Optically driven microtools with an antibody-immobilised surface for on-site cell assembly.

To enable the accurate reproduction of organs in vitro, and improve drug screening efficiency and regenerative medicine research, it is necessary to assemble cells with single-cell resolution to form cell clusters. However, a method to assemble such forms has not been developed. In this study, a platform for on-site cell assembly at the single-cell level using optically driven microtools in a microfluidic device is developed. The microtool was fabricated by SU-8 photolithography, and antibodies were immobilised on its surface. The cells were captured by the microtool through the bindings between the antibodies on the microtool and the antigens on the cell membrane. Transmembrane proteins, CD51/61 and CD44 that facilitate cell adhesion, commonly found on the surface of cancer cells were targeted. The microtool containing antibodies for CD51/61 and CD44 proteins was manipulated using optical tweezers to capture HeLa cells placed on a microfluidic device. A comparison of the adhesion rates of different surface treatments showed the superiority of the antibody-immobilised microtool. The assembly of multiple cells into a cluster by repeating the cell capture process is further demonstrated. The geometry and surface function of the microtool can be modified according to the cell assembly requirements. The platform can be used in regenerative medicine and drug screening to produce cell clusters that closely resemble tissues and organs in vivo.

AC-Electroosmosis-Assisted Surface Plasmon Resonance Sensing for Enhancing Protein Signals with a Simple Kretschmann Configuration.

A surface plasmon resonance (SPR) sensor chip fabricated with a comb-shaped microelectrode array to supply alternating current (AC) voltage is reported. The chip induces circulating flow near the surface (i.e., AC electroosmosis). The circulating flow provides a mixing effect, which enhances the binding of the analyte molecules. We evaluated the SPR characteristics of the chip and demonstrated an improvement in protein binding to the chip surface. SPR sensor chips with comb-shaped microelectrodes were fabricated using standard UV lithography. Sensing experiments were conducted using a standard Kretschmann-type SPR measurement system. To demonstrate the mixing effect of AC electroosmosis, we evaluated the binding of immunoglobulin G molecules onto the sensor surface where anti-immunoglobulin G antibodies were covalently immobilized. The result indicates that the amount of binding increases by a factor of 1.7 above that achieved by using a conventional chip, suggesting enhancement of the protein signal.

On-site processing of single chromosomal DNA molecules using optically driven microtools on a microfluidic workbench.

We developed optically driven microtools for processing single biomolecules using a microfluidic workbench composed of a microfluidic platform that functions under an optical microscope. The optically driven microtools have enzymes immobilized on their surfaces, which catalyze chemical reactions for molecular processing in a confined space. Optical manipulation of the microtools enables them to be integrated with a microfluidic device for controlling the position, orientation, shape of the target sample. Here, we describe the immobilization of enzymes on the surface of microtools, the microfluidics workbench, including its microtool storage and sample positioning functions, and the use of this system for on-site cutting of single chromosomal DNA molecules. We fabricated microtools by UV lithography with SU-8 and selected ozone treatments for immobilizing enzymes. The microfluidic workbench has tool-stock chambers for tool storage and micropillars to trap and extend single chromosomal DNA molecules. The DNA cutting enzymes DNaseI and DNaseII were immobilized on microtools that were manipulated using optical tweezers. The DNaseI tool shows reliable cutting for on-site processing. This pinpoint processing provides an approach for analyzing chromosomal DNA at the single-molecule level. The flexibility of the microtool design allows for processing of various samples, including biomolecules and single cells.

Capture and elongation of single chromosomal DNA molecules using optically driven microchopsticks.

DNA analysis based on the observation of single DNA molecules has been a key technology in molecular biology. Several techniques for manipulating single DNA molecules have been proposed for this purpose; however, these techniques have limits on the manipulatable DNA. To overcome this, we demonstrate a method of DNA manipulation using microstructures captured by optical tweezers that allow the manipulation of a chromosomal DNA molecule. For proper DNA handling, we developed microstructures analogous to chopsticks to capture and elongate single DNA molecules under an optical microscope. Two microstructures (i.e., microchopsticks) were captured by two focused laser beams to pinch a single yeast chromosomal DNA molecule between them and thereby manipulate it. The experiments demonstrated successful DNA manipulation and revealed that the size and geometry of the microchopsticks are important factors for effective DNA handling. This technique allows a high degree of freedom in handling single DNA molecules, potentially leading to applications in the study of chromosomal DNA.

