Ryuji Yokokawa

We have established an orientation technique of microtubules and evaluated their polarities by the movement of kinesin-coated beads quantitatively in poly(dimethyl siloxane) (PDMS) channels. More than 95% of beads moved to the desired direction; this indicates almost all the microtubules were functionally oriented including plus and minus polarities. The technique is essential for fabricating a bio-hybrid nanotransport system, in which the kinesin−microtubule system combined with microfluidic st

We have demonstrated a novel micro/nanotransport system using biomolecular motors driven by adenosine triphosphate (ATP). For the driving mechanism, microtubule-kinesin system, which is one of the linear biomolecular motor systems was investigated. ATP dissolved in an aqueous condition is hydrolyzed to adenosine diphosphate (ADP) to energize the bionanoactuators in this mechanism. This means the system does not require an external electrical or mechanical energy source. Therefore, a purely chemi

Artificial nanotransport systems inspired by intracellular transport processes have been investigated for over a decade using the motor protein kinesin and microtubules. However, only unidirectional cargo transport has been achieved for the purpose of nanotransport in a microfluidic system. Here, we demonstrate bidirectional nanotransport by integrating kinesin and dynein motor proteins. Our molecular system allows microtubule orientation of either polarity in a microfluidic channel to construct

Artificial nano-scale transportation is demonstrated by reconstructing the intracellular transport in a living cell. The transport system is established on two novel techniques: one is the introduction of a single microtubule filament with controlled polarity in a microfabricated submicrometre channel; the other is the immobilization technique of microtubules by a mercury lamp. To transport a kinesin-coated bead in a designated direction, each microtubule filament is polarly oriented by in vitro

Kinesin-driven microtubules have been focused on to serve as molecular transporters, called "molecular shuttles," to replace micro/nanoscale molecular manipulations necessitated in micro total analysis systems. Although transport, concentration, and detection of target molecules have been demonstrated, controllability of the transport directions is still a major challenge. Toward broad applications of molecular shuttles by defining multiple moving directions for selective molecular transport, we

Kinesin is a motor protein that plays important roles in a variety of cellular functions. In vivo, multiple kinesin molecules are bound to cargo and work as a team to produce larger forces or higher speeds than a single kinesin. However, the coordination of kinesins remains poorly understood because of the experimental difficulty in controlling the number and arrangement of kinesins, which are considered to affect their coordination. Here, we report that both the number and spacing significantly

On-chip microsyringes are developed by integrating parallel micro actuators and a microfluidic chip. Sliders of an Electrostatically Controlled Linear Inchworm Actuator (ECLIA) are applied to manipulate microsyringes in the nanometer range, which allows liquid control on the picoliter scale. ECLIA drives sliders in parallel with high accuracy and a large stroke. The requirements for syringe performance, such as parallel and precise liquid control, can be satisfied by the above features of ECLIA.

Blood vessel morphology is dictated by mechanical and biochemical cues. Flow-induced shear stress and pericytes both play important roles, and they have previously been studied using on-chip vascular networks to uncover their connection to angiogenic sprouting and network stabilization. However, it is unknown which shear stress values promote angiogenesis, how pericytes are directed to sprouts, and how shear stress and pericytes affect the overall vessel morphology. Here, we employed a microflui

The lack of functional vascular system in stem cell-derived cerebral organoids (COs) limits their utility in modeling developmental processes and disease pathologies. Unlike other organs, brain vascularization is poorly understood, which makes it particularly difficult to mimic <i>in vitro</i>. Although several attempts have been made to vascularize COs, complete vascularization leading to functional capillary network development has only been achieved <i>via</i> transplantation into a mouse bra

We have investigated a novel material, thin-film aerogel, for use in microelectromechanical systems (MEMS) and determined its mechanical properties on the microscale. Fabrication processes, including photolithography and etching, have been developed to build the overhanging cantilevers and bridges used for the measurement. Young's modulus for the deposited aerogel material has been measured from the resonant frequencies of those microstructures. A nanoindentation test allows us to estimate not o

Three-dimensional (3D) tissue culture is a powerful tool for understanding physiological events. However, 3D tissues still have limitations in their size, culture period, and maturity, which are caused by the lack of nutrients and oxygen supply through the vasculature. Here, we propose a new method for culturing a 3D tissue-a spheroid-directly on an 'on-chip vascular bed'. The method can be applied to any 3D tissue because the vascular bed is preformed, so that angiogenic factors from the tissue

Single-molecule fluorescence observation of adenosine triphosphate (ATP) is a powerful tool to elucidate the chemomechanical coupling of ATP with a motor protein. However, in total internal reflection fluorescence microscopy (TIRFM), available ATP concentration is much lower than that in the in vivo environment. To achieve single-molecule observation with a high signal-to-noise ratio, zero-mode waveguides (ZMWs) are utilized even at high fluorescent molecule concentrations in the micromolar rang

We developed an integrated protein assay device, "Single Molecule MicroTAS (SMM)," which enables cell lysis, protein extraction, purification, and activity assay. The assay was achieved at the single-molecule scale for a genetically engineered protein, F(1)-ATPase, which is the smallest known rotary motor. A cell lysis condition, with a wide range of applied voltages (50-250 V) and other optimized values (pulse width: 50 micros; duty: 0.01%; electrode gap: 25 microm; total flow rate: 5 microL mi