Ryuji Yokokawa

Hybrid nanotransport system by biomolecular linear motors

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 chemical system which is similar to the in vivo transport will be realized. This paper reports some fundamental studies to integrate biomaterials and MEMS. The microtubules, or rail molecules, were patterned on a glass substrate with poly(dimethyl siloxane) (PDMS) using a regular soft lithography technique. Microbeads (320 nm in diameter) and a micromachined structure (2/spl times/3 /spl mu/m, 2 /spl mu/m in thickness) coated with kinesin molecules were transported along the microtubules at an average speed of 476/spl plusmn/56 and 308 nm/s, respectively. While ATP injection activated the transport system we have also managed to provide repetitive on/off control using hexokinase as an inhibitor. For the minimum response time in the repetitive control, the optimized concentration for ATP was 10/sup 2/ /spl mu/M and 10/sup 3/ U/L for hexokinase.

Simultaneous and bidirectional transport of kinesin-coated microspheres and dynein-coated microspheres on polarity-oriented microtubules.

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 a transport track. Each motor protein acts as a nanoactuators that transports microspheres in opposite directions determined by the polarity of the oriented microtubules: kinesin‐coated microspheres move toward the plus end of microtubules, whereas dynein‐coated microspheres move toward the minus end. We demonstrate both unidirectional and bidirectional transport using kinesin‐ and dynein‐coated microspheres on microtubules oriented and glutaraldehyde‐immobilized in a microfluidic channel. Tracking and statistical analysis of microsphere movement demonstrate that 87–98% of microspheres move in the designated direction at a mean velocity of 0.22–0.28 µm/s for kinesin‐coated microspheres and 0.34–0.39 µm/s for dynein‐coated microspheres. This bidirectional nanotransport goes beyond conventional unidirectional transport to achieve more complex artificial nanotransport in vitro. Biotechnol. Biotechnol. Bioeng. 2008;101: 1–8.

Unidirectional transport of a bead on a single microtubule immobilized in a submicrometre channel

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 gliding assay, in which microtubules are carried by the movement of kinesin immobilized on the channel surface. Then, microtubules are immobilized by exposing kinesin to the mercury lamp, which inactivates kinesin but not microtubules. A kinesin-coated bead is newly introduced and carried on the microtubule from one end of the channel to the other as designed. This is an essential component to build up an integrated nano-scale transport system driven by motor proteins.

Constant flow-driven microfluidic oscillator for different duty cycles.

This paper presents microfluidic devices that autonomously convert two constant flow inputs into an alternating oscillatory flow output. We accomplish this hardware embedded self-control programming using normally closed membrane valves that have an inlet, an outlet, and a membrane-pressurization chamber connected to a third terminal. Adjustment of threshold opening pressures in these 3-terminal flow switching valves enabled adjustment of oscillation periods to between 57 and 360 s with duty cycles of 0.2-0.5. These values are in relatively good agreement with theoretical values, providing the way for rational design of an even wider range of different waveform oscillations. We also demonstrate the ability to use these oscillators to perform temporally patterned delivery of chemicals to living cells. The device only needs a syringe pump, thus removing the use of complex, expensive external actuators. These tunable waveform microfluidic oscillators are envisioned to facilitate cell-based studies that require temporal stimulation.

Colocalization of Quantum Dots by Reactive Molecules Carried by Motor Proteins on Polarized Microtubule Arrays

The field of microfluidics has drastically contributed to downscale the size of benchtop experiments to the dimensions of a chip without compromising results. However, further miniaturization and the ability to directly manipulate individual molecules require a platform that permits organized molecular transport. The motor proteins and microtubules that carry out orderly intracellular transport are ideal for driving in vitro nanotransport. Here, we demonstrate that a reconstruction of the cellular kinesin/dynein-microtubule system in nanotracks provides a molecular total analysis system (MTAS) to control massively parallel chemical reactions. The mobility of kinesin and a microtubule dissociation method enable orientation of a microtubule in an array for directed transport of reactive molecules carried by kinesin or dynein. The binding of glutathione S-transferase (GST) to glutathione (GSH) and the binding of streptavidin to biotin are visualized as colocalizations of quantum dots (Q-dots) when motor motilities bring them into contact. The organized nanotransport demonstrated here suggests the feasibility of using our platform to perform parallel biochemical reactions focused at the molecular level.

