Algal growth inhibition test with TEMPO-oxidized cellulose nanofibers.

Ecotoxicity data on cellulose nanofibers (CNFs) are limited despite their wide potential applications prospects, such as structural and packaging materials, filters, coatings, foods, and cosmetics. In this study, toxicity tests of 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-oxidized CNFs (TEMPO-CNFs), which are one of the major CNF products commercially available in Japan, on the green alga Raphidocelis subcapitata were conducted. As nanomaterials are considered difficult-to-test substances, the Organisation for Economic Co-operation and Development has released a guidance document that provides considerations regarding ecotoxicity tests of nanomaterials. In the algal growth inhibition tests of TEMPO-CNFs, there were specific issues to be examined, including the effects of medium components on the characteristics of TEMPO-CNFs, CNF interference with algal density measurements, algal interference with CNF measurements, and the effects of ion concentration changes in the test medium by the addition of CNFs on algal growth. To examine these issues, we conducted preliminary studies and established a suitable test method for algal growth inhibition tests of TEMPO-CNFs. We confirmed that the components in the medium for algal growth inhibition tests had negligible effects on the characteristics (zeta-potential, viscosity, and morphology) and concentration stability of TEMPO-CNFs and that in vitro and in vivo fluorescence measurements were applicable for estimating the algal densities, without interference by TEMPO-CNFs. In contrast, we observed that the grown algae interfered with the CNF concentration measurements. Therefore, we established a method to correct the measured CNF concentrations by estimating the algal contribution. Furthermore, we found that the nutrient salt concentrations in the medium changed due to interactions with CNFs; however, this change did not affect algal growth. Based on the results of the preliminary studies, algal growth inhibition tests of TEMPO-CNFs were conducted using in vitro and in vivo fluorescence measurements, along with measurements of CNFs and ion concentrations in the test dispersions. The test results showed that no growth inhibition was observed on growth rate or yield even at the maximum CNF concentration of 100 mg/L, suggesting that the ecological effect of TEMPO-CNFs on algae was relatively low. The results of this study will be valuable for conducting ecotoxicity assessments on additional CNFs and comparable nanomaterials in future studies.

Size distributions of cellulose nanocrystals in dispersions using the centrifugal sedimentation method.

Nanocellulose is a remarkable biomaterial. It is a plastic alternative with significance from the viewpoint of carbon offset and neutrality. To efficiently develop nanocellulose-based functional materials, it is imperative to evaluate their dispersion states. In this study, the sedimentation equivalent diameter distributions of cellulose nanocrystals (CNC) are analyzed by centrifugal sedimentation. The diameter distribution is well correlated with that estimated from the widths and the lengths of the CNCs obtained by transmission electron microscopy. Hence, centrifugal sedimentation has the potential to assess the dispersion states of nanocellulose on the nanometer scale and should contribute to basic research and applications.

Elevated Myl9 reflects the Myl9-containing microthrombi in SARS-CoV-2-induced lung exudative vasculitis and predicts COVID-19 severity.

The mortality of coronavirus disease 2019 (COVID-19) is strongly correlated with pulmonary vascular pathology accompanied by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-triggered immune dysregulation and aberrant activation of platelets. We combined histological analyses using field emission scanning electron microscopy with energy-dispersive X-ray spectroscopy analyses of the lungs from autopsy samples and single-cell RNA sequencing of peripheral blood mononuclear cells to investigate the pathogenesis of vasculitis and immunothrombosis in COVID-19. We found that SARS-CoV-2 accumulated in the pulmonary vessels, causing exudative vasculitis accompanied by the emergence of thrombospondin-1-expressing noncanonical monocytes and the formation of myosin light chain 9 (Myl9)-containing microthrombi in the lung of COVID-19 patients with fatal disease. The amount of plasma Myl9 in COVID-19 was correlated with the clinical severity, and measuring plasma Myl9 together with other markers allowed us to predict the severity of the disease more accurately. This study provides detailed insight into the pathogenesis of vasculitis and immunothrombosis, which may lead to optimal medical treatment for COVID-19.

