Ken Hirano

Indirect micromanipulation of single molecules in water-in-oil emulsion.

Based on real‐time observation and micromanipulation, analytical methods for single DNA molecules have been under development for some time. Precise manipulation, however, is still difficult because single molecules are too small for conventional techniques. We have developed a chemical reaction system that uses water droplets in oil as containers of materials. The water droplets can be manipulated by optical force. The manipulation of the water droplets permits the fusion of two selected droplets. This process corresponds to mixing of different samples. We designate this system as “w/o (water‐in‐oil emulsion) microreactor system”, and each droplet can be thought of as a “microreactor”. In this system, single molecules can be manipulated readily, as a molecule can be contained in a μm‐sized microreactor. The microreactor utilizes extremely small quantities of samples, therefore, reactions are rapid, as diffusion times in the microreactor are very short. The manipulation technique of the microreactors based on optical force has been applied to induce fusion between microreactors loaded with DNA and YOYO, a fluorescent dye that binds to DNA. This fusion induced a rapid binding of YOYO.

Near-infrared laser-triggered carbon nanohorns for selective elimination of microbes

Carbon nanomaterials, such as carbon nanohorns and carbon nanotubes, have attracted considerable attention for their biomedical applications. We report here the first application of carbon nanohorns (CNHs) as potent laser therapeutic agents for highly selective elimination of microorganisms. This is the first report, supported by direct observations, of the highly selective elimination of yeast and bacteria (Saccharomyces cerevisiae and Escherichia coli) by employing molecular recognition element–CNH complexes and a near-infrared laser.

Automated manipulation of non-spherical micro-objects using optical tweezers combined with image processing techniques

Automated optical trapping of non-spherical objects offers great flexibility as a non-contact micromanipulation tool in various research fields. Computer vision control enables fruitful applications of automated manipulation in biology and material science. Here we demonstrate fully-automated, simultaneous, independent trapping and manipulation of multiple non-spherical objects using multiple-force optical clamps. Customized real-time feature recognition and trapping beam control algorithms are also presented.

Evaluation of cell characteristics by step-wise orientational rotation using optoelectrostatic micromanipulation

The characteristics of a single cell can be studied using optoelectrostatic micromanipulation (OEMM) techniques. Escherichia coli (E. coli) and Schizosaccharomyces pombe (S. Pombe) cells were characterized by step-wise orientational rotation driven by a high-frequency alternating electric field. The peak voltage and frequency of the electric field ranged from 2.5 to 80 V (average electric field: 0.25/spl times/10/sup 5/-0.8/spl times/10/sup 6/ V/m) and from 5 kHz to 2 MHz, respectively. The characteristics were measured by a microelectrode system installed on a microscope stage. An yttrium aluminum garnet (YAG) laser was introduced to hold a cell at its focal point. The characteristics show that a live cell has a peak critical orientational rotation frequency (PCRF) at 1 MHz and the PCRF of a dead cell depends on the process of killing, i.e., thermal, chemical, ultraviolet light or electric pulse. The orientational rotation can be utilized to investigate the dielectric properties of an individual cell.

Carbon Nanotube–Polymer Composite for Light‐Driven Microthermal Control

Carbon nanotubes (CNTs) have attracted considerable attention because of their various applications. In particular, the development of functional CNT–polymer composites has been a hot research topic in the last years. However, contrary to many theoretical expectations, the physical potential of CNT–polymer composites has not been fully utilized because of the low dispersibility of CNTs in polymer matrices. Therefore, surface engineering of the CNTs is considered to be indispensable for exploiting their physical potential. Here, we present a novel organic-solvent-dispersible single-walled CNT (SWNT) complex that has good dispersibility in poly(dimethylsiloxane) (PDMS)—a model polymer matrix which represents an attractive material for lab-on-a-chip technologies, such as microor nanofabrication. Controlling the temperature of a reaction mixture on a chip is of particular importance for many such applications. A light-driven PDMS microchip that encapsulates the SWNT complexes was shown to be capable of ultrarapid temperature control in a microspace. Covalent and noncovalent functionalizations of SWNTs are useful techniques for improving the dispersibility of the nanotubes in organic solvents. Covalent functionalization, however, disrupts the one-dimensional electronic structure and the desirable optical properties of the SWNTs. The noncovalent approach, on the other hand, is considered to be a promising technique because it results in better retention of the electronic structure of the CNTs. We therefore synthesized a phospholipid, PL, bovine serum albumin, BSA, functionalized single-walled nanotube, SWNT, (PL– BSA–SWNT) complex by using a noncovalent technique (see Figure 1a and the Supporting Information for details). The BSA molecules bind noncovalently to the surface of the SWNTs through hydrophobic interactions, p–p interactions, and interactions via the amine functionalities of the protein. The hydrophobic alkyl chains of the PL increase the dispersibility of the BSA-functionalized SWNT (BSA– SWNT) complexes in both nonaqueous solvents and the PDMS polymer matrix. Pristine SWNTs and the BSA– SWNT complex are not dispersible in dichloromethane (Figure 1b, 1 and 2), whereas the PL–BSA–SWNT complex is readily dispersible in various organic solvents, but not in water (Figure 1b, 3–7). Bundle-free or isolated SWNTs have been reported to exhibit characteristic signals in the visible (Vis) and near-infrared (NIR) regions of their optical absorbance spectra as a result of van Hove transitions. The Vis/NIR optical absorption spectrum of a dispersion of the PL–BSA–SWNT complex in dichloromethane showed first metallic (M11) and second semiconducting (S22) bands in the ranges 440–600 nm and 550–800 nm, respectively (Figure 1c). In addition, we structurally characterized the PL– BSA–SWNT complex by means of atomic force microscopy Figure 1. Organic-solvent-dispersible SWNT complex. a) Image of the PL–BSA–SWNT complex. b) Photographs of dispersions of various SWNT constructs (1: SWNT, 2: BSA–SWNT, 3–7: PL–BSA–SWNT) in organic solvents (1–3: dichloromethane, 4: chloroform, 5: toluene, 6: ethyl acetate) and in water (7). c) Vis/NIR spectrum of a PL–BSA– SWNT/dichloromethane solution (350 mgmL ). d) AFM image of PL– BSA–SWNT complexes deposited on a mica substrate (left), and height profiles [nm] along lines 1–3 (right).