Molecular ring toss of circular BAC DNA using micropillar array for single-molecule studies.

This paper reports a method for trapping circular DNA molecules and imaging the dynamics with high spatial resolution using a micropillar-array device. We successfully trapped circular bacterial artificial chromosome DNA molecules at a micropillar-based "ring toss" in the laminar flow of a microchannel under a fluorescence microscope and demonstrated the imaging of their extension by flow and condensation process induced by spermine solution. DNA molecules were visualized in an extended loop conformation, allowing high spatial resolution, and the results showed that the dynamics is induced by the microfluidic control of the surrounding chemical environment. The method is expected to lead to the elucidation of the physical characteristics and the dynamics of circular DNA molecules.

Characterisation of optically driven microstructures for manipulating single DNA molecules under a fluorescence microscope.

Optical tweezers are powerful tools for manipulating single DNA molecules using fluorescence microscopy, particularly in nanotechnology-based DNA analysis. We previously proposed a manipulation technique using microstructures driven by optical tweezers that allows the handling of single giant DNA molecules of millimetre length that cannot be manipulated by conventional techniques. To further develop this technique, the authors characterised the microstructures quantitatively from the view point of fabrication and efficiency of DNA manipulation under a fluorescence microscope. The success rate and precision of the fabrications were evaluated. The results indicate that the microstructures are obtained in an aqueous solution with a precision ∼50 nm at concentrations in the order of 10(6) particles/ml. The visibility of these microstructures under a fluorescence microscope was also characterised, along with the elucidation of the fabrication parameters needed to fine tune visibility. Manipulating yeast chromosomal DNA molecules with the microstructures illustrated the relationship between the efficiency of manipulation and the geometrical shape of the microstructure. This report provides the guidelines for designing microstructures used in single DNA molecule analysis based on on-site DNA manipulation, and is expected to broaden the applications of this technique in the future.

Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells.

In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca(2+)] change in the ß-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of ß-cells.

Signal enhancement of protein binding by electrodeposited gold nanostructures for applications in Kretschmann-type SPR sensors.

We propose a novel Kretschmann-type surface plasmon resonance (SPR) sensor chip having a surface covered with electrodeposited gold nanostructures to enhance the sensitivity of SPR biosensing. The nanostructure is three-dimensional and has a larger surface area than a conventional flat surface chip, which increases the amount of protein binding and also induces a large change in the effective dielectric constant of the sensing area. The gold nanostructures were formed by electrodeposition under galvanostatic conditions, so their size could be controlled by manipulating the deposition time and current. The sensing characteristics, including the concentration dependence and noise level, indicated that the performance of the resulting chip (called a Au-black chip) was equivalent to that of a conventional sensor chip. We also determined the optimal electrodeposition conditions to obtain a sharp SPR curve for protein detection assay; the optimal thickness of the gold layer was 50-60 nm. Enhanced protein sensing was demonstrated by using a binding assay of anti-BSA antibody and BSA molecules. The protein binding signal was several times higher than that of the conventional assay. The insights into electrodeposition for SPR sensing presented here will enable improved sensitivity for detecting low-concentration and small proteins.

A perfusable microfluidic device with on-chip total internal reflection fluorescence microscopy (TIRFM) for in situ and real-time monitoring of live cells.

A microfluidic device integrated with a Total Internal Reflection (TIR)-based chip for cell observation and analysis was developed. This integrated device enables in situ Total Internal Reflection Fluorescence Microscopy (TIRFM) on adherent cells cultured under continuous medium perfusion. This TIR-based chip, allows TIRFM to be easily performed on cells without the assembly of complicated optical components and cell culture chambers. The integrated device was evaluated by tracking the movement of fluorescent beads and monitoring the location of insulin granules in mouse pancreatic ß-cells. This system offers higher signal-to-noise (S/N) ratio than epi-fluorescence microscopy (EPIFM), and comparable image quality to commercial TIRFM systems when imaging insulin granules. We also detected repetitive changes in intracellular Ca(2+) concentration in MIN6-m9 cells stimulated with KCl, which demonstrates quick perfusion for cell analysis while maintaining high S/N ratio.