Patterning of graphene using wet etching with hypochlorite and UV light.

Graphene patterning via etching is important for enhancing or controling the properties of devices and supporting their applications in micro- and nano-electronic fields. Herein, we present a simple, low-cost, and scalable wet etching method for graphene patterning. The technique uses hypochlorite solution combined with ultraviolet light irradiation to rapidly remove unwanted graphene areas from the substrate. Raman spectroscopy, atomic force microscopy, scanning electron microscopy, and optical microscopy results showed that well-patterned graphene with micrometer scale regions was successfully prepared. Furthermore, graphene field effect transistor arrays were fabricated, and the obtained devices exhibited good current-voltage characteristics, with maximum mobility of ~ 1600 cm2/Vs, confirming the feasibility of the developed technique.

Single-Walled Carbon Nanotube Membranes Accelerate Active Osteogenesis in Bone Defects: Potential of Guided Bone Regeneration Membranes.

Carbon nanotubes (CNTs) are potentially important biomaterials because of their chemical, physical, and biological properties. Our research indicates that CNTs exhibit high compatibility with bone tissue. The guided bone regeneration (GBR) technique is commonly applied to reconstruct alveolar bone and treat peri-implant bone defects. In GBR, bone defects are covered with a barrier membrane to prevent the entry of nonosteogenic cells such as epithelial cells and fibroblasts. The barrier membrane also maintains a space for new bone formation. However, the mechanical and biological properties of materials previously used in clinical practice sometimes delayed bone regeneration. In this study, we developed a CNT-based membrane for GBR exhibiting high strength to provide a space for bone formation and provide cellular shielding to induce osteogenesis. The CNT membrane was made via the dispersion of single-walled CNTs (SWCNTs) in hyaluronic acid solution followed by filtration. The CNT membrane assumed a nanostructure surface due to the bundled SWCNTs and exhibited high strength and hydrophilicity after oxidation. In addition, the membrane promoted the proliferation of osteoblasts but not nonosteogenic cells. CNT membranes were used to cover experimental bone defects made in rat calvaria. At 8 weeks after surgery, more extensive bone formation was observed in membrane-covered defects compared with bone defects not covered with membrane. Almost no diffusion of CNTs was observed around the membrane. These results indicate that the CNT membrane has adequate strength, stability, and surface characteristics for osteoblasts, and its shielding properties promote bone formation. Demonstration of the safety and osteogenic potential of the CNT membranes through further animal studies should facilitate their clinical application in GBR.

Streptavidin-Conjugated Oxygen-Doped Single-Walled Carbon Nanotubes as Near-Infrared Labels for Immunoassays.

Single-walled carbon nanotubes (CNTs) are promising candidates for near-infrared (NIR) fluorescent labels in diagnostic fields. We report a complex of oxygen-doped CNT (o-CNT) and streptavidin (SA) for preparing CNT-based NIR labels with a high reaction efficiency in immunoassays. This complex specifically binds to biotin molecules by conjugating a linker molecule of phospholipid polyethylene glycol (PL-PEG) to SA (o-CNT-SA). The immunoprecipitation reaction efficiency between o-CNT-SA and biotin reaches 79.3% when the surface of o-CNTs is uniformly covered with SA-conjugated PL-PEG. The strong affinity between SA and biotin is useful for preparing CNT-based sensitive NIR fluorescent labels.

N2 Gas Adsorption Sites of Single-Walled Carbon Nanotube Bundles: Identifying Interstitial Channels at Very Low Relative Pressure.

Utilizing the nanoscale space created by carbon nanotubes (CNTs) is of importance for applications like energy storage devices, sensors, and functional materials. Gas adsorption is a versatile, quantitative characterization method to analyze nanoscale pore sizes and volumes. Here, we inspected N2 adsorption to the nanospace formed by the bundles of single-walled CNTs with an average nanotube diameter of ca. 2.0 nm and its distributions of 0.7-4.1 nm. Based on comparisons among the as-grown, purified (opened), and heat-treated (closed) CNTs with similar geometric bundle structures, we found that the interstitial channels emerged from a very low relative pressure of approximately 10-8 by removing the impurities from the CNT bundles, which is the first empirical demonstration. These findings can not only be utilized to understand the structures of CNT films, fibers, and bulks but also applied to porous materials science.