Manipulation of single coiled DNA molecules by laser clustering of microparticles

We have developed a method of manipulating single DNA molecules for application in single-molecule analysis. Using a bead cluster formed by laser trapping, the technique allows single DNA molecules to be manipulated at any point on the molecule without the need for prior chemical modification as in DNA-bead complex techniques. We describe the method and the characteristics of cluster formation, and present examples of actual DNA molecule manipulation.

Pinophilins A and B, Inhibitors of Mammalian A-, B-, and Y-Family DNA Polymerases and Human Cancer Cell Proliferation

Pinophilins A (1) and B (2), new hydrogenated azaphilones, and Sch 725680 (3) were isolated from cultures of a fungus (Penicillium pinophilum Hedgcok) derived from a seaweed, and their structures were determined using spectroscopic analyses. These compounds selectively inhibited the activities of mammalian DNA polymerases (pols), A (pol γ), B (pols α, δ, and ε), and Y (pols η, ι, and κ) families, but did not influence the activities of the four X-family pols (pols β, λ, μ, and terminal deoxynucleotidyl transferase). Compound 1 was the strongest inhibitor, with IC₅₀ values of 48.6 to 55.6 μM. Kinetic analysis showed that compound 1 is a noncompetitive inhibitor of both pol α and κ activities with the DNA template-primer substrate, and a competitive inhibitor with the nucleotide substrate. In contrast, compounds 1-3 showed no effect on the activities of plant and prokaryotic pols or any other DNA metabolic enzymes tested. The compounds suppressed cell proliferation and growth in five human cancer cell lines, but had no effect on the viability of normal human cell lines.

Photoinduced antiviral carbon nanohorns

Nanocarbons, such as carbon nanohorns (CNH) and carbon nanotubes, are materials of interest in many fields of science and technology because of their remarkable physical properties. We report here a novel approach for using NIR laser-driven CNH as an antiviral agent. NIR laser-driven functional CNH complexes could open the way to a new range of antiviral materials.

Development of a new detection method for DNA molecules

A highly sensitive analysis method for biological molecules is required because of recent developments in molecular biology. Conventional highly sensitive detection methods are based on labelling techniques using fluorescent dyes or enzymes. However, since those techniques involved some problems with signal instability, a new technology has been sought. Because the superconducting interference device (SQUID) is an extremely highly sensitive magnetic sensor, it can be applied to the highly sensitive detection of DNA labelled with small magnetic particles. The signal from SQUID is stable in contrast to fluorescent dyes and enzymes, therefore it permits highly sensitive measurement over long periods of time. Sample coverslips on which the small magnetic particles were anchored using biotin labelled DNA were prepared to demonstrate the availability of this method. Scanning the high-Tc SQUID sensor on the coverslip demonstrated that the magnetic flux on the coverslip agreed well with the pattern of labelled DNA anchored on the coverslip. This result suggests that SQUID can be applied for the specific detection of DNA molecules, especially for the detection of DNA chips.

3-O-Methylfunicone, a Selective Inhibitor of Mammalian Y-Family DNA Polymerases from an Australian Sea Salt Fungal Strain

We isolated a pol inhibitor from the cultured mycelia extract of a fungal strain isolated from natural salt from a sea salt pan in Australia, which was identified as 3-O-methylfunicone by spectroscopic analyses. This compound selectively inhibited the activities of mammalian Y-family DNA polymerases (pols) (i.e., pols η, ι and κ). Among these pols, human pol κ activity was most strongly inhibited, with an IC50 value of 12.5 μM. On the other hand, the compound barely influenced the activities of the other families of mammalian pols, such as A-family (i.e., pol γ), B-family (i.e., pols α, δ and ɛ) or X-family (i.e., pols β, λ and terminal deoxynucleotidyl transferase), and showed no effect on the activities of fish pol δ, plant pols, prokaryotic pols and other DNA metabolic enzymes, such as calf primase of pol α, human immunodeficiency virus type-1 (HIV-1) reverse transcriptase, human telomerase, T7 RNA polymerase, mouse IMP dehydrogenase (type II), human topoisomerases I and II, T4 polynucleotide kinase or bovine deoxyribonuclease I. This compound also suppressed the growth of two cultured human cancer cell lines, HCT116 (colon carcinoma cells) and HeLa (cervix carcinoma cells), and UV-treated HeLa cells exhibited lower clonogenic survival in the presence of inhibitor.