Size-exclusion SPR sensor chip: application to detection of aggregation and disaggregation of biological particles.

We propose a novel surface plasmon resonance (SPR) sensor chip with a microfabricated slit array. The microslit excludes micrometre-size objects larger than its slit size from the SPR sensing area, so that it functions as an in situ filter. We demonstrated the sensing of microparticles of different diameters using the chip, and the results show a successful size-exclusion effect. As a demonstration of the biological application, we performed the detection of aggregation and disaggregation of biological particles using sugar-chain-immobilized gold nanoparticles as a test sample.

Evaluation of electrodeposited gold nanostructures for applications in QCM sensing.

The electrodeposition of gold nanostructures increases the surface area of a biosensor, which brings an enhancement of the sensitivity by increasing the amount of analyte binding to the surface. To evaluate the relationship among the surface structure, the area and the analyte binding, we quantitatively analyzed them for quartz crystal microbalance (QCM) sensing by scanning electron microscopy and cyclic voltammetry measurements. The results indicate a several-times increase of analyte bindings, and also the limitation of the sensing performance.

Open-access and multi-directional electroosmotic flow chip for positioning heterotypic cells.

We propose a novel method of cell positioning using electroosmotic flow (EOF) to analyze cell-cell interactions. The EOF chip has an open-to-air configuration, is equipped with four electrodes to induce multi-directional EOF, and allows access of tools for liquid handling and of physical probes for cell measurements. Evaluation of the flow within this chip indicated that it controlled hydrodynamic transport of cells, in terms of both speed and direction. We also evaluated cell viability after EOF application and determined appropriate conditions for cell positioning. Two cells were successively positioned in pocket-like microstructures, one in each micropocket, by controlling the EOF direction. As an experimental demonstration, we observed contact interactions between two individual cells through gap junction channels. The EOF chip should provide ways to elucidate various cell-cell interactions between heterotypic cells.

Pressure-mediated transfection of murine spleen and liver.

Extension of in vivo nucleic acid transfection techniques and increased information about those transfection properties and side effects are urgently needed to advance biological research and drug therapy. Tissue pressure-mediated transfection, involving lightly pressing the target tissue after intravenous injection of plasmid DNA or small-interfering RNA (siRNA), is a promising approach because of its high transfection efficiency and resulting low tissue damage. In this study, the gene expression/silencing properties and proinflammatory cytokine production associated with tissue pressure-mediated transfection were evaluated to extend its application. We have found that tissue pressure-mediated transfection can be applied to plasmid DNA and siRNA transfection to the spleen and siRNA transfection to the liver. In addition, we have demonstrated that these methods induce little production of proinflammatory cytokines, including tumor necrosis factor-alpha, interleukin (IL)-6, IL-12, and interferon-gamma. Moreover, we succeeded in controlling and quantifying the degree of pressure on the spleen and kidney and found that 0.59 N/cm(2) is sufficient for efficient and highly reproducible plasmid DNA transfection to the spleen and kidney in mice. Tissue pressure-mediated transfection of the kidney, liver, and spleen exhibits well-balanced characteristics including (1) simple and convenient manipulation, (2) tissue-specific, effective broad transfection properties, and (3) a low inflammatory response. Therefore, this information could be useful for a molecular-level mechanism analysis of diseases at an individual level in mammals, exploration of therapeutic target molecules and evaluation of gene therapy and nucleic acid-based therapy approaches, as well as potential clinical applications.

On-site manipulation of single chromosomal DNA molecules by using optically driven microstructures.

We report a novel method for manipulation of single giant DNA molecules under a video microscope. Using optically driven microstructures, we manipulated chromosomal DNA of length in the order of millimetres, extended by electroosmotic flow without DNA breakage in aqueous solution: we picked up DNA, using microfabricated hooks and wound it around microfabricated bobbins.