Modulation of viability of live cells by focused ion-beam exposure.

Introduction of membrane-impermeant substances into living cells is the key method to understand contemporary cellular processes by investigating cellular responses and phenotypes. Here, we performed gold ion beam exposure into live cells by using the focused ion beam implantation method, which was originally developed to precisely control semiconductor device performances. We evaluated the viability of the gold-irradiated cells by measuring the concentration of adenosine triphosphate (ATP), which is an intracellular energy source produced in the mitochondrial membrane. The viability of the irradiated cells was found to be 20% higher than that of the unirradiated control cells. The atoms might promote the energy generating processes within the mitochondrion. Our results suggest that the viability of living cells can be modulated by accurately controlling the dopant atom numbers. Our technique may be considered as a potential tool in life and medical sciences to quantitatively elucidate the dose-dependent effects of dopants.

Single-molecule study on the decay process of the football-shaped GroEL-GroES complex using zero-mode waveguides.

It has been widely believed that an asymmetric GroEL-GroES complex (termed the bullet-shaped complex) is formed solely throughout the chaperonin reaction cycle, whereas we have recently revealed that a symmetric GroEL-(GroES)(2) complex (the football-shaped complex) can form in the presence of denatured proteins. However, the dynamics of the GroEL-GroES interaction, including the football-shaped complex, is unclear. We investigated the decay process of the football-shaped complex at a single-molecule level. Because submicromolar concentrations of fluorescent GroES are required in solution to form saturated amounts of the football-shaped complex, single-molecule fluorescence imaging was carried out using zero-mode waveguides. The single-molecule study revealed two insights into the GroEL-GroES reaction. First, the first GroES to interact with GroEL does not always dissociate from the football-shaped complex prior to the dissociation of a second GroES. Second, there are two cycles, the "football cycle " and the "bullet cycle," in the chaperonin reaction, and the lifetimes of the football-shaped and the bullet-shaped complexes were determined to be 3-5 s and about 6 s, respectively. These findings shed new light on the molecular mechanism of protein folding mediated by the GroEL-GroES chaperonin system.

Adsorption mechanism of ribosomal protein L2 onto a silica surface: a molecular dynamics simulation study.

A large-scale molecular dynamics simulation was carried out in order to investigate the adsorption mechanism of ribosomal protein L2 (RPL2) onto a silica surface at various pH values. RPL2 is a constituent protein of the 50S large ribosomal subunit, and a recent experimental report showed that it adsorbs strongly to silica surfaces and that it can be used to immobilize proteins on silica surfaces. The simulation results show that RPL2, especially domains 1 (residues 1-60) and 3 (residues 203-273), adsorbed more tightly to the silica surface above pH 7. We found that a major driving force for the adsorption of RPL2 onto the silica surface is the electrostatic interaction and that the structural flexibility of domains 1 and 3 may further contribute to the high affinity.

Green tea epigallocatechin gallate exhibits anticancer effect in human pancreatic carcinoma cells via the inhibition of both focal adhesion kinase and insulin-like growth factor-I receptor.

The exact molecular mechanism by which epigallocatechin gallate (EGCG) suppresses human pancreatic cancer cell proliferation is unclear. We show here that EGCG-treated pancreatic cancer cells AsPC-1 and BxPC-3 decrease cell adhesion ability on micro-pattern dots, accompanied by dephosphorylations of both focal adhesion kinase (FAK) and insulin-like growth factor-1 receptor (IGF-1R) whereas retained the activations of mitogen-activated protein kinase and mammalian target of rapamycin. The growth of AsPC-1 and BxPC-3 cells can be significantly suppressed by EGCG treatment alone in a dose-dependent manner. At a dose of 100 µM which completely abolishes activations of FAK and IGF-1R, EGCG suppresses more than 50% of cell proliferation without evidence of apoptosis analyzed by PARP cleavage. Finally, the MEK1/2 inhibitor U0126 enhances growth-suppressive effect of EGCG. Our data suggests that blocking FAK and IGF-1R by EGCG could prove valuable for targeted therapy, which can be used in combination with other therapies, for pancreatic cancer.

Measuring relative-story displacement and local inclination angle using multiple position-sensitive detectors.

