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.

Characterization of an Electrode-Type Tactile Display Using Electrical and Electrostatic Friction Stimuli.

Unlike tactile displays that use mechanical actuators, electrode-type tactile displays can be easily integrated and miniaturized because they consist of electrodes and insulators. Electrical tactile displays only require electrodes and use an electric current to stimulate vibration or pressure. Likewise, electrostatic friction tactile displays also only require electrodes and an insulator and can induce changes in friction between the display and a fingerpad. We have developed a tactile display that integrates electrical and electrostatic friction stimulation owing to their affinity to microfabrication techniques. This tactile display can provide both pressure and friction at the same time. In this study, we presented an elongated bar shape via the tactile display to experimental participants. The experimental results showed that a tactile display employing multiple stimuli as opposed to a single stimulus can induce the perception of larger shapes.

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.

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.

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.

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.