassay (FEI) is used in only limited conditions in spite of its simplicity, because the homogeneous reagent cannot be reused. In the present study, a new FEI was evaluated with an immobilized reagent. FITC was used to label protein A, which has specific reactivity with IgG. The FITC-labeled protein A was immobilized on nylon membrane and placed in a fluorometer cell. It produced fluorescence enhancement corresponding to IgG concentration in phosphate-buffered saline (PBS) and bovine serum solution, and it responded to IgG also in human plasma. Thus the IgG concentration can be determined directly using the reagentless immunosensor based on FEI.

ToF-SIMS analysis of amyloid beta aggregation on different lipid membranes

Amyloid beta (Aβ) peptides are considered to be strongly related to Alzheimers disease. Aβ peptides form a β-sheet structure on hard lipid membranes and it would aggregate to form amyloid fibrils, which are toxic to cells. However, the aggregation mechanism of Aβ is not fully understood. To evaluate the influence of the lipid membrane condition for Aβ aggregation, the adsorption forms of Aβ (1-40) on mixture membranes of lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol β-d-glucoside (β-CG) were investigated by time-of-flight secondary ion mass spectrometry. As a result, Aβ adsorbed along the localized DMPC lipid on the mixture lipid membranes, whereas it was adsorbed homogeneously on the pure DMPC and β-CG membranes. Moreover, amino acid fragments that mainly existed in the n-terminal of Aβ (1-40) peptide were strongly detected on the localized DMPC region. These results suggested that the Aβ was adsorbed along the localized DMPC lipid with a characteristic orientation. These findings suggest that the hardness of the membrane is very sensitive to coexisting materials and that surface hardness is important for aggregation of Aβ.

Structural analysis of the outermost hair surface using TOF-SIMS with gas cluster ion beam sputtering

A hair cuticle, which consists of flat overlapping scales that surround the hair fiber, protects inner tissues against external stimuli. The outermost surface of the cuticle is covered with a thin membrane containing proteins and lipids called the epicuticle. In a previous study, the authors conducted a depth profile analysis of a hair cuticles amino acid composition to characterize its multilayer structure. Time-of-flight secondary ion mass spectrometry with a bismuth primary ion source was used in combination with the C60 sputtering technique for the analysis. It was confirmed that the lipids and cysteine-rich layer exist on the outermost cuticle surface, which is considered to be the epicuticle, though the detailed structure of the epicuticle has not been clarified. In this study, depth profile analysis of the cuticle surface was conducted using the argon gas cluster ion beam (Ar-GCIB) sputtering technique, in order to characterize the structure of the epicuticle. The shallow depth profile of the cuticle surface was investigated using an Ar-GCIB impact energy of 5 keV. Compared to the other amino acid peaks rich in the epicuticle, the decay of 18-methyleicosanic acid (18-MEA) thiolate peak was the fastest. This result suggests that the outermost surface of the hair is rich in 18-MEA. In conclusion, our results indicate that the outermost surfaces of cuticles have a multilayer (lipid and protein layers), which is consistent with the previously proposed structure.

Development of a new endotoxin sensor with intermittent injection of limulus reagent for continuous monitoring of dialysate fluid

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 microliters/min and mixed with 30 microliters 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 microliter. 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 concentration 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 microliters. In conclusion, to measure ET concentrations below 0.1 EU/ml, the cell volume of a metal free spectrophotometer should be minimal.