Characterization and Biodistribution Analysis of Oxygen-Doped Single-Walled Carbon Nanotubes Used as in Vivo Fluorescence Imaging Probes.

Single-walled carbon nanotubes (SWCNTs) show strong fluorescence in the 1000-1700 nm second near-infrared (NIR-II) wavelength range and are considered promising candidates for angiographic imaging probes. Oxygen-doped SWCNTs coated with phospholipid-polyethylene glycol (o-SWCNT-PEG) show exceptional potential, as they emit fluorescence at ∼1300 nm through excitation with 980 nm light. Here, with the aim of putting o-SWCNTs to practical use as an angiographic agent in animal experiments, the retention time after intravenous administration in the vasculature of mice and the biodistribution were studied. To provide bio affinity, the o-SWCNTs were coated with phospholipid polyethylene glycol. The intravenously injected o-SWCNT-PEG circulated within the vasculature for 3 h and cleared within 1 day. There was prominent fluorescence and Raman signals from the SWCNTs in the liver and spleen early in the experiment; the signals remained for 1 month. No apparent abnormalities in weight or appearance were observed after 2 months, suggesting low toxicity of o-SWCNT-PEG. These characteristics of o-SWCNT-PEG would make it useful as an angiographic imaging probe in the NIR-II wavelength range.

Delayed Increase in Near-Infrared Fluorescence in Cultured Murine Cancer Cells Labeled with Oxygen-Doped Single-Walled Carbon Nanotubes.

The labeling technique for cells with over-thousand-nanometer near-infrared (OTN-NIR) fluorescent probes has attracted much attention for in vivo deep imaging for cell tracking and cancer metastasis, because of low scattering and absorption of OTN-NIR light by biological tissues. However, the intracellular behavior following the uptake of the single-walled carbon nanotubes (SWCNTs), an OTN-NIR fluorophore, remains unknown. The aim of this study is to investigate the time-dependent change in OTN-NIR fluorescence images of cultured murine cancer cells (Colon-26) following treatment with a recently developed OTN-NIR fluorescent probe, epoxide-type oxygen-doped SWCNTs (o-SWCNTs). The o-SWCNTs were synthesized by oxygenation of SWCNTs by ozone under ultraviolet irradiation and were dispersed in an aqueous solution of N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl- sn-glycero-3-phosphoethanolamine to prepare biocompatible o-SWCNTs (o-SWCNT-PEG). OTN-NIR fluorescent o-SWCNT-PEG showed an abnormal behavior following cellular uptake. OTN-NIR fluorescence was not observed in the cells after 24 h incubation with the o-SWCNT-PEG, but clearly increased with longer incubation time from three days after the treatment. This result was further confirmed by Raman microscopy, suggesting that OTN-NIR fluorescence intensity was associated with the cellular uptake of the o-SWCNT-PEG. These results suggest that the Colon-26 cells were successfully labeled by the o-SWCNT-PEG that emit OTN-NIR fluorescence. The o-SWCNT-PEG may aggregate in the cells over time, which could favor their internalization. This delayed concentration followed by a long retention of the o-SWCNT-PEG in cells will facilitate further biotechnological applications of the o-SWCNTs to in vivo deep OTN-NIR fluorescent imaging.

Oxygen-doped carbon nanotubes for near-infrared fluorescent labels and imaging probes.

Chemical modification of carbon nanotube surface can controllably modulate their optical properties. Here we report a simple and effective synthesis method of oxygen-doped single-walled carbon nanotubes (o-SWCNTs), in which a thin film of SWCNTs is just irradiated under the UV light for a few minutes in air. By using this method, the epoxide-type oxygen-adducts (ep-SWCNTs) were produced in addition to the ether-type oxygen-adducts (eth-SWCNTs). The Treated (6, 5) ep-SWCNTs show a red-shifted luminescence at ~1280 nm, which corresponds to the most transparent regions for bio-materials. Immunoassay, fluorescence vascular angiography and observation of the intestinal contractile activity of mice were demonstrated by using the produced o-SWCNTs as infrared fluorescent labels and imaging agents.

