Contractile and Tensile Measurement of Molecular Artificial Muscles for Biohybrid Robotics.

A printable artificial muscle assembled from biomolecular motors, which we have recently developed, showed great potential in overcoming the design limitations of conventional biohybrid robots as a new bio-actuator. Characterizing its contractility for extending its applicability is important. However, conventional measurement methods are composed of complex operations with poor reproducibility, flexibility, and real-time responsiveness. This study presents a new method for measuring the contractile force generated by artificial muscles. A measurement system was constructed, wherein artificial muscles were patterned by UV laser scanning in an oil-sealed microchamber, and the contractile force was measured in real time using a microforce sensor extended by a 3D-printed microcantilever. The measurement accuracy of the sensor was ensured through calibration and correction. For demonstration purposes, a series of contractile measurements were carried out using the proposed system. The relationship between contractile force and the dimensions of the activation space of the artificial muscles, as well as the tensile properties of the contracted muscle chain were evaluated. The results will help characterize the contractile properties of the artificial muscle and lay the foundations for its further application in biohybrid robotics.

In situ integrated microrobots driven by artificial muscles built from biomolecular motors.

Microrobots have been developed for applications in the submillimeter domain such as the manipulation of micro-objects and microsurgery. Rapid progress has been achieved in developing miniaturized components for microrobotic systems, resulting in a variety of functional microactuators and soft components for creating untethered microrobots. Nevertheless, the integration of microcomponents, especially the assembly of actuators and mechanical components, is still time-consuming and has inherent restrictions, thus limiting efficient fabrications of microrobots and their potential applications. Here, we propose a method for fabricating microrobots in situ inspired by the construction of microsystems in living organisms. In a microfluidic chip, hydrogel mechanical components and artificial muscle actuators are successively photopatterned from hydrogel prepolymer and biomolecular motors, respectively, and integrated in situ into functional microrobots. The proposed method allows the fast fabrication of microrobots through simple operations and affordable materials while providing versatile functions through the precise spatiotemporal control of in situ integration and reconfiguration of artificial muscles. To validate the method, we fabricated microrobots to elicit different motions and on-chip robots with unique characteristics for microfluidic applications. This study may establish a new paradigm for microrobot integration and lead to the production of unique biohybrid microrobots with various advantages.

Quick and Mild Isolation of Intact Lysosomes Using Magnetic-Plasmonic Hybrid Nanoparticles.

Rapid and efficient isolation of intact lysosomes is necessary to study their functions and metabolites by proteomic analysis. We developed a swift and robust nanoparticle-based magnetic separation method in which magnetic-plasmonic hybrid nanoparticles (MPNPs) conjugated with amino dextran (aDxt) were targeted to the lumen of lysosomes via the endocytosis pathway. For well-directed magnetic separation of the lysosomes, it is important to trace the intracellular trafficking of the aDxt-conjugated MPNPs (aDxt-MPNPs) in the endocytosis pathway. Therefore, we analyzed the intracellular transport process of the aDxt-MPNPs by investigating the time-dependent colocalization of plasmonic scattering of aDxt-MPNPs and immunostained marker proteins of organelles using the threshold Manders' colocalization coefficient (Rt). Detailed analysis of time variations of Rt for early and late endosomes and lysosomes allowed us to derive the transport kinetics of aDxt-MPNPs in a cell. After confirming the incubation time required for sufficient accumulation of aDxt-MPNPs in lysosomes, the lysosomes were magnetically isolated as intact as possible. By varying the elapsed time from homogenization to complete isolation of lysosomes (tdelay) and temperature (T), the influences of tdelay and T on the protein composition of the lysosomes were investigated by polyacrylamide gel electrophoresis and amino acid analysis. We found that the intactness of lysosomes could become impaired quite quickly, and to isolate lysosomes as intact as possible with high purity, tdelay = 30 min and T = 4 °C were optimal settings.

A printable active network actuator built from an engineered biomolecular motor.

Leveraging the motion and force of individual molecular motors in a controlled manner to perform macroscopic tasks can provide substantial benefits to many applications, including robotics. Nonetheless, although millimetre-scale movement has been demonstrated with synthetic and biological molecular motors, their efficient integration into engineered systems that perform macroscopic tasks remains challenging. Here, we describe an active network capable of macroscopic actuation that is hierarchically assembled from an engineered kinesin, a biomolecular motor, and microtubules, resembling the contractile units in muscles. These contracting materials can be formed in desired areas using patterned ultraviolet illumination, allowing their incorporation into mechanically engineered systems, being also compatible with printing technologies. Due to the designed filamentous assembly of kinesins, the generated forces reach the micronewton range, enabling actuation of millimetre-scale mechanical components. These properties may be useful for the fabrication of soft robotic systems with advanced functionalities.