We propose a novel sensor system for monitoring the structural health of a building. The system optically measures the relative-story displacement during earthquakes for detecting any deformations of building elements. The sensor unit is composed of three position sensitive detectors (PSDs) and lenses capable of measuring the relative-story displacement precisely, even if the PSD unit was inclined in response to the seismic vibration. For verification, laboratory tests were carried out using an Xθ-stage and a shaking table. The static experiment verified that the sensor could measure the local inclination angle as well as the lateral displacement. The dynamic experiment revealed that the accuracy of the sensor was 150 µm in the relative-displacement measurement and 100 µrad in the inclination angle measurement. These results indicate that the proposed sensor system has sufficient accuracy for the measurement of relative-story displacement in response to the seismic vibration.

Development of an ion beam alignment system for real-time scanning tunneling microscope observation of dopant-ion irradiation.

An ion beam alignment system has been developed in order to realize real-time scanning tunneling microscope (STM) observation of "dopant-ion" irradiation that has been difficult due to the low emission intensity of the liquid-metal-ion-source (LMIS) containing dopant atoms. The alignment system is installed in our original ion gun and STM combined system (IG/STM) which is used for in situ STM observation during ion irradiation. By using an absorbed electron image unit and a dummy sample, ion beam alignment operation is drastically simplified and accurized. We demonstrate that sequential STM images during phosphorus-ion irradiation are successfully obtained for sample surfaces of Si(111)-7x7 at room temperature and a high temperature of 500 degrees C. The LMIS-IG/STM equipped with the developed ion beam alignment system would be a powerful tool for microscopic investigation of the dynamic processes of ion irradiation.

Real-time imaging of single-molecule fluorescence with a zero-mode waveguide for the analysis of protein-protein interaction.

Real-time imaging of single-molecule fluorescence with a zero-mode waveguide (ZMW) was achieved. With modification of the ZMW geometry, the signal-to-background ratio is twice that obtainable with a conventional ZMW. The improved signal-to-background ratio makes it possible to visualize individual binding-release events between chaperonin GroEL and cochaperonin GroES at a concentration of 5 microM. Two rate constants representing two-timer kinetics in the release of GroES from GroEL were measured with the ZMW, and the measurements agreed well with those made with a total internal reflection fluorescence microscopy. These results indicate that the novel ZMW makes feasible the direct observation of protein-protein interaction at an intracellular concentration in real time.

Effect of the C-terminal truncation on the functional cycle of chaperonin GroEL: implication that the C-terminal region facilitates the transition from the folding-arrested to the folding-competent state.

To elucidate the exact role of the C-terminal region of GroEL in its functional cycle, the C-terminal 20-amino acid truncated mutant of GroEL was constructed. The steady-state ATPase rate and duration of GroES binding showed that the functional cycle of the truncated GroEL is extended by approximately 2 s in comparison with that of the wild type, without interfering with the basic functions of GroEL. We have proposed a model for the functional cycle of GroEL, which consists of two rate-limiting steps of approximately 3- and approximately 5-s duration (Ueno, T., Taguchi, H., Tadakuma, H., Yoshida, M., and Funatsu, T. (2004) Mol. Cell 14, 423-434 g). According to the model, detailed kinetic studies were performed. We found that a 20-residue truncation of the C terminus extends the time until inorganic phosphate is generated and the time for arresting protein folding in the central cavity, i.e. the lifetime of the first rate-limiting step in the functional cycle, to an approximately 5-s duration. These results suggest that the integrity of the C-terminal region facilitates the transition from the first to the second rate-limiting state.

A molecular simulation study of an organosilane self-assembled monolayer/SiO2 substrate interface.

The bonding network of an alkylsilane self-assembled monolayer (SAM)SiO(2) substrate interface is investigated by means of canonical Monte Carlo (MC) simulations. SAMSiO(2) systems with different interfacial bonding topologies are sampled by the Metropolis MC method, and the AMBER potential with a newly developed organosilicon parameters are used to obtain an optimized structure with a given bonding topology. The underlying substrates are modeled as hydroxy-terminated (100) or (111) cristobalites. The SAMSiO(2) interface is characterized by a polysiloxane bonding network which comprises anchoring bonds and cross-linking bonds, namely, molecule-substrate and molecule-molecule Si-O-Si bonds, respectively. We show that at thermal equilibrium, the ratio of the number of anchoring bonds to cross-linking bonds decreases as a total Si-O-Si bond density increases, and that nevertheless, number of anchoring bonds always dominate over that of cross-linking bonds. Moreover we show that the total Si-O-Si bond density strongly affects the lateral ordering of the alkylsilane molecules, and that increase in the Si-O-Si bond density disorders the molecular packing. Our results imply that a lab-to-lab variation in the experimentally prepared SAMs can be attributed to different Si-O-Si bond densities at the SAMSiO(2) interface.

