Formation of nanofilms on cell surfaces to improve the insertion efficiency of a nanoneedle into cells.

A nanoneedle, an atomic force microscope (AFM) tip etched to 200 nm in diameter and 10 µm in length, can be inserted into cells with the aid of an AFM and has been used to introduce functional molecules into cells and to analyze intracellular information with minimal cell damage. However, some cell lines have shown low insertion efficiency of the nanoneedle. Improvement in the insertion efficiency of a nanoneedle into such cells is a significant issue for nanoneedle-based cell manipulation and analysis. Here, we have formed nanofilms composed of extracellular matrix molecules on cell surfaces and found that the formation of the nanofilms improved insertion efficiency of a nanoneedle into fibroblast and neural cells. The nanofilms were shown to improve insertion efficiency even in cells in which the formation of actin stress fibers was inhibited by the ROCK inhibitor Y27632, suggesting that the nanofilms with the mesh structure directly contributed to the improved insertion efficiency of a nanoneedle.

Evaluation of the insertion efficiencies of tapered silicon nanoneedles and invasiveness of diamond nanoneedles in manipulations of living single cells.

We have been developing a low invasive cell manipulation technology based on inserting an ultra-thin needle--"nanoneedle"--into a living cell by using an atomic force microscope (AFM). The nanoneedle, made from a silicon AFM tip by focused-ion-beam etching, has a diameter of several hundred nanometers and a length of about 10 microns. Successful insertion of the nanoneedle into the cell can be confirmed by the appearance of a steep relaxation of repulsive force in the force-distance curve as monitored by the AFM system. This technology, termed "cell surgery", can be applied for the detection of intracellular proteins in a living cell or for highly efficient gene transfer. The present study shows that the durability of a tapered nanoneedle is superior to that of a cylindrical nanoneedle, and that a proper aspect ratio for the tapered nanoneedle must be chosen to maintain sufficient insertion efficiency for a particular target cell: tapered nanoneedles of an aspect ratio over 20 showed high insertion efficiency for various kinds of mammalian cells. We then used diamond for the material of the nanoneedle because its specific properties, such as high stiffness, heat conductivity, and electrical conductivity capacitated by boron doping, were deemed useful for the analysis and manipulation of intracellular phenomena. We compared the capability of the diamond nanoneedle in cell manipulation with that of the silicon nanoneedle. Evaluation of the effect of the former on transcription efficiency and localization analysis of p53 expression revealed the low invasiveness for cell manipulation as was also the case for the silicon nanoneedle. We also succeeded in achieving highly efficient plasmid DNA delivery into a mouse fibroblast C3H10T1/2 using the diamond nanoneedle. The diamond nanoneedle is expected to contribute to the versatility of "cell surgery" technology.

Controlled formation of magnetite crystal by partial oxidation of ferrous hydroxide in the presence of recombinant magnetotactic bacterial protein Mms6.

Mms6 is a small acidic protein that is tightly associated with bacterial magnetite in Magnetospirillum magneticum AMB-1. This protein has previously shown iron binding activity, allowing it to generate uniform magnetic crystals by co-precipitation of ferrous and ferric ions. Here, magnetite crystals were formed by the partial oxidation of ferrous hydroxide in the presence and absence of Mms6. The crystals synthesised were systematically characterised according to their sizes and morphologies using high-resolution transmission electron microscopy. Mms6-mediated synthesis of magnetite by this methods produced crystals of a uniform size and narrow size distribution with a cubo-octahedral morphology, similar to bacterial magnetite observed in M. magneticum AMB-1. The crystals formed in the absence of Mms6 were octahedral, larger with an increased size distribution. Protein quantification analysis of Mms6 in the synthesised particles indicated tight association of this protein onto the crystal. Furthermore, high affinities to iron ions and a highly charged electrostatic quality suggest that the protein acts as a template for the nucleus formation and/or acts as a growth regulator by recognising crystal faces. The method introduced in this study presents an alternative route for controlling the size and shape of magnetite crystals without the use of organic solvent and high temperatures.

