Yasuhiro Horiike

Light-Regulated Activation of Cellular Signaling by Gold Nanoparticles That Capture and Release Amines

A photoresponsive nanocarrier for amines based on gold nanoparticles (GNPs) having a photocleavable succinimidyl ester has been developed. It offers a useful platform for the synthesis of caged compounds. Using the GNPs, we have developed caged histamine for the first time and applied it to evoke intracellular signaling by controlled near-UV irradiation. The present work will allow for new possibilities in studies of inter- and intracellular signaling networks.

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.

Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

Abstract The development of methods for the off–on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG7). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni2+-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII7–10) to the irradiated regions. In contrast, when bovine serum albumin—a major serum protein—was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

Influences of electroosmotic flows in nanopillar chips on DNA separation: Experimental results and numerical simulations

The various potential factors affecting the performance of nanopillar chips on DNA separation were investigated from the viewpoints of both numerical calculations and actual experiments. To yield higher performance and replace the conventional DNA separation techniques such as microchip electrophoresis, the phenomenon specific to the nanopillar chips should be deeply understood. In this paper, although various factors affecting the performance of the nanopillar chips are considered, we focused on the effect of electroosmotic flow, which is particularly noticeable in quartz-made nanopillar chips. High-resolution separation of DNA was realized when an electroosmotic flow was suppressed by simply using a higher concentration of buffer, but DNA separation failed in the presence of an electroosmotic flow. It was confirmed from the numerical simulations and the direct observations that the deformation of DNA band during the injection process was induced by electroosmotic flow and consequently led to a poor resolution.

Silane coupling agent bearing a photoremovable succinimidyl carbonate for patterning amines on glass and silicon surfaces with controlled surface densities

Patterned immobilization of synthetic and biological ligands on material surfaces with controlled surface densities is important for various bioanalytical and cell biological purposes. This paper describes the synthesis, characterization, and application of a novel silane coupling agent bearing a photoremovable succinimidyl carbonate, which enables the photopatterning of various primary amines on glass and silicon surfaces. The silane coupling agent is 1-[5-methoxy-2-nitro-4-(3-trimethoxysilylpropyloxy)phenyl]ethyl N-succinimidyl carbonate. The distinct feature of this molecule is that it has a photocleavable 2-nitrobenzyl switch between a trimethoxysilyl group and a succinimidyl carbonate, each reactive to the hydroxy groups of inorganic oxides and primary amines. Based on this molecular design, the compound allows for the one-step introduction of succinimidyl carbonates onto the surface of glass and silicon, immobilization of primary amines, and region-selective and dose-dependent release of the amines by near-UV irradiation. Therefore, we were able to pattern amine ligands on the substrates in given surface densities and arbitrary geometries by controlling the doses and regions of photoirradiation. These features were verified by UV-vis spectroscopy, contact angle measurements, infrared (IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), ellipsometry, and atomic force microscopy (AFM). The compound was applied to form a chemical density gradient of amino-biotin on a silicon substrate in a range of 0.87-0.12 chains/nm(2) by controlling photoirradiation under a standard fluorescence microscope. Furthermore, we also succeeded in forming a chemical density gradient at a lower surface density range (0.15-0.011 chains/nm(2)) on the substrate by diluting the feed amino-biotin with an inert control amine.

Battery-less wireless communication system through human body for in-vivo healthcare chip

This paper proposes a battery-less wireless communication system for in-vivo healthcare chip. We measured attenuation characteristics through a human body equivalent for six frequencies. Measured attenuation of 13.56 MHz is 47 dB through 15 cm thickness of human body equivalent. It is too difficult to use the usual modulations under such low power consumption. Then, we implemented the proposed system using the 13-56 MHz band with pulse interval modulation (PIM). In the simulated result, 16 mV of output voltage can be obtained at the outside receiver when coupling factor is 0.1. We also investigate antenna structure, and a tablet structure is suitable for the proposed system.

In Vivo Batteryless Wireless Communication System for Bio-MEMS Sensors

We propose a batteryless wireless communication system for in vivo healthcare chips. The system uses inductive coupling at 13.56 MHz with internal and external coils, and employs pulse interval modulation (PIM) to endure the large attenuation in the human body. A wireless communication circuit is presented in this paper, and 16 mV of output voltage can be obtained at the external receiver. The antenna coil structure is investigated, and it is found that a tablet structure is suitable for the proposed system.

Precise patterning of photoactivatable glass coverslip for fluorescence observation of shape-controlled cells

The shape of cells is a key determinant of cellular fates and activities. In this study, we demonstrate a method for controlling the cellular shape on a chemically modified glass coverslip with micropatterned cell adhesiveness. The glass surface was chemically modified with an alkylsiloxane monolayer having a caged carboxyl group, where single-cell-sized hydrophilic islands with hydrophobic background were created by irradiating the substrate in contact with a photomask to produce the carboxyl group. Thus, the created surface hydrophilicity pattern was converted to a negative pattern of a protein-repellent amphiphilic polymer, Pluronic F108, according to its preferential adsorption to the hydrophobic surfaces. The following addition of a cell-adhesive protein, fibronectin, resulted in its selective adsorption to the irradiated regions. In this way, cell-adhesive islands were produced reproductively, and the cells formed a given shape on the islands. As examples of the cell-shape control, we seeded HeLa cells and NIH3T3 cells to an array of triangular spots, and fluorescently imaged the dynamic motions of cell protrusions extended from the periphery of the cells. The present method will not only be useful for studying the molecular mechanism of cell polarity formation, but also for studying other shape-related cellular events such as apoptosis, differentiation and migration.

RF attenuation characteristics for in vivo wireless healthcare chip

We investigate a wireless communication system for an in vivo healthcare chip. In this paper, we present measured attenuation characteristics through the human body at several frequencies. In the measurement, we use physiological saline and fresh meat instead of a real human body. From the measured results, we found that 13.56 MHz has an attenuation of 47 dB and is suitable for the proposed system.

One-Chip Integration of Rapid Diagnosis Infectious-Disease Chip Based on New Phenomena of DNA Trap and Denature in Nanogaps

We demonstrate the concept of simple and rapid diagnosis chips for infectious diseases by analyzing viral DNA from the plasma of patients. DNA was manipulated simply in a chip-integrated pretreatment device and nanogap array. A simple and rapid detection of target DNA by purification and hybridization with a number of DNA trapped in a pretreatment device and nanogap array was performed. T4DNA was immobilized and purified on micropillars in the pretreatment device. Purified T4DNA was further transported to the nanogap array and fixed by stretching. The fixed T4DNA was denatured into single-stranded (ss) DNA in the array. When a probe DNA was introduced into the nano-gap array to hybridize the trapped ssDNA, an intense luminescence was observed in the array. These results provide a simple and rapid detection method of target DNA of various infectious diseases.