A novel approach to Au nanoparticle-based identification of DNA nanoarrays.

The combination of electron beam lithography and gold nanoparticle-based detection method is subject to a novel high-resolution approach to detecting DNA nanoarrays. In this work, gold nanoparticle-based detection of DNA hybridization on nanostructured arrays is presented. The nanostructured arrays were created by electron beam lithography of a self-assembled monolayer. Amine groups, which are active moieties and are used for attachment of DNA, were introduced to the nanostructures, and the amine-modified structures were characterized by scanning Maxwell-stress microscopy (SMM) for seeing the modification process. The DNA probe covalently immobilized within the nanostructures was hybridized with a biotinylated target DNA. Streptavidin-gold conjugate was then bound to the biotin, thereby assembling inside the nanostructured arrays. The sequence-specific hybridization was imaged by atomic force microscopy (AFM). On the other hand, the activity of the DNA molecules within the nanostructured arrays was verified by fluorescence microscopy using streptavidin-Cy 5 conjugate instead of streptavidin-gold nanoparticles conjugate. On the basis of fluorescent detection, an alternative method has been developed for detection of DNA nanostructures, which will benefit the development of DNA chips.

Production of nanopatterns by a combination of electron beam lithography and a self-assembled monolayer for an antibody nanoarray.

We report on a flexible method of producing antibody (IgG) nanopatterns by combining electron beam (EB) lithography and a perfluorodecyltriethoxysilane (FDTES) self-assembled monolayer (SAM). Using EB lithography of the FDTES SAM, we easily fabricated IgG patterns with feature sizes on the order of 100 nm. The patterned IgG retained its ability to interact specifically with an anti-LgG. The influence of different concentrations of the IgG and anti-IgG on the resulting fluorescent IgG arrays was investigated. These IgG nanopatterns appeared to be remarkably well controlled and showed almost no detectable nonspecific binding of proteins on a hydrophobic SAM under a suitable incubation condition, characterized by atomic force microscopy, and epi-fluorescence microscopy. The technique enables the realization of high-throughput protein nanoscale arrays with high specificity.

Characterization of DNA hybridization on partially aminated diamond by aromatic compounds.

Here, we report a novel method of micropatterning oligonucleotides via aromatic groups as linkers on partially amino-terminated diamond and the inherence on subsequent hybridization. The covalent immobilization of probe oligonucleotides and characterization of immobilized probe oligonucleotides with carboxylic compounds were investigated by X-ray photoelectron spectroscopy (XPS). To confirm the effects of linker flexibility in a low amino group on diamond for probe oligonucleotides, three kinds of dicarboxylic compound--adipic acid, terephthalic acid, and trimesic acid--were used for immobilization of probe oligonucleotides, like linkers; and these oligonucleotides were hybridized with target oligonucleotides labeled with Cy 5 on the micropatterned diamond surface. The hybridization intensities determined by epifluorescence microscopy were compared and analyzed.

Label-free DNA sensors using ultrasensitive diamond field-effect transistors in solution.

Charge detection biosensors have recently become the focal point of biosensor research, especially field-effect-transistors (FETs) that combine compactness, low cost, high input, and low output impedances, to realize simple and stable in vivo diagnostic systems. However, critical evaluation of the possibility and limitations of charge detection of label-free DNA hybridization using silicon-based ion-sensitive FETs (ISFETs) has been introduced recently. The channel surface of these devices must be covered by relatively thick insulating layers ( SiO2, Si3N4, Al2O3, or Ta2O5) to protect against the invasion of ions from solution. These thick insulating layers are not suitable for charge detection of DNA and miniaturization, as the small capacitance of thick insulating layers restricts translation of the negative DNA charge from the electrolyte to the channel surface. To overcome these difficulties, thin-gate-insulator FET sensors should be developed. Here, we report diamond solution-gate FETs (SGFETs), where the DNA-immobilized channels are exposed directly to the electrolyte solution without gate insulator. These SGFETs operate stably within the large potential window of diamond (>3.0 V). Thus, the channel surface does not need to be covered by thick insulating layers, and DNA is immobilized directly through amine sites, which is a factor of 30 more sensitive than existing Si-ISFET DNA sensors. Diamond SGFETs can rapidly detect complementary, 3-mer mismatched (10 pM) and has a potential for the detection of single-base mismatched oligonucleotide DNA, without biological degradation by cyclically repeated hybridization and denature.