Micrometer-sized molecular robot changes its shape in response to signal molecules.

Rapid progress in nanoscale bioengineering has allowed for the design of biomolecular devices that act as sensors, actuators, and even logic circuits. Realization of micrometer-sized robots assembled from these components is one of the ultimate goals of bioinspired robotics. We constructed an amoeba-like molecular robot that can express continuous shape change in response to specific signal molecules. The robot is composed of a body, an actuator, and an actuator-controlling device (clutch). The body is a vesicle made from a lipid bilayer, and the actuator consists of proteins, kinesin, and microtubules. We made the clutch using designed DNA molecules. It transmits the force generated by the motor to the membrane, in response to a signal molecule composed of another sequence-designed DNA with chemical modifications. When the clutch was engaged, the robot exhibited continuous shape change. After the robot was illuminated with light to trigger the release of the signal molecule, the clutch was disengaged, and consequently, the shape-changing behavior was successfully terminated. In addition, the reverse process-that is, initiation of shape change by input of a signal-was also demonstrated. These results show that the components of the robot were consistently integrated into a functional system. We expect that this study can provide a platform to build increasingly complex and functional molecular systems with controllable motility.

Self-organized optical device driven by motor proteins.

Protein molecules produce diverse functions according to their combination and arrangement as is evident in a living cell. Therefore, they have a great potential for application in future devices. However, it is currently very difficult to construct systems in which a large number of different protein molecules work cooperatively. As an approach to this challenge, we arranged protein molecules in artificial microstructures and assembled an optical device inspired by a molecular system of a fish melanophore. We prepared arrays of cell-like microchambers, each of which contained a scaffold of microtubule seeds at the center. By polymerizing tubulin from the fixed microtubule seeds, we obtained radially arranged microtubules in the chambers. We subsequently prepared pigment granules associated with dynein motors and attached them to the radial microtubule arrays, which made a melanophore-like system. When ATP was added to the system, the color patterns of the chamber successfully changed, due to active transportation of pigments. Furthermore, as an application of the system, image formation on the array of the optical units was performed. This study demonstrates that a properly designed microstructure facilitates arrangement and self-organization of molecules and enables assembly of functional molecular systems.

Utilization of myosin and actin bundles for the transport of molecular cargo.

The utilization of motor proteins for the movement and assembly of synthetic components is currently a goal of nanoengineering research. Application of the myosin actin motor system for nanotechnological uses has been hampered due to the low flexural rigidity of individual F-actin filaments. Here it is demonstrated how actin bundling can be used to affect the translational behavior of myosin-propelled filaments, transport molecules across a motor-patterned surface, and that the movement of bundled actin can be regulated photonically. These data suggest that actin bundling may significantly improve the applicability of the myosin motor for future nanotechnological applications.

Reduction of the damping on an AFM cantilever in fluid by the use of micropillars.

In single molecule force measurements with soft atomic force microscope (AFM) cantilevers, the force sensitivity is limited by the Brownian motion of the cantilever. When a cantilever is close to the surface, the hydrodynamic interaction between the cantilever beam and the surface, called the "squeezing effect", becomes significant, and the resonance peak of the thermal oscillation of the cantilever is heavily broadened and shifted to lower frequency which makes it difficult to eliminate the thermal noise by low-pass filtering. In this study, we propose an easy and low-cost method to improve the force sensitivity. We demonstrate that by bringing a tip of a cantilever onto the edge of a micropillar structure a significant reduction of the damping and an enhancement of force sensitivity are achieved.

[Development of bio-hybrid micro machines].

Living system use many types of micro or nano-mechanical systems, which are called "motor protein". Those biological motors have unique features, such as nano-meter scaled molecular motor, high efficiently energy transduction from chemical energy or having a capacity of self-assembly. The realization of bio-hybrid micro-machines to integrate such motor proteins and micro-or nano-structures fabricated of inorganic materials, would have some potential values that are not achieved by traditional electronic, magnetic or optical devices. In this paper, we discuss a possibility of motor proteins to use as driving unit for micro analysis systems, such as Lab on a chip or microTAS (micro Total Analysis System) devices.

A microrotary motor powered by bacteria.

