Highly sensitive near-infrared fluorescent probes for nitric oxide and their application to isolated organs

Novel near-infrared (NIR) fluorescent probes for nitric oxide (NO) have been designed, synthesized, and evaluated. Their NIR fluorescence was increased in an NO concentration-dependent manner under physiological conditions, and their reaction efficiency with NO was at least 53 times higher than that of a widely used NO probe, DAF-2. They were confirmed to function in isolated intact rat kidneys. Because NIR light can penetrate deeply into tissues, these probes may have potential for in vivo NO imaging.     

Integration of immunoassay into extended nanospace

We report on the first successful miniaturization of sandwich immunoassay into an extended nanospace channel for the detection of alpha-fetoprotein. A fused silica microchip with a nanochannel flanked by two microchannels, to facilitate liquid handling, was constructed using photo- and electron beam lithography, ICP and thermal bonding. Reagents could be selectively introduced into the nanochannel and the flow be stopped by using a pressure controller that alternatively opened and closed inlet and outlet ports. The construction and handling of the nano-in-microchip device for immunoassay is described.     

Design of Photostable,Activatable Near‐Infrared Photoacoustic Probes Using Tautomeric Benziphthalocyanine as a Platform

Near‐infrared (NIR) imaging techniques have attracted significant attention for biological and medicinal applications due to the ability of NIR to penetrate deeply into tissues. However, there are very few stable, activatable molecular probes that can utilize NIR light in the wavelength range beyond 800 nm. Herein, we report a new activatable NIR system for photoacoustic imaging based on tautomeric benziphthalocyanines (BPcs). We found that the existence of a free hydroxyl group is crucial for NIR absorption of BPcs. Synthesized water‐soluble hydroxy BPcs exhibited high photostability and no fluorescence, which are desirable features for photoacoustic imaging. We synthesized BPcs in which the free hydroxyl group was masked by an esterase‐labile or an H₂O₂‐labile group. The photoacoustic signals of these hydroxy‐masked BPcs were increased upon NIR excitation at 880 nm in the presence of esterase or H₂O₂, respectively. These are rare examples of activatable probes utilizing NIR light at around 900 nm.     

A Novel Transparent Polyacetylene Bearing a Triphenylamine Moiety Prepared with a [Rh(norbornadiene)Cl]2 Catalyst

Novel soluble polyacetylenes having a triphenylamine moiety were synthesized by a [Rh(norbornadiene)Cl]₂ catalyst under quite mild conditions in fairly high yields. The obtained polymer showed to have trans-transoid sequence as the major main-chain structure which was generated through the so-called cis-to-trans isomerization was already induced even during the polymerization. The HCl doping of the polymer was resulted in the formation of the oxidized polymer where the so-called polaron or bipolaron was produced. In addition, we found that in the case of the pristine polymer unpaired electrons mainly localized on the triphenylamine moiety as the side chain. On the other hand, after the doping the unpaired electron delocalized on the main chain of the polymer.     

Inter-lamellar interactions modulated by addition of guest components

We have investigated the effects of a guest component (polymer or spherical colloidal particle) confined between flexible lamellar slits on the inter-lamellar interaction by means of a small-angle X-ray scattering technique and a neutron spin echo technique. The dominant interaction between flexible lamellar membranes without guest components is the Helfrich mechanism. The addition of a neutral polymer into the lamellar phase induces an attractive inter-lamellar interaction and finally destabilizes the lamellar phase. On the other hand, spherical colloidal particles confined between flexible lamellar membranes reduce the undulational fluctuations of lamellae and bring a repulsive inter-lamellar interaction. The behavior of the layer compression modulus of the lamellar membrane containing colloidal particles is well described by the entropical repulsive inter-lamellar interaction driven by steric hindrance.Received: 26 March 2004, Published online: 4 May 2004PACS: 82.70.Uv Surfactants, micellar solutions, vesicles, lamellae, amphiphilic systems, (hydrophilic and hydrophobic interactions) - 61.25.Hq Macromolecular and polymer solutions; polymer melts; swelling - 83.80.Hj Suspensions, dispersions, pastes, slurries, colloids - 89.75.Fb Structures and organization in complex systems     

Arsenic metabolism in a freshwater food chain: Blue–green alga (Nostoc sp.)→ shrimp (Neocaridina denticulata)→ carp (Cyprinus carpio)

