Toshinari Isono

Selective detection of sub-atto-molar Streptavidin in 10 13 -fold impure sample using photonic crystal nanolaser sensors

Biosensors selectively detecting a very small amount of biomarker protein in human blood are desired for early and reliable diagnoses of severe diseases. This paper reports the detection of protein (streptavidin: SA) in ultra-low concentration, with an ultra-high selectivity against contaminants, using photonic crystal nanolasers. For biotin-modified nanolasers in pure water with SA, an extremely-low detection limit of 16 zM is evaluated. Even in a mixture with 1 μM bovine serum albumin as the contaminant, 100 zM SA is detected, meaning a selectivity of 10(13). These are remarkable capabilities that are promising for practical biosensing in the medical applications mentioned above.

Amyloid-β25–35 induces impairment of cognitive function and long-term potentiation through phosphorylation of collapsin response mediator protein 2

Alzheimers disease (AD) is characterized by amyloid-β (Aβ) protein and tau deposition in the brain. Numerous studies have reported a central role of Aβ in the development of AD, but the pathogenesis is not well understood. Collapsin response mediator protein 2 (CRMP2), an intracellular protein mediating a repulsive axon guidance molecule, Semaphorin3A, is also accumulated in neurofibrillary tangles in AD brains. To gain insight into the role of CRMP2 phosphorylation in AD pathogenesis, we investigated the effects of Aβ neurotoxicity in CRMP2 phosphorylation-deficient knock-in (crmp2(ki/ki)) mice, in which the serine residue at 522 was replaced with alanine. Intracerebroventricular (i.c.v.) injection of Aβ₂₅₋₃₅ peptide, a neurotoxic fragment of Aβ protein, to wild-type (wt) mice increased hippocampal phosphorylation of CRMP2. Behavioral assessment revealed that i.c.v. injection of Aβ₂₅₋₃₅ peptide caused impairment of novel object recognition in wt mice, while the same peptide did not in crmp2(ki/ki) mice. In electrophysiological recording, wt and crmp2(ki/ki) mice have similar input-output basal synaptic transmission and paired-pulse ratios. However, long-term potentiation was impaired in hippocampal slices of Aβ₂₅₋₃₅ peptide-treated wt but not those of crmp2(ki/ki). Our findings indicate that CRMP2 phosphorylation is required for Aβ-induced impairment of cognitive memory and synaptic plasticity.

Selective adsorption of protein molecules on phase-separated sapphire surfaces

Site-selective adsorption of protein molecules was found on sapphire surfaces that exhibit a phase separation into two domains: weakly charged hydrophobic domain and negatively charged hydrophilic one. Ferritin and bovine serum albumin molecules, which are negatively charged in a buffer solution, are adsorbed to the hydrophobic domains. Avidin molecules, which are positively charged, are adsorbed to the other domain. Fibrinogen molecules, which consist of both negative and positive modules, are adsorbed to the whole sapphire surface. Hemoglobin molecules, whose net charge is almost zero, are also adsorbed to the whole surfaces. These results indicate that electrostatic double layer interaction is the primary origin of the observed selectivity. Dependence of protein adsorption or desorption behaviors on the pH value can also be interpreted by the proposed model.

Evolution of supported planar lipid bilayers on step-controlled sapphire surfaces.

Self-organized step/terrace structures on a sapphire surface were used to investigate interface properties between a solid surface and a supported planar lipid bilayer (SPB). We prepared random-stepped, single-stepped and multistepped sapphire surfaces. Some multistepped surfaces covered with crossing steps exhibit phase-separation into hydrophilic and hydrophobic domains. We studied evolution of self-spreading lipid bilayers that are subject to the atomic structures and chemical states on the surfaces. The growth direction of SPBs in the self-spreading method is regulated by the atomic steps. While the SPBs were apparently uniform after a 1 h self-spreading, a density gradient of the lipid molecules was observed even after 24 h spreading. We found that various patterns of the SPBs that depend on the density of the lipid molecules are self-assembled on the phase-separated surfaces. Although the SPB is supported on the sapphire surface via an about 1 nm water layer, the self-spreading direction and the morphology of the SPBs are affected by the atomic steps, whose height is much smaller than that of the water layer.

Amino- and carboxyl-terminal domains of Filamin-A interact with CRMP1 to mediate Sema3A signalling.

