Assessing seizure liability in vitro with voltage-sensitive dye imaging in mouse hippocampal slices.

Non-clinical toxicology is a major cause of drug candidate attrition during development. In particular, drug-induced seizures are the most common finding in central nervous system (CNS) toxicity. Current safety pharmacology tests for assessing CNS functions are often inadequate in detecting seizure-inducing compounds early in drug development, leading to significant delays. This paper presents an in vitro seizure liability assay using voltage-sensitive dye (VSD) imaging techniques in hippocampal brain slices, offering a powerful alternative to traditional electrophysiological methods. Hippocampal slices were isolated from mice, and VSD optical responses evoked by stimulating the Schaffer collateral pathway were recorded and analyzed in the stratum radiatum (SR) and stratum pyramidale (SP). VSDs allow for the comprehensive visualization of neuronal action potentials and postsynaptic potentials on a millisecond timescale. By employing this approach, we investigated the in vitro drug-induced seizure liability of representative pro-convulsant compounds. Picrotoxin (PiTX; 1-100 µM), gabazine (GZ; 0.1-10 µM), and 4-aminopyridine (4AP; 10-100 µM) exhibited seizure-like responses in the hippocampus, but pilocarpine hydrochloride (Pilo; 10-100 µM) did not. Our findings demonstrate the potential of VSD-based assays in identifying seizurogenic compounds during early drug discovery, thereby reducing delays in drug development and providing insights into the mechanisms underlying seizure induction and the associated risks of pro-convulsant compounds.

In situ synchrotron X-ray scission of polytetrafluoroethylene chains and elucidation of dry etching.

We investigated the mechanism of polytetrafluoroethylene (PTFE) chain scission through in situ hard X-ray photoelectron spectroscopy at room temperature, 200 °C, and 230 °C. The C-C bonds in the main chain and C-F bonds in the side chains were broken, and F desorption from the PTFE surface was observed at room temperature. The formation of CF3 was also observed from the recombination of broken C-C bonds in the main chain and detached F, which were not induced by soft X-rays. In contrast, when the PTFE substrate was irradiated with hard X-rays at 200 °C, the CF3 intensity initially produced by recombination reactions decreased with irradiation time, and the photoelectron spectrum retained the original PTFE spectrum. Under these conditions, the F1s/C1s intensity ratio did not change with the irradiation time; hence, the fragment containing only CF2, the chemical composition of the original PTFE, was desorbed. When the substrate temperature was 230 °C, the CF3 intensity increased in relation to that at 200 °C. This result indicated that the formation of CF3 via recombination reactions of broken molecular chains is enhanced by thermal assistance. These phenomena were considered to be based on the balance between recombination and desorption by photochemical and pyrochemical reactions. These results will lead to a better understanding of the use of X-ray-irradiated fluorine resins and PTFE in potential space-based environments. This study will also promote the improvement of PTFE microfabrication methods and thin-film formation using synchrotron radiation.

In situ fluorescence yield soft X-ray absorption spectroscopy of electrochemical nickel deposition processes with and without ethylene glycol.

The electrochemical Ni deposition at a platinum electrode was investigated in a plating nickel bath in the presence and absence of ethylene glycol (EG) using fluorescence yield soft X-ray absorption spectroscopy (FY-XAS) in the Ni L2,3-edge and O K-edge regions under potential control. At ≤+0.35 V vs. the reversible hydrogen electrode (RHE), the electrochemical Ni deposition was detected by the Ni L2,3-edge FY-XAS in the presence of EG whereas almost no such event was observed in the absence of EG. A drastic decrease of FY-XAS intensities in the O K-edge region was also observed in the presence of EG at >+0.35 V vs. RHE, suggesting that the nano-/micro-structured Ni deposition initiated by the removal of water molecules occurs on the Pt electrode. The complex formation of Ni2+ with EG and the adsorption of EG on the Ni surface could play an important role in the Ni deposition. This study demonstrates that the in situ FY-XAS is a powerful and surface-sensitive technique to understand (electro)chemical reactions including polyol synthesis and electrocatalysis at solid-liquid interfaces.

X-ray Photoemission Spectroscopy Study of Uniaxial Magnetic Anisotropy Induced in a Ni Layer Deposited on a LiNbO3 Substrate.

The competition between magnetic shape anisotropy and the induced uniaxial magnetic anisotropy in the heterojunction between a ferromagnetic layer and a ferroelectric substrate serves to control magnetic domain structures as well as magnetization reversal characteristics. The uniaxial magnetic anisotropy, originating from the symmetry breaking effect in the heterojunction, plays a significant role in modifying the characteristics of magnetization dynamics. Magnetoelastic phenomena are known to generate uniaxial magnetic anisotropy; however, the interfacial electronic states that may contribute to the uniaxial magnetic anisotropy have not yet been adequately investigated. Here, we report experimental evidence concerning the binding energy change in the ferromagnetic layer/ferroelectric substrate heterojunction using X-ray photoemission spectroscopy. The binding energy shifts, corresponding to the chemical shifts, reveal the binding states near the interface. Our results shed light on the origin of the uniaxial magnetic anisotropy induced from the heterojunction. This knowledge can provide a means for the simultaneous control of magnetism, mechanics, and electronics in a nano/microsystem consisting of ferromagnetic/ferroelectric materials.

A lab in a bento box: an autonomous centrifugal microfluidic system for an enzyme-linked immunosorbent assay.

In this paper, we report on the demonstration of a portable immunoassay system consisting of a small centrifugal microfluidic device driver (bento box) and a centrifugal microfluidic device made of polypropylene and fabricated by injection molding. The bento box consists of a cheap DC motor and an Arduino microcontroller. It has a simple structure and is the size of a bento box, that is, 150 × 150 × 100 (W × D × H) mm3. The developed device can automatically execute an enzyme-linked immunosorbent assay (ELISA) process under a steady rotating condition because it was designed based on the principle of CLOCK, which we previously presented. Here, we first executed an ELISA using a system consisting of the bento box and a device made of polydimethylsiloxane (PDMS) and compared it with a servo-controlled device driver. It was confirmed that the results of the bento box were consistent with those of the servo-controlled device driver. The limit of detection (LOD) using the bento box was 0.759 ng ml-1. Therefore, the controllability of the bento box was demonstrated. Next, we evaluated the injection-molded device through multi-step fluid control. We confirmed, through real-time observation of the device, that accurate flow control in the designed ELISA procedure was executed. Lastly, ELISA was employed for the measurements of mouse IgG using the system consisting of the bento box and the polypropylene device. The system performed all fluidic controls within 12 min; we confirmed the specificity of the system, and the LOD was 0.320 ng ml-1.

Solid/liquid-interface-dependent synthesis and immobilization of copper-based particles nucleated by X-ray-radiolysis-induced photochemical reaction.

X-ray-radiolysis-induced photochemical reaction of a liquid solution enables the direct synthesis and immobilization of nano/micro-scale particles and their aggregates onto a desired area. As is well known, the synthesis, growth and aggregation are dependent on the pH, additives and X-ray irradiation conditions. In this study, it was found that the topography and composition of synthesized particles are also dependent on the types of substrate dipped in an aqueous solution of Cu(COOCH3)2 in the X-ray-radiolysis-induced photochemical reaction. These results are attributed to the fact that a secondary electron induced by the X-ray irradiation, surface or interface on which the particles are nucleated and grown influences the particle shape and composition. This study will shed light on understanding a novel photochemical reaction route induced under X-ray irradiation. The development of this process using the X-ray-radiolysis-induced photochemical reaction in aqueous liquids enables us to achieve the rapid and easy operation of the synthesis, growth and immobilization of special nano/micro-scale complex materials or multifunctional composites.

Controllability of cupric particle synthesis by linear alcohol chain number as additive and pH control in cupric acetate solution using X-ray radiolysis.

Synthesis and immobilization of caltrop cupric particles onto a Si substrate using X-ray radiolysis directly from a liquid solution of Cu(COOCH3)2 is demonstrated. Caltrop cupric oxide particles are formed in the X-ray radiolysis of aqueous solutions of Cu(COOCH3)2, which also contain methanol, ethanol, 2-propanol or 1-propanol as ^\bulletOH scavenger. The blade lengths of the caltrop particles are dependent on the alcohol chain length. In particular, it was found that an alkyl alcohol whose chain length is longer than four is unable to synthesize any particles in aqueous solutions of Cu(COOCH3)2 in X-ray radiolysis. These results are attributed to the alkyl alcohol chain length influencing the rate of reaction of radicals and determines the solvable ratio of its alcohol into water. In addition, it was found that the synthesized particle geometric structure and composition can also be controlled by the pH of the aqueous solution in the X-ray radiolysis. This study may open a door to understanding and investigating a novel photochemical reaction route induced under X-ray irradiation. The development of the X-ray radiolysis process enables us to achieve the rapid and easy process of synthesis and immobilization of higher-order nano/microstructure consisting of various materials.

Deep X-ray lithography system with a uniform and high-accuracy fabrication area established in beamline BL11 at NewSUBARU.

A new lithography system to fabricate high-aspect-ratio 3D microstructures was developed at the NewSUBARU synchrotron radiation facility (University of Hyogo, Japan). The X-ray beam generated by this system has high parallelism (horizontal and vertical divergence angles of 278 µrad and 14 µrad, respectively) and high photon flux (31 mW mm-2 at a beam current of 300 mA). The high photon flux and exposure area of the system were validated and a beam-scan method for a large exposure area with a uniform dose distribution has been proposed. In addition, the deep X-ray lithography performance was characterized using a conventional photosensitive material and the synchrotron-radiation-induced direct etching of polytetrafluoroethylene (PTFE) was demonstrated. An enlargement of the microfabrication area up to 100 mm × 100 mm while contemporarily ensuring high uniformity was achieved.

Caltrop particles synthesized by photochemical reaction induced by X-ray radiolysis.

X-ray radiolysis of a Cu(CH3COO)2 solution was observed to produce caltrop-shaped particles of cupric oxide (CuO, Cu2O), which were characterized using high-resolution scanning electron microscopy and micro-Raman spectrometry. X-ray irradiation from a synchrotron source drove the room-temperature synthesis of submicrometer- and micrometer-scale cupric oxide caltrop particles from an aqueous Cu(CH3COO)2 solution spiked with ethanol. The size of the caltrop particles depended on the ratio of ethanol in the stock solution and the surface of the substrate. The results indicated that there were several synthetic routes to obtain caltrop particles, each associated with electron donation. The technique of X-ray irradiation enables the rapid synthesis of caltrop cupric oxide particles compared with conventional synthetic methods.

Surface characteristics and electrical properties of PMMA chips for incubation-type planar-patch-clamp biosensors.

Polymethylmethacrylate (PMMA) plates were successfully applied as sensor chips in an incubation-type planar patch clamp (IPPC). Hot embossing both sides formed the PMMA plates, and a focused ion beam realized micropores. The low seal resistance of the IPPC was investigated by analyzing the surface roughness of the chips. Atomic force microscopy (AFM) showed that the chip surface had a roughness of several nanometers due to the molding process. Coating the molded surface with an anti-adhesive compound further increased the surface roughness of the PMMA chip because the anti-adhesive compound itself had a large roughness and in some case, the compound partially peeled off while detaching the mold. Similarly, coating a chip with extracellular matrix (ECM) poly-l-lysine (PLL) also increased the surface roughness. The measured seal resistance of the PMMA chip for an HEK293 cell was in the range of 4-15 MΩ. The low seal resistance was attributed to the sharp-edge structure of the micropore and the surface roughness of the chip. Nevertheless, the whole cell current was successfully recorded from HEK293 cells expressing channel rhodopsin wide receiver (ChRWR) using salt-bridge-type stable Ag/AgCl electrodes. Another advantage of the PMMA sensor chip was the small parasitic capacitance.

Immunoassay using microfluid filters constructed by deep X-ray lithography.

Microfluid filters were fabricated, which possessed 2,100 cylindrical through-bores (psi 40 microm) in 200 microm-thickness polymethylmethacrylate (PMMA) sheets (psi 3 mm), by deep X-ray lithography using synchrotron radiation. To evaluate the microfluid filters as a device for an immunoassay, we bound the goat anti-mouse immunoglobulin G (IgG) antibody to the surface of the filters, and set the filters between reaction vessels stacked vertically in a microreactor. An enzyme-linked immunosorbent assay (ELISA) of mouse IgG using the goat anti-mouse IgG/horseradish-peroxidase (HRP) conjugate indicated that mouse IgG could be quantitatively detected in the range of 0-100 ng/ml, demonstrating the applicability of vertical microfluidic operation to the immunoassay.