New linear-parabolic rate equation for thermal oxidation of silicon.

We propose a new oxidation rate equation for silicon supposing only a diffusion of oxidizing species but not including any rate-limiting step by interfacial reaction. It is supposed that diffusivity is suppressed in a strained oxide region near SiO(2)/Si the interface. The expression of a parabolic constant in the new equation is the same as that of the Deal-Grove model, while a linear constant makes a clear distinction with that of the model. The estimated thickness using the new expression is close to 1 nm, which compares well with the thickness of the structural transition layers.

DNA micropatterning on polycrystalline diamond via one-step direct amination.

We report a novel method of one-step direct amination on polycrystalline diamond to produce functionalized surfaces for DNA micropatterning by photolithography. Polycrystalline diamond was exposed to UV irradiation in ammonia gas to generate amine groups directly. After patterning, optical microscopy confirmed that micropatterns covered with an Au mask were regular in size and shape. The regions outside the micropatterns were passivated with fluorine termination by C3F8 plasma, and the chemical changes on the two different surfaces--the amine groups inside the patterned regions by one-step direct amination and fluorine termination outside the patterned regions--were characterized by spatially resolved X-ray photoelectron spectroscopy (XPS). The patterned areas terminated with active amine groups were then immobilized with probe DNA via a bifunctional molecule. The sequence specificity was conducted by hybridizing fluorescently labeled target DNA to both complementary and noncomplementary probe DNA attached inside the micropatterns. The fluorescence micropatterns observed by epifluorescence microscopy corresponded to those imaged by optical microscopy. DNA hybridization and denaturation experiments on a DNA-modified diamond show that the diamond surfaces reveal superior stability. The influence of a different amination time on fluorescence intensity was compared. Different terminations as passivated layers were investigated, and as a result, fluorine termination points to the greatest signal-to-noise ratio.

Enhancing semiconductor device performance using ordered dopant arrays.

As the size of semiconductor devices continues to shrink, the normally random distribution of the individual dopant atoms within the semiconductor becomes a critical factor in determining device performance--homogeneity can no longer be assumed. Here we report the fabrication of semiconductor devices in which both the number and position of the dopant atoms are precisely controlled. To achieve this, we make use of a recently developed single-ion implantation technique, which enables us to implant dopant ions one-by-one into a fine semiconductor region until the desired number is reached. Electrical measurements of the resulting transistors reveal that device-to-device fluctuations in the threshold voltage (Vth; the turn-on voltage of the device) are less for those structures with ordered dopant arrays than for those with conventional random doping. We also find that the devices with ordered dopant arrays exhibit a shift in Vth, relative to the undoped semiconductor, that is twice that for a random dopant distribution (- 0.4 V versus -0.2 V); we attribute this to the uniformity of electrostatic potential in the conducting channel region due to the ordered distribution of dopant atoms. Our results therefore serve to highlight the improvements in device performance that can be achieved through atomic-scale control of the doping process. Furthermore, ordered dopant arrays of this type may enhance the prospects for realizing silicon-based solid-state quantum computers.

Selective deposition of polystyrene nanoparticles in a nanoetchpit array on a silicon substrate.

Nanometer-sized polystyrene particles were selectively deposited by interfacial tension in nanometer-sized etchpit arrays made on a silicon substrate.

Patterning of DNA nanostructures on silicon surface by electron beam lithography of self-assembled monolayer.

Nanoscale patterns of modified oligonucleotides are produced on octadecyltrimethoxysilane self-assembled monolayers at a silicon surface by electron beam lithography. DNA structures with feature sizes of the order of 250 nm were detected by epi-fluorescence microscopy.