Pause of the target gliding microtuble on the virtual cathode.

We report the transient response of gliding microtubules on a virtual cathode. In vivo activities, microtubule-kinesin systems are known to act as motor proteins with respect to cell motility cytokinesis and cellular transport by hydrolyzing ATP molecules. With development of in vitro assays, motor proteins have been attracting much attention as a key component for highly efficient nano-transportation systems. The molecular functions based on structural states are affected by changing the ionic condition of the molecular functions and by changing the electrical field in solution because of electrical charges of the molecules. The virtual cathode, which was generated on the SiN display surface by a low energy electron beam, locally induced electrochemical reactions and electric field around the targeted molecules on the display surface, and then the gliding motions of the targeted microtubules were regulated. In this study, we demonstrated that the virtual cathode display temporally stops a selected gliding microtubule by only applying the virtual cathode to the microtubule. The pause mode of the microtubule was easily canceled by simply turning the virtual cathode off, and then the gliding motion was restarted.

Spatiotemporal Control of Electrokinetic Transport in Nanofluidics Using an Inverted Electron-Beam Lithography System.

Manipulation techniques of biomolecules have been proposed for biochemical analysis which combine electrokinetic dynamics, such as electrophoresis or electroosmotic flow, with optical manipulation to provide high throughput and high spatial degrees of freedom. However, there are still challenging problems in nanoscale manipulation due to the diffraction limit of optics. We propose here a new manipulation technique for spatiotemporal control of chemical transport in nanofluids using an inverted electron-beam (EB) lithography system for liquid samples. By irradiating a 2.5 keV EB to a liquid sample through a 100-nm-thick SiN membrane, negative charges can be generated within the SiN membrane, and these negative charges can induce a highly focused electric field in the liquid sample. We showed that the EB-induced negative charges could induce fluid flow, which was strong enough to manipulate 240 nm nanoparticles in water, and we verified that the main dynamics of this EB-induced fluid flow was electroosmosis caused by changing the zeta potential of the SiN membrane surface. Moreover, we demonstrated manipulation of a single nanoparticle and concentration patterning of nanoparticles by scanning EB. Considering the shortness of the EB wavelength and Debye length in buffer solutions, we expect that our manipulation technique will be applied to nanomanipulation of biomolecules in biochemical analysis and control.

L-2L ladder digital-to-analogue converter for dynamics generation of chemical concentrations.

Cellular response to dynamic chemical stimulation encodes rich information about the underlying reaction pathways and their kinetics. Microfluidic chemical stimulators play a key role in generating dynamic concentration waveforms by mixing several aqueous solutions. In this article, we propose a multi-layer microfluidic chemical stimulator capable of modulating chemical concentrations by a simple binary logic based on the electronic-hydraulic analogy of electronic R-2R ladder circuits. The proposed device, which we call L-2L ladder digital-to-analogue converter (DAC), allows us to systematically modulate 2 n levels of concentrations from single sources of solution and solvent by a single operation of 2n membrane valves, which contrasts with existing devices that require complex channel geometry with multiple input sources and valve operations. We fabricated the L-2L ladder DAC with n = 3 bit resolution and verified the concept by comparing the generated waveforms with computational simulations. The response time of the proposed DAC was within the order of seconds because of its simple operation logic of membrane valves. Furthermore, detailed analysis of the waveforms revealed that the transient concentration can be systematically predicted by a simple addition of the transient waveforms of 2n = 6 base patterns, enabling facile optimization of the channel geometry to fine-tune the output waveforms.

Explicit calculation method for cell alignment in non-circular geometries.

The alignment of spindle-shaped cells in two-dimensional geometries induces singular points called topological defects, at which the alignment angle of the cell cannot be defined. To control defects related to biological roles such as cell apoptosis, calculation methods for predicting the defect positions are required. This study proposes an explicit calculation method for predicting cell alignment and defect positions in non-circular geometries. First, a complex potential is introduced to describe the alignment angles of cells, which is used to derive an explicit formula for cell alignment in a unit disc. Then, the derived formula for the unit disc is extended to the case for non-circular geometries using a numerical conformal mapping. Finally, the complