Mizuo Maeda

Rapid naked-eye detection of mercury ions based on non-crosslinking aggregation of double-stranded DNA-carrying gold nanoparticles

Colorimetric detection of mercury ions (Hg(2+)) with the naked eye was accomplished within 1 min by a combination of non-crosslinking aggregation of double-stranded DNA-carrying gold nanoparticles and complex formation of thymine-Hg(2+)-thymine.

Cyclic RGD peptide-labeled upconversion nanophosphors for tumor cell-targeted imaging

One of the great challenges of oncology is to improve methods for early tumor detection. Thus tumor cell-targeted optical imaging has been intensively studied. Bioimaging with upconversion (UC) phosphors (UCPs) is of considerable interest due to a variety of possible applications taking advantage of infrared-to-visible luminescence. Here we report for the first time tumor cell-targeted UC imaging using UCPs modified with cyclic RGD peptide (RGD-Y2O3). Cyclic RGD peptide binds specifically to integrin alphavbeta3 which is highly expressed in a tumor cell surface of certain cancer types but not in normal tissues. Since UC emission from RGD-Y2O3 was observed for U87MG cancer cell (high integrin alphavbeta3 expression), but not for MCF-7 cancer cell (low integrin alphavbeta3 expression), this UC imaging is considered to be integrin alphavbeta3 specific. The non-invasive imaging of integrin alphavbeta3 expression using UCP-based probes will have great potential in cancer imaging in general in living subjects.

Power-free sequential injection for microchip immunoassay toward point-of-care testing

This paper presents a simple fluid handling technique for microchip immunoassay. Necessary solutions were sequentially injected into a microchannel by air-evacuated poly(dimethylsiloxane), and were passively regulated by capillary force at the inlet opening. For heterogeneous immunoassay, microchips are potentially useful for reduction of sample consumption and assay time. However, most of the previously reported microchips have limitations in their use because of the needs for external power sources for fluid handling. In this paper, an on-chip heterogeneous immunofluorescence assay without such an external power source is demonstrated. The microchip consisting of poly(dimethylsiloxane) (PDMS) and glass has a simple structure, and therefore is suitable for single-use applications. Necessary solutions were sequentially injected into a microchannel in an autonomous fashion with the power-free pumping technique, which exploits the high solubility and the rapid diffusion of air in PDMS. For deionized water, this method yielded flow rates of 3-5 nL s-1 with reproducibility of 4-10%. The inlet opening of the microchannel functioned as a passive valve to hold the solution when the flow was finished. Rabbit immunoglobulin G (rIgG) and human C-reactive protein (CRP) were detected using the microchannel walls as reaction sites. With the sample consumption of 1 microL and the assay time of approximately 20 min including the antibody immobilization step, the sandwich immunoassay methods for rIgG and CRP exhibited the limits of detection of 0.21 nM (0.21 fmol) and 0.42 nM (0.42 fmol), respectively.

Bovine Insulin Filaments Induced by Reducing Disulfide Bonds Show a Different Morphology, Secondary Structure, and Cell Toxicity from Intact Insulin Amyloid Fibrils

Amyloid fibrils are associated with more than 20 diseases, including Alzheimers disease and type II diabetes. Insulin is a 51-residue polypeptide hormone, with its two polypeptide chains linked by one intrachain and two interchain disulfide bonds, and has long been known to self-assemble in vitro into amyloid fibrils. We demonstrate here that bovine insulin forms flexible filaments in the presence of a reducing agent, Tris (2-carboxyethyl) phosphine. The insulin filaments, possibly formed due to partial reduction of S-S bonds in insulin molecules, differ from intact insulin fibrils in terms of their secondary structure. The insulin filaments were determined to have an antiparallel beta-sheet structure, whereas the insulin fibrils have a parallel beta-sheet structure. Of importance, the cell toxicity of the insulin filaments was remarkably lower than that of the insulin fibrils. This finding supports the idea that cell toxicity of amyloids correlates with their morphology. The remarkably low toxicity of the filamentous structure should shed new light on possible pharmacological approaches to the various diseases caused by amyloid fibrils.

Controlling the number and positions of oligonucleotides on gold nanoparticle surfaces

We describe a new method to immobilize a given number of oligonucleotides (ODNs) on gold nanoparticles (AuNPs) in a specific arrangement directed by a geometrical template made of DNA. The basic strategy is as follows. First, a set of thiolated ODNs for immobilization and a nonthiolated template are hybridized to make a DNA nanostructure. Next, the DNA nanostructure is reacted with AuNPs via the thiol groups to form a complex of the AuNP and the DNA nanostructure. Finally, the intended AuNP/ODN conjugate is obtained by removing the template from the complex. The above strategy enables us to make various formats of AuNP/ODN conjugates simply by changing the design of the DNA nanostructure. We demonstrate proof-of-concept experiments using a linear design of the DNA nanostructure.

MEASUREMENTS OF ELECTRON TEMPERATURE, ELECTRON DENSITY, AND NEUTRAL DENSITY IN A RADIO-FREQUENCY INDUCTIVELY COUPLED PLASMA

Electron temperature, electron density, and neutral atom density were measured in a radio‐frequency (rf) inductively coupled plasma using Thomson and Rayleigh scattering of laser radiation. Measurements were made in an argon discharge for pressures from 1 to 20 mTorr and input rf powers from 100 to 500 W. Spatial distribution profiles were measured for discharges with different aspect ratios. Electron temperature was found to depend on pressure but only weakly on power. Electron density depended strongly on both pressure and power. The neutral density was found to be significantly depleted in the plasma center and this depletion was attributed to heating of the neutrals by charged particle collisions. These results were compared to a simple model of inductively coupled plasmas.

Easy design of logic gates based on aptazymes and noncrosslinking gold nanoparticle aggregation

We have developed an easy method for constructing aptazyme-based logic gates using noncrosslinking gold nanoparticle aggregation.

Two‐dimensional laser‐induced fluorescence imaging of a pulsed‐laser deposition process of YBa2Cu3O7−x

Two‐dimensional time‐resolved density distributions of ground state barium (Ba) and copper (Cu) atoms as well as ground state yttrium oxide (YO) molecules have been measured by two‐dimensional laser‐induced fluorescence during a pulsed‐laser deposition (PLD) process of YBa2Cu3O7−x. The gas phase of PLD has been investigated at ambient oxygen gas pressures between vacuum and 1 Torr. Characteristic behaviors have been observed for each of the measured ground state species. This is due to different oxidation schemes with the ambient oxygen gas. Whereas YO molecules mainly formed near the expanding front of the ablation plume, Ba atoms immediately oxidized at the plume front. Cu atoms, however, did not react during the gas phase of PLD. In addition, a well defined boundary of the expanding front of the ablation plume has been observed at oxygen gas pressures above 100 mTorr. Measurements of density distributions of emissive Ba, Cu, and YO are also discussed.

Photoinduced hydrogen production by direct electron transfer from photosystem I cross-linked with cytochrome c3 to [NiFe]-hydrogenase.

Abstract The photosynthetic reaction center is an efficient molecular device for the conversion of light energy to chemical energy. In a previous study, we synthesized the hydrogenase/photosystem I (PSI) complex, in which Ralstonia hydrogenase was linked to the cytoplasmic side of Synechocystis PSI, to modify PSI so that it photoproduced molecular hydrogen (H2). In that study, hydrogenase was fused with a PSI subunit, PsaE, and the resulting hydrogenase-PsaE fusion protein was self-assembled with PsaE-free PSI to give the hydrogenase/PSI complex. Although the hydrogenase/PSI complex served as a direct light-to-H2 conversion system in vitro, the activity was totally suppressed by adding physiological PSI partners, ferredoxin (Fd) and ferredoxin-NADP+-reductase (FNR). In the present study, to establish an H2 photoproduction system in which the activity is not interrupted by Fd and FNR, position 40 of PsaE from Synechocystis sp. PCC6803, corresponding to the Fd-binding site on PSI, was selected and targeted for the cross-linking with cytochrome c3 (cytc3) from Desulfovibrio vulgaris. The covalent adduct of cytc3 and PsaE was stoichiometrically assembled with PsaE-free PSI to form the cytc3/PSI complex. The NADPH production by the cytc3/PSI complex coupled with Fd and FNR decreased to approximately 20% of the original activity, whereas the H2 production by the cytc3/PSI complex coupled with hydrogenase from Desulfovibrio vulgaris was enhanced 7-fold. Consequently, in the simultaneous presence of hydrogenase, Fd, and FNR, the light-driven H2 production by the hydrogenase/cytc3/PSI complex was observed (0.30 μmol H2/mg chlorophyll/h). These results suggest that the cytc3/PSI complex may produce H2 in vivo.

Arraying heterotypic single cells on photoactivatable cell-culturing substrates

This article describes a photochemical method for the site-selective assembly of heterotypic cells on a glass substrate modified with a silane coupling agent having a caged functional group. Silane coupling agents having a carboxyl (COOH), amino (NH 2), hydroxyl (OH), or thiol (SH) group protected by a photocleavable 2-nitrobenzyl group were synthesized to modify the surfaces of glass coverslips. The caged substrates were first coated by the adsorption of a blocking agent, bovine serum albumin (BSA), to make the entire surface non-cell-adhesive and then irradiated at 365 nm under a standard fluorescence microscope. The photocleavage reaction on the surface was followed by contact angle measurements and X-ray photoelectron spectroscopy. When COS7, NIH3T3, and HEK293 cells were seeded onto these substrates in a serum-free medium, the cells adhered selectively and efficiently to the irradiated regions on the caged NH 2 substrate, whereas the other caged COOH, SH, and OH substrates were nonphotoactivatable for cell adhesion. Qualitative and quantitative analysis of BSA adsorbed to the uncaged substrates revealed that this highly efficient photoactivation on the caged NH 2 substrate arose because of the following reasons: (i) upon photoactivation, BSA adsorbed in advance on the 2-nitrobenzyl groups was readsorbed onto the uncaged functional groups and (ii) BSA readsorbed onto the NH 2 groups became unable to passivate the surface against cell adhesion whereas BSA on the other groups still had normal passivating activity. It was also demonstrated that heterotypic single COS7, NIH3T3, and HEK293 cells were positioned at any desired arrangement on the caged NH 2 substrate by repeating the UV irradiation at optimized array spot sizes and cell seeding in optimized cell concentrations. The present method will be particularly useful in studying the dynamic processes of cell-cell interactions at a single-cell level.