Novel detection system for biomolecules using nano-sized bacterial magnetic particles and magnetic force microscopy.

A system for streptavidin detection using biotin conjugated to nano-sized bacterial magnetic particles (BMPs) has been developed. BMPs, isolated from magnetic bacteria, were used as magnetic markers for magnetic force microscopy (MFM) imaging. The magnetic signal was obtained from a single particle using MFM without application of an external magnetic field. The number of biotin conjugated BMPs (biotin-BMPs) bound to streptavidin immobilized on the glass slides increased with streptavidin concentrations up to 100 pg/ml. The minimum streptavidin detection limit using this technique is 1 pg/ml, which is 100 times more sensitive than a conventional fluorescent detection system. This is the first report using single domain nano-sized magnetic particles as magnetic markers for biosensing. This assay system can be used for immunoassay and DNA detection with high sensitivities.

Evaluation of the actin cytoskeleton state using an antibody-functionalized nanoneedle and an AFM.

A cell diagnosis technique was developed, which uses an Atomic Force Microscope (AFM) and an ultra-thin AFM probe sharpened to a diameter of 200 nm (nanoneedle). Due to the high aspect ratio of the nanoneedle, it was successfully inserted into a living cell without affecting its viability. Furthermore, by functionalizing the nanoneedle with specific antibodies and measuring the unbinding forces ('fishing forces') during evacuation of the nanoneedle from the cell, it was possible to measure specific mechanical interactions between the antibody-functionalized nanoneedle and the intracellular contents of the cell. In this study, an anti-actin-antibody-functionalized nanoneedle was used to evaluate the actin cytoskeleton state in living cells. To examine the effect of cytoskeleton condition on the measured fishing forces, the cytoskeleton-disrupting drugs cytochalasin D (cytD) and Y-27632 were used, showing a marked decrease in the measured fishing forces following incubation with either of the drugs. Furthermore, the technique was used to measure the time course changes in a single cell during incubation with cytD, showing a gradual time-dependent decrease in fishing forces. Even minute doses of the drugs, the effects of which were hardly evident by optical and fluorescence methods, could be clearly detected by the measurement of nanoneedle-protein fishing forces, pointing to the high sensitivity of this detection method. This technique may prove beneficial for the evaluation of cytoskeleton conditions in health and disease, and for the selection of specific cells according to their intracellular protein contents, without the need for introduction of marker proteins into the cell.

Mechanical force-based probing of intracellular proteins from living cells using antibody-immobilized nanoneedles.

We developed a method combining atomic force microscopy (AFM) and antibody-immobilized nanoneedles to discriminate living cells by probing intracellular cytoskeletal proteins without the need for cell labeling. The nanoneedles are ultra-thin AFM probes sharpened to 200 nm in diameter. While retracting a nanoneedle inserted into a cell, we measured the mechanical force needed to unbind the antibody-target protein complex. Using this method, the intermediate filament protein, nestin and neurofilament were successfully detected in mouse embryonic carcinoma P19 cells and rat primary hippocampal cells within a minute for a single cell and cell differentiation states could be determined. Additionally, the measured magnitude of the force detecting nestin was indicative of the malignancy of breast cancer cells. This method was shown to affect neither the doubling time of cells nor does it leave extrinsic antibodies within the examined cells, allowing to be used in subsequent analyses in their native state.

Analysis of the unbinding force between telomestatin derivatives and human telomeric G-quadruplex by atomic force microscopy.

The force analysis between a macrocyclic hexazole (6OTD) monomer/dimer and telomeric DNA using atomic force microscopy revealed the difference in their binding modes. The 6OTD dimer bound to the G-quadruplex more strongly than the monomer by sandwiching the G-quadruplex.

Control of the morphology and size of magnetite particles with peptides mimicking the Mms6 protein from magnetotactic bacteria.