Biomolecular linear motors confined to move upon micro-patterns on glass

Biomolecular linear motor proteins - kinesin and microtubules - are finely patterned on a conventional, flat glass substrate using parylene lift-off process; these patterns are useful for single molecule analysis and biohybrid transportation. The patterning was performed from 5 /spl mu/m - 40 /spl mu/m in width with minimal nonspecific binding of the proteins. A conventional gliding assay was realized using this glass substrate and the behaviors of the single proteins were analyzed.

Multiple independent autonomous hydraulic oscillators driven by a common gravity head

Self-switching microfluidic circuits that are able to perform biochemical experiments in a parallel and autonomous manner similar to instruction-embedded electronics, are rarely implemented. Here, we present design principles and demonstrations for gravity-driven, integrated, microfluidic pulsatile flow circuits. With a common gravity-head as the only driving force, these fluidic oscillator arrays realize a wide range of periods (0.4 s – 2 h) and flow rates (0.10 – 63 μL min−1) with completely independent timing between the multiple oscillator sub-circuits connected in parallel. As a model application, we perform systematic, parallel analysis of endothelial cell elongation response to different fluidic shearing patterns generated by the autonomous microfluidic pulsed flow generation system.

Design, simulation and fabrication of a total internal reflection (TIR)-based chip for highly sensitive fluorescent imaging

This paper presents a total internal reflection based chip which generates evanescent waves for highly sensitive fluorescent imaging. The chip is monolithically, massively cast in polydimethylsiloxane (PDMS) at a very low cost using a Si mold fabricated by Si anisotropic wet etching and deep reactive ion etching (DRIE). Our method integrates all miniaturized optical components, namely cylindrical microlens, prism and waveguide, into one monolithic PDMS chip; thus assembly is unnecessary, and misalignment is eliminated. The slide-format and monolithic chip can be used with both upright and inverted fluorescent microscopes with flexible sample delivery platforms. The flexibility of sample delivery platforms facilitates various surface treatment/immobilization techniques required in fluorescent imaging. Moreover, the fiberoptics coupling into the chip allows a broad choice of wavelengths and types of laser sources ranging from UV to IR. We have successfully demonstrated the capability of the chip in highly sensitive imaging of tetramethylrhodamine (TMR) fluorescent dye and immobilized fluorescent nanobeads. Our monolithic, miniaturized TIR-based chip could potentially serve as an evanescent excitation-based platform integrated into a micro-total analysis system (μ-TAS).

Microfluidic oscillators with widely tunable periods

We present experiments and theory of a constant flow-driven microfluidic oscillator with widely tunable oscillation periods. This oscillator converts two constant input-flows from a syringe pump into an alternating, periodic output-flow with oscillation periods that can be adjusted to between 0.3 s to 4.1 h by tuning an external membrane capacitor. This capacitor allows multiple adjustable periods at a given input flow-rate, thus providing great flexibility in device operation. Also, we show that a sufficiently large external capacitance, relative to the internal capacitance of the microfluidic valve itself, is a critical requirement for oscillation. These widely tunable microfluidic oscillators are envisioned to be broadly useful for the study of biological rhythms, as on-chip timing sources for microfluidic logic circuits, and other applications that require variation in timed flow switching.

Microfluidic Automation Using Elastomeric Valves and Droplets: Reducing Reliance on External Controllers

This paper gives an overview of elastomeric valve- and droplet-based microfluidic systems designed to minimize the need of external pressure to control fluid flow. This Concept article introduces the working principle of representative components in these devices along with relevant biochemical applications. This is followed by providing a perspective on the roles of different microfluidic valves and systems through comparison of their similarities and differences with transistors (valves) and systems in microelectronics. Despite some physical limitation of drawing analogies from electronic circuits, automated microfluidic circuit design can gain insights from electronic circuits to minimize external control units, while implementing high-complexity and high-throughput analysis.