Carbon Nanotubes as Fluorescent Labels for Surface Plasmon Resonance-Assisted Fluoroimmunoassay.

The photoluminescence properties of carbon nanotubes (CNTs), including the large Stokes shift and the absence of fluorescent photobleaching, can be used as a fluorescent label in biological measurements. In this study, the performance of CNTs as a fluorescent label for surface plasmon resonance (SPR)-assisted fluoroimmunoassay is evaluated. The fluorescence of (8, 3) CNTs with an excitation wavelength of 670 nm and an emission wavelength of 970 nm is observed using a sensor chip equipped with a prism-integrated microfluidic channel to excite the SPR. The minimum detectable concentration of a CNT dispersed in water using a visible camera is 0.25 µg/mL, which is equivalent to 2 × 1010 tubes/mL. The target analyte detection using the CNT fluorescent labels is theoretically investigated by evaluating the detectable number of CNTs in a detection volume. Assuming detection of virus particles which are bound with 100 CNT labels, the minimum number of detectable virus particles is calculated to be 900. The result indicates that CNTs are effective fluorescent labels for SPR-assisted fluoroimmunoassay.

Single atom spectroscopy: Decreased scattering delocalization at high energy losses, effects of atomic movement and X-ray fluorescence yield.

Single atom localization and identification is crucial in understanding effects which depend on the specific local environment of atoms. In advanced nanometer scale materials, the characteristics of individual atoms may play an important role. Here, we describe spectroscopic experiments (electron energy loss spectroscopy, EELS, and Energy Dispersed X-ray spectroscopy, EDX) using a low voltage transmission electron microscope designed towards single atom analysis. For EELS, we discuss the advantages of using lower primary electron energy (30 keV and 60 keV) and higher energy losses (above 800 eV). The effect of atomic movement is considered. Finally, we discuss the possibility of using atomically resolved EELS and EDX data to measure the fluorescence yield for X-ray emission.

Preparation of small-sized graphene oxide sheets and their biological applications.

By using carbon nanohorns as starting materials, small- and uniform-sized graphene oxide (S-GO) sheets can be prepared in high yields via an oxidation method. The obtained S-GO sheets have a band-like structure with a length of 20-50 nm, a width of 2-10 nm, and a thickness of 0.5-5 nm. S-GO sheets are hydrophilic due to abundant oxygenated groups on the surfaces and edges; hence, this nanomaterial is highly dispersive in aqueous solutions and some hydrophilic organic solvents. Additionally, like other S-GO samples, the S-GO sheets prepared here are strongly fluorescent over the visible light wavelength region. These characteristics underscore the high potential of S-GO sheets for nanomedical and diagnostic applications. In proof-of-concept experiments, the S-GO sheets were conjugated with an arginine-glycine-aspartic acid derivative for tumour-targeting drug delivery applications, and with an immunoglobulin G antibody for immunoassay applications.

Single molecular spectroscopy: identification of individual fullerene molecules.

We report the molecule-by-molecule spectroscopy of individual fullerenes by means of electron spectroscopy based on scanning transmission electron microscopy. Electron energy-loss fine structure analysis of carbon 1s absorption spectra is used to discriminate carbon allotropes with known symmetries. C(60) and C(70) molecules randomly stored inside carbon nanotubes are successfully identified at a single-molecular basis. We show that a single molecule impurity is detectable, allowing the recognition of an unexpected contaminant molecule with a different symmetry. Molecules inside carbon nanotubes thus preserve their intact molecular symmetry. In contrast, molecules anchored at or sandwiched between atomic BN layers show spectral modifications possibly due to a largely degraded structural symmetry. Moreover, by comparing the spectrum from a single C(60) molecule and its molecular crystal, we find hints of the influence of solid-state effects on its electronic structure.