Biological molecular motors have a number of unique advantages over artificial motors, including efficient conversion of chemical energy into mechanical work and the potential for self-assembly into larger structures, as is seen in muscle sarcomeres and bacterial and eukaryotic flagella. The development of an appropriate interface between such biological materials and synthetic devices should enable us to realize useful hybrid micromachines. Here we describe a microrotary motor composed of a 20-mum-diameter silicon dioxide rotor driven on a silicon track by the gliding bacterium Mycoplasma mobile. This motor is fueled by glucose and inherits some of the properties normally attributed to living systems.

Selective detection and transport of fully matched DNA by DNA-loaded microtubule and kinesin motor protein.

DNA-loaded microtubules (MTs) moving on a kinesin motor protein-coated substrate can selectively hybridize with a target fully matched DNA over single-base mismatched DNA and transport it. This technique is capable of collecting target biomolecules toward one point site to design new methodology of DNA analysis.

Malachite green-conjugated microtubules as mobile bioprobes selective for malachite green aptamers with capturing/releasing ability.

We have developed a novel mobile bioprobe using a conjugate of a kinesin-driven microtubule (MT) and malachite green (MG) as a platform for capturing MG RNA aptamers. The fluorescence of MG increases when it is bound to an MG aptamer, allowing MT-MG conjugates to work as sensors of RNA transcripts containing the MG aptamer sequence. Kinesin motor proteins provide an effective driving force to create mobile bioprobes without any manipulation. Although the fluorescence of a small number of MG-binding aptamers is low, the self-organization of tubulins into MTs enables the microscopic observation of the bound aptamers by collecting them on MTs. We demonstrate that MT-MG conjugates can select target aptamers from a transcription mixture and transport them without losing their inherent motility. Because the MG aptamer binds MG in a reversible manner, MT-MG conjugates can conditionally load and unload the target aptamers. This is one advantage of this system over the molecular probes developed previously in which reversible unloading is impossible due to high-affinity binding, such as between avidin and biotin. Furthermore, an MT-MG conjugate can be used as a platform for other MG aptameric sensors with recognition regions for various target analytes optimized by further selection procedures. This is the first step to applying living systems to in vitro devices. This technique could provide a new paradigm of mobile bioprobes establishing high-throughput in vitro selection systems using microfluidic devices operating in parallel.

Motor protein nano-biomachine powered by self-supplying ATP.

A new nano-biomachine has been created from microtubules (MTs) and hetero-bifunctional polymer particles bearing pyruvate kinase, which is propelled on glass surfaces coated with kinesin by use of self-supplying ATP.

Living microtransporter by uni-directional gliding of Mycoplasma along microtracks.

The gliding bacterium Mycoplasma mobile adheres to plastic surfaces and moves around vigorously. However, it has not been possible to control the direction of movements on plain surfaces. Here we report that, on patterned lithographic substrates, M. mobile cells are unable to climb tall walls and move along the bottom edge of the walls. This property to move persistently along walls enabled us to design patterns that control direction of movements, resulting in uni-directional circling or one-way gating between two areas. Furthermore, cells loaded with streptavidin beads following biotinylation of surface proteins moved at normal speeds. These bacteria could be useful as living microtransporters, carrying cargo around within micrometer-scale spaces.

Amino acids 519-524 of Dictyostelium myosin II form a surface loop that aids actin binding by facilitating a conformational change.

Residues 519-524 of Dictyostelium myosin II form a small surface loop on the actin binding face, and have been suggested to bind directly to actin through high affinity hydrophobic interactions. To test this hypothesis, we have characterized mutant myosins that lack this loop in vivo and in vitro. A mutant myosin in which this loop was replaced by an Ala residue (delta519-524/+A) was non-functional in vivo. Replacement with a single Gly residue instead of Ala yielded partial function, suggesting that structural flexibility, rather than hydrophobicity, is the key feature of the loop. The in vivo phenotype of the mutant enabled us to identify a number of additional amino acid changes that restore function to the delta519-524/+A mutation. Intriguingly, many of these, including L596S, were located at some distances away from the 519-524 loop. We have also isolated suppressors for the L596S mutant myosin, which was not functional in vivo. The suppressors for delta519-524/+A and those for L596S showed complementary charge patterns. In ATPase assays, delta519-524/+A S1 showed very low activity and little enhancement by actin, whereas L596S S1 was hyper active and displayed enhanced affinity for actin. In motility assays, delta519-524/+A myosin released actin filaments upon addition of ATP and was unable to support movements. L596S myosin was also inactive, but in this case actin filaments stayed immobile even after the addition of ATP. Transient kinetic measurements demonstrated that delta519-524/+A S1 is not only slower than wild type to bind actin filaments, but also slower to dissociate from actin filaments. Based on these results, we concluded that the 519-524 loop is not a major actin binding site but aids actin binding by facilitating a critical conformational change.