Mms6 is a dominant protein that tightly associates with the surface of bacterial magnetites in Magnetospirillum magneticum AMB-1. The protein has previously been shown to mediate the formation of uniform magnetite crystals of cubo-octahedral morphology consisting of (1 1 1) and (1 0 0) crystal faces with a narrow size distribution during chemical magnetite synthesis. In order to understand the role of this protein in chemical magnetite synthesis, magnetite formation was investigated using synthetic peptides mimicking the Mms6 protein. Particles that were synthesized in the presence of short peptides harbouring the C-terminal acidic region of Mms6 exhibited a spherical morphology with circularities of 0.70-0.90 similar to those of bacterial magnetites and particles formed in the presence of the Mms6 protein. In contrast, a rectangular morphology with circularities of 0.60-0.85 were obtained when other peptides were used for synthesis. The results indicated that the C-terminal region of the Mms6 protein has significant control over the morphology of magnetite crystals in the chemical synthetic method. This method can, therefore, be useful as an alternative method of controlling the size and morphology of magnetite crystals under ambient conditions.

Aminosilane multilayer formed on a single-crystalline diamond surface with controlled nanoscopic hardness and bioactivity by a wet process.

Diamond could be an excellent support for nanodevices utilizing biomolecules if it is covered with a polymer layer immobilizing a variety of biomolecules. We report a wet method to form a 3-aminopropyltriethoxysilane (APTES) multilayer with a controlled hardness, roughness, and capacity for immobilizing protein. The method is feasible in typical biochemical laboratories where biomolecules are prepared. Atomic force microscopy (AFM) revealed that the surface geometries and nanoscopic hardness of the multilayers on an oxygen-terminated single-crystalline diamond surface depended on the dielectric constant of the solvent; the smaller the constant, the harder the layer. The hard multilayers had holes and APTES aggregates on the surfaces, while less hard ones had homogeneous surfaces with rare holes and little aggregates. The secondary deposition of APTES in a solvent with a large dielectric constant on a hard multilayer removed the holes, and further treatment of the multilayer in acidic ethanol solution diminished the aggregates. Such a surface can immobilize streptavidin with enough specificity against nonspecific adsorption using a combination of polyethylene glycol reagents. The results of a scratching test and nanoindentation test with AFM provided consistent results, suggesting some universality of the scratching test independent of the tip structure of the cantilever. The mechanism of formation of multilayers on the diamond surface and their binding to it is discussed.

Self-assembled bifunctional surface mimics an enzymatic and templating protein for the synthesis of a metal oxide semiconductor.

The recent discovery and characterization of silicatein, a mineral-synthesizing enzyme that assembles to form the filamentous organic core of the glassy skeletal elements (spicules) of a marine sponge, has led to the development of new low-temperature synthetic routes to metastable semiconducting metal oxides. These protein filaments were shown in vitro to catalyze the hydrolysis and structurally direct the polycondensation of metal oxides at neutral pH and low temperature. Based on the confirmation of the catalytic mechanism and the essential participation of specific serine and histidine residues (presenting a nucleophilic hydroxyl and a nucleophilicity-enhancing hydrogen-bonding imidazole nitrogen) in silicatein's catalytic active site, we therefore sought to develop a synthetic mimic that provides both catalysis and the surface determinants necessary to template and structurally direct heterogeneous nucleation through condensation. Using lithographically patterned poly(dimethylsiloxane) stamps, bifunctional self-assembled monolayer surfaces containing the essential catalytic and templating elements were fabricated by using alkane thiols microcontact-printed on gold substrates. The interface between chemically distinct self-assembled monolayer domains provided the necessary juxtaposition of nucleophilic (hydroxyl) and hydrogen-bonding (imidazole) agents to catalyze the hydrolysis of a gallium oxide precursor and template the condensed product to form gallium oxohydroxide (GaOOH) and the defect spinel, gamma-gallium oxide (gamma-Ga(2)O(3)). Using this approach, the production of patterned substrates for catalytic synthesis and templating of semiconductors for device applications can be envisioned.