Diameter selective electron transfer from encapsulated ferrocenes to single-walled carbon nanotubes.

The diameter selective photoluminescence quenching of single-walled carbon nanotubes (SWCNTs) is observed upon ferrocene encapsulation, which can be attributed to electron transfer from the encapsulated ferrocenes to the SWCNTs. Interestingly, the dependence of the electron transfer process on the nanotube diameter is governed by the molecular orientation of the ferrocenes in the SWCNT rather than the reduction potentials of the SWCNT.

Electron diffraction and electron energy-loss spectroscopy studies of a hybrid material composed of coronene molecules encapsulated in single-walled carbon nanotubes.

Electron diffraction and electron energy-loss spectroscopy (EELS) studies were conducted on bundles of single-walled carbon nanotubes encaging coronene molecules (coronenes@SWCNTs). Selected area electron diffraction (SAED) pattern of the coronenes@SWCNTs suggests that the coronene molecules inside the SWCNTs are separated into segments. Each segment is a stack consisting of ∼ 10 molecules and has a different tilted condition with respect to the nanotube axis. EELS spectra of the coronenes@SWCNTs show characteristic structures due to interband transitions between the van Hove singularities of the SWCNTs, and also π-plasmon and π + σ plasmon (volume plasmon) peaks. The volume plasmon energy of 23.0 eV for the coronene@SWCNTs is larger than that of an empty SWCNT bundle, indicating a contribution from the valence electrons of the coronene molecules. This value for the volume plasmon energy was reproduced using a model with an average of 85% filling of the SWCNTs by the coronene molecules. Therefore, both the SAED and EELS observations suggest that the SWCNTs are highly filled with coronene molecules. Further indication was that interband transition energies of coronene molecules of the present coronenes@SWCNTs material may be different from those in isolated ones and/or in solid state.

Immunoassay with single-walled carbon nanotubes as near-infrared fluorescent labels.

The intrinsic photoluminescence of single-walled carbon nanotubes (CNTs) in the near-infrared (NIR) above 1000 nm makes them promising candidates for biological probes owing to low interference by bioorganic molecules and deep tissue penetration. We here demonstrate an immunoassay by using a NIR CNT labels conjugated to immunoglobulin G (IgG) antibodies. Most of the CNT-conjugated IgG was successfully immunoprecipitated with protein G-attached magnetic beads and eluted from them, which was confirmed by the NIR emission of the conjugated CNTs at 1000-1200 nm. The photoluminescence intensity of the CNT labels was strong enough to detect antigens at 600 pM by our simple procedures.

Single-molecule sensing electrode embedded in-plane nanopore.

Electrode-embedded nanopore is considered as a promising device structure for label-free single-molecule sequencing, the principle of which is based on nucleotide identification via transverse electron tunnelling current flowing through a DNA translocating through the pore. Yet, fabrication of a molecular-scale electrode-nanopore detector has been a formidable task that requires atomic-level alignment of a few nanometer sized pore and an electrode gap. Here, we report single-molecule detection using a nucleotide-sized sensing electrode embedded in-plane nanopore. We developed a self-alignment technique to form a nanopore-nanoelectrode solid-state device consisting of a sub-nanometer scale electrode gap in a 15 nm-sized SiO(2) pore. We demonstrate single-molecule counting of nucleotide-sized metal-encapsulated fullerenes in a liquid using the electrode-integrated nanopore sensor. We also performed electrical identification of nucleobases in a DNA oligomer, thereby suggesting the potential use of this synthetic electrode-in-nanopore as a platform for electrical DNA sequencing.

Microwave assisted covalent functionalization of C(60)@SWCNT peapods.

The covalent functionalization of the external wall of C(60)@SWCNT peapods, by in situ generated aryl diazonium salts, assisted by microwave irradiation is reported. Spectroscopic, thermal and microscopy characterization was performed. Electrochemistry revealed the three reversible reductions of encapsulated C(60), however, shifted towards positive potentials when compared with those of intact C(60).