Bioaccumulation and biomethylation of inorganic arsenic were investigated in a three-step fresh-water food chain consisting of an autotroph (blue- green alga: Nostoc sp.), a herbivore (shrimp: Neocaridina denticulata) and a carnivore (carp: Cyprinus carpio). The autotroph, herbivore and carnivore survived in arsenic-containing water below 1000, 2 and 60 mg As(V) dm^?3, respectively. Bioaccumulation of arsenate by Nostoc sp. was decreased with an increase in the nitrogen concentration of the medium. Arsenic(V) was accumulated from the water phase and part-methylated by the carp, as well as by the algae and shrimp. Arsenic was mostly accumulated in the gut of the carp. The predominant arsenical in the guts was the monomethylarsenic species. Arsenic accumulation via food in the above three-step food chain decreased by one order of magnitude and the relative concentration of methylated arsenic to the total arsenic accumulated increased successively with an elevation in the trophic level. When arsenicals were transferred via the food chain, no monomethylarsenic, or only a trace amount, was detected in the three organisms. Dimethylarsenic in the alga, both dimethyl- and trimethyl-arsenic in shrimp, and trimethyl-arsenic in carp, were the predominant methylated arsenic species, respectively.     

An enzymatically activated fluorescence probe for targeted tumor imaging

Beta-galactosidase is a widely used reporter enzyme, but although several substrates are available for in vitro detection, its application for in vivo optical imaging remains a challenge. To obtain a probe suitable for in vivo use, we modified our previously developed activatable fluorescence probe, TG-betaGal (J. Am. Chem. Soc. 2005, 127, 4888-4894), on the basis of photochemical and photophysical experiments. The new probe, AM-TG-betaGal, provides a dramatic fluorescence enhancement upon reaction with beta-galactosidase, and further hydrolysis of the ester moiety by ubiquitous intracellular esterases affords a hydrophilic product that is well retained within the cells without loss of fluorescence. We used a mouse tumor model to assess the practical utility of AM-TG-betaGal, after confirming that tumors in the model could be labeled with an avidin-beta-galactosidase conjugate. This conjugate was administered to the mice in vivo, followed by AM-TG-betaGal, and subsequent ex vivo fluorescence imaging clearly visualized intraperitoneal tumors as small as 200 microm. This strategy has potential clinical application, for example, in video-assisted laparoscopic tumor resection.     

β-Galactosidase fluorescence probe with improved cellular accumulation based on a spirocyclized rhodol scaffold

We identified a rhodol bearing a hydroxymethyl group (HMDER) as a suitable scaffold for designing fluorescence probes for various hydrolases. HMDER shows strong fluorescence at physiological pH, but phenolic O-alkylation of HMDER results in a strong preference for the spirocyclic form, which has weak fluorescence. As a proof of concept, we utilized this finding to develop a new fluorescence probe for β-galactosidase. This probe has favorable characteristics for imaging in biological samples: it has good cellular permeability, and its hydrolysis product is well-retained intracellularly. It could rapidly and clearly visualize β-galactosidase activity in cultured cells and in Drosophila melanogaster tissue, which has rarely been achieved with previously reported fluorescence probes.     

Detection of nonfluorescent molecules using differential interference contrast thermal lens microscope for extended nanochannel chromatography

An ultrasensitive absorbance detector, the differential interference contrast thermal lens microscope (DIC-TLM), was employed for a chromatography system using silica nanochannel. Recently, separation of ultrasmall volume sample has been strongly required for single-cell biological and chemical analysis. Previously, we have developed a chromatography system using nanochannels of ~100 nm scale (extended nanochannels) fabricated on a silica substrate. The extended nanochromatography realized highly efficient separation of samples <1 fL without packing materials. However, its detection method was limited to fluorescence method due to the small volume, and a new detector based on absorbance has been required. On the contrary, we have also developed DIC-TLM, a photothermal spectrometer based on absorption and thermal relaxation of sample for determination of concentration of nonfluorescent molecules in extended nanochannel. In this paper, we combined the extended nanochromatography and the DIC-TLM for separation and detection of nonfluorescent dyes. Particularly, basic performances of the DIC-TLM including quantitative performance and sensitivity were deliberated for injected samples of ~fL volume.