Reorganization of the actin cytoskeleton is an early cellular response to various extracellular signals. Sema3A, a repulsive axon guidance molecule, induces the reorganization of actin cytoskeleton in the growth cones. Collapsin response mediator protein 1 (CRMP1) mediates the intracellular Sema3A signalling through its Ser522 phosphorylation. Here we show that UNC-33, CRMP1 C. elegans homologue, interacts with FLN-1, an actin-binding Filamin-A orthologue. In nematodes, this interaction participates in the projection of DD/VD motor neurons. CRMP1 binds both the actin-binding domain and the last immunoglobulin-like repeat of Filamin-A. The alanine mutants of Filamin-A or CRMP1 in their interacting residues suppress the Sema3A repulsion in neurons. Conversely, a phosphor-mimicking mutant CRMP1(Ser522Asp) enhances the Sema3A response. Atomic-force microscopy analysis reveals that the V-shaped Filamin-A changes to a condensed form with CRMP1(Ser522Asp). CRMP1(Ser522Asp) weakens the F-actin gelation crosslinked by Filamin-A. Thus, phosphorylated CRMP1 may remove Filamin-A from the actin cytoskeleton to facilitate its remodelling.

Photonic crystal nanolaser sensors with ALD coating

Nanolaser sensors achieve ultrahigh sensitivity for biomolecules while unexpected noise was an issue. In this study, it was suppressed by ALD coating. Higher permittivity coating improved the sensitivity, suggesting unknown principles of this sensor.

Selective detection of sub-atto-molar streptavidin in 10^13 fold impure sample using nanoslot photonic crystal nanolaser

Biosensors which can selectively detect a very small amount of biomarker protein in human blood are desired toward early diagnoses of severe diseases. However, no methods simultaneously satisfy the requirements such as high sensitivity, high selectivity, simple detection, and immediacy. We succeeded in detecting ultra-low-concentration streptavidin (SA) even in a highly impure sample using nanoslot photonic crystal (PC) nanolasers. This nanolaser consists of GaInAsP semiconductor slab with a periodic airhole array. Since the total device area is no larger than 20 × 20 μm2, highthroughput fabrication is possible even using e-beam lithography. Moreover, it is easy to operate by photopumping through free-space optics. Since the evanescent wave of the laser mode penetrates from the PC slab, the laser wavelength changes sensitively to the environmental index. In the sensing experiment, we first functionalized the devices with biotin, and then measured the wavelength in ultrapure water before and after immersing in the solutions with various concentrations of SA. As a result, we evaluated that the detection limit of SA is 16 zM. In another experiment, we put 1 μM BSA into the solution as a contaminant, and repeated the same measurement. We detected 100 zM SA even in the impure solution only when biotin is functionalized in advance, meaning a selectivity ratio (BSA / SA) of 1013. Thus this device achieves unprecedentedly high sensitivity and selectivity in addition to the simple fabrication and fast sensing. It is very promising as a point of care device for medical diagnoses.

Photonic crystal nanoslot nanolaser for ultra-high sensitivity and selectivity protein sensing

GaInAsP photonic crystal nanoslot nanolaser with strong mode localization was used for specific detection of streptavidin. A record low detection limit of 16 zM and an ultra-high selectivity of 1012 against high-concentration contaminants were observed.

Fabrication of Carbon Nanotube Films for Electric Packaging

Printed wiring board (PWB) for electric packaging requires high density and microscaled wiring. As miniaturization of the Cu wiring proceeds, an increase in the resistance of Cu wires and a decrease in the reliability due to electro-migration will be more and more serious problems. This drawback is similar to one of the problems in large scale integrations (LSIs). Carbon nanotubes (CNTs) are promising material for LSI interconnects, because CNTs have very unique properties, such as a capability for a high current density, mechanical strength, and high thermal conductivity. CNTs are ideal materials for wiring or reinforcement of the Cu interconnects in the packaging technology, because interconnects with a low resistance and a high current density is realized. Up to now, multiwalled carbon nanotubes (MWNTs) have been applied to the via hole interconnects. In this study, MWNTs are synthesized by a remote plasma chemical vapor deposition (PCVD) at low temperature [1]. Because interconnect process in the LSI process is performed below 400°C the PCVD technique can be used. When CNTs are applied to the PWB, on the other hand, a PCVD is unsuitable because the materials used in the PWBs are generally low-heat-resistance materials. We have to develop a new technique suitable for the PWB technology. In this paper, we describe a new fabrication technique of CNTs wiring on PWBs, in which the whole process is performed at room temperature or at least below 150°C and basically in liquid phase. Therefore the present technique is compatible with the conventional PWB process.

Fabrication of carbon nanotube thin films by surface engineering

We have investigated uniform carbon nanotube (CNT) dispersion in a solution and fabrication of CNT thin films on solid surfaces. To prevent bundle formation, we have examined surface modification of both the CNT surfaces and the substrate surfaces. To enhance the interaction between surfaces of the substrate and the CNTs, the substrate surfaces were made hydrophobic, because CNTs are generally hydrophobic. When CNTs were dispersed on the hydrophobic surfaces, relatively uniform CNT thin films were formed. To weaken the interaction between the individual CNTs, CNT surfaces were coated with synthetic polymers or biomolecules. Amphiphilic, ionic polymers with pi-stacking are most suitable for CNT solubilization in a polar solvent. High-density well-dispersed CNT thin films were fabricated by modifying CNT surfaces with single-stranded DNAs.