Enzyme-linked immunosorbent assay for nonylphenol using antibody-bound microfluid filters in vertical fluidic operation.

We developed an enzyme-linked immunosorbent assay for an endocrine disrupter, nonylphenol, using a microreactor composed of two reaction vessels stacked vertically through a microfluid filter. The filters constructed by deep X-ray lithography possessed 2100 through-bores (phi40 x 200 microm) in polymethylmethacrylate sheets (phi3 mm), which are appropriate for biochemical reactions. Through the optimization of the immunoassay, nonylphenol was quantitatively detected at the range of 0.1-10 ng/ml.

Microfluidic chip toward cellular ATP and ATP-conjugated metabolic analysis with bioluminescence detection.

In this article, a microfluidic platform integrating capillary electrophoresis and bioluminescence (BL) detection that was fabricated in poly(dimethylsiloxane) (PDMS) with lab-on-a-chip technology was demonstrated for cellular metabolic analyses. A microchannels network, "cross combining with Y", was designed to perform on-chip sample preparation, separation, and BL detection of ATP and ATP-conjugated metabolites, using firefly luciferin-luciferase BL system. A dynamic modification of the channel wall of PDMS proved to be crucial to reverse the direction of electroosmotic flow (EOF), which was uniquely achieved by a prewash cycle with a cationic surfactant didodecyldimethylammonium bromide. The influences of surfactant on the EOF and BL reaction were also investigated. Quantitative analyses revealed a dynamic linear range over 2 orders of magnitude for ATP, with a detection limit down to submicromolar (midattomole). The method was validated by measuring cellular ATP of E. coli. with direct on-chip cell lysis. Further work was emphasized on ATP-conjugated metabolite analysis, using galactose as an example. Assays of galactose in human urine samples confirmed the reliability of the protocol, which revealed good prospect of this platform for ATP-conjugated submetabolomic profiling.

Characterization of molecular transport in poly(dimethylsiloxane) microchannels for electrophoresis fabricated with synchrotron radiation-lithography and UV-photolithography.

In the present paper, a study was undertaken of molecular transport in ploy(dimethylsiloxane) microchannels that were fabricated by ultraviolet (UV)-photolithography and synchrotron radiation (SR)-lithography characterized and compared for microchip capillary electrophoresis by evaluating in-channel molecular dispersion. A fluorescent tag, sulforhodamine B was used as the probing molecule. It was found that microchannels made by SR-lithography fabrication were superior to those made by UV-photolithography fabrication in terms of molecular transport performance. A deep insight into surface conditions characterized by scanning electron microscopy suggested it was related to the difference in surface roughness. Chromatographic retention in electropherograms further supported such a conclusion, which depended on the phase ratio of the channel surface. The results revealed for PDMS microchannels in this work were in good agreement with the phenomenon found for glass microchannels in the literature.

Chemiluminescence detection for a microchip capillary electrophoresis system fabricated in poly(dimethylsiloxane).

Chemiluminescence (CL) detection integrated with a microchip capillary electrophoresis (MCE) system that was fabricated in poly(dimethylsiloxane) was demonstrated for chemical and biochemical analyses. Two model CL systems were involved here: metal ion-catalyzed luminol-peroxide reaction and dansyl species conjugated peroxalate-peroxide reaction. Different strategies based on three chip patterns (cross, cross combining with Y, and cross combining with V) to perform on-line CL detection for MCE were evaluated and compared in terms of sensitivity, reproducibility, and peak symmetry. The chip pattern of cross combining with Y proved to be promising for the luminol-peroxide CL system, while the chip pattern of cross combining with V was preferred for the peroxalate-peroxide system where CL reagent could not be effectively transported by electroosmotic flow. A detection limit down to submicromolar concentrations (midattomole) was achieved with good reproducibility and symmetric peak shape. Successful separation of three metal cations such as Cr(III), Co(II), and Cu(II) and chiral recognition of dansyl phenylalanine enantiomers within 1 min revealed distinct advantages of combining MCE with CL detection for rapid and sensitive analyses.