Detection of Interfering Ions Using Ion Flux Phenomena in Flow-Through Cl-ISEs with Ion Exchange Membranes.

Ion-selective electrodes (ISEs) are among the most successful electrochemical sensors used in various applications because of their ability to measure electrolyte concentrations in liquids easily. It is common practice to suppress ion fluxes through the ion-sensitive membranes in ISEs because such fluxes worsen the lower limit of detection. In this study, we propose a method to detect interfering ions using this ion flux phenomenon. As a proof of principle, a flow-type Cl-ISE based on an ion exchange membrane loaded with the target ion chloride was used to acquire transient potential profiles during standstill after the introduction of liquids containing various ion species. When the target ion of the ion-sensitive membrane was measured, there was almost no change in potential over time. In contrast, when hydrophilic interfering ions were measured, the potential gradually decreased, and when hydrophobic interfering ions were measured, the potential gradually increased. The direction and intensity of these changes over time depended on the ion species and concentrations. The main reason for these potential changes is presumed to be the change in the local ionic composition of the sample near the sensing membrane due to ion exchange between the sample and membrane. This phenomenon could not be observed in a hydrophobic ion exchanger membrane doped with a quaternary ammonium salt and was characteristically observable using hydrophilic ion exchange membranes with a high charge density and a high ion diffusion rate. Finally, using a high-throughput flow-type system, we demonstrated the detection of interfering ions in solutions containing multiple ion species by using the ion flux phenomenon.

Single-molecule motions of MHC class II rely on bound peptides.

The major histocompatibility complex (MHC) class II protein can bind peptides of different lengths in the region outside the peptide-binding groove. Peptide-flanking residues (PFRs) contribute to the binding affinity of the peptide for MHC and change the immunogenicity of the peptide/MHC complex with regard to T cell receptor (TCR). The mechanisms underlying these phenomena are currently unknown. The molecular flexibility of the peptide/MHC complex may be an important determinant of the structures recognized by certain T cells. We used single-molecule x-ray analysis (diffracted x-ray tracking (DXT)) and fluorescence anisotropy to investigate these mechanisms. DXT enabled us to monitor the real-time Brownian motion of the peptide/MHC complex and revealed that peptides without PFRs undergo larger rotational motions than peptides with PFRs. Fluorescence anisotropy further revealed that peptides without PFRs exhibit slightly larger motions on the nanosecond timescale. These results demonstrate that peptides without PFRs undergo dynamic motions in the groove of MHC and consequently are able to assume diverse structures that can be recognized by T cells.

Diffracted X-ray tracking for monitoring intramolecular motion in individual protein molecules using broad band X-ray.

Diffracted X-ray tracking (DXT) enables the tilting and twisting motions of single protein molecules to be monitored with micro- to milliradian resolution using a highly brilliant X-ray source with a wide energy bandwidth. We have developed a technique to monitor single molecules using gold nanocrystals attached to individual protein molecules using the BL28B2 beamline at SPring-8. In this paper we present the installation of a single toroidal X-ray mirror at BL28B2 to focus X-rays in an energy range of 10-20 keV (ΔE/E = 82% for an X-ray with a wide energy bandwidth). With this beamline we tracked diffraction spots from gold nanocrystals over a wide angle range than that using quasi-monochromatic X-rays. Application of the wide angle DXT technique to biological systems enabled us to observe the on-site motions of single protein molecules that have been functionalized in vivo. We further extend the capability of DXT by observing the fractional tilting and twisting motions of inner proteins under various conditions. As a proof of this methodology and to determine instrumental performance the intramolecular motions of a human serum albumin complex with 2-anthracenecarboxylic acid was investigated using the BL28B2 beamline. The random tilting and twisting intramolecular motions are shown to be directly linked to the movement of individual protein molecules in the buffer solution.

Tracking 3D picometer-scale motions of single nanoparticles with high-energy electron probes.

We observed the high-speed anisotropic motion of an individual gold nanoparticle in 3D at the picometer scale using a high-energy electron probe. Diffracted electron tracking (DET) using the electron back-scattered diffraction (EBSD) patterns of labeled nanoparticles under wet-SEM allowed us to super-accurately measure the time-resolved 3D motion of individual nanoparticles in aqueous conditions. The highly precise DET data corresponded to the 3D anisotropic log-normal Gaussian distributions over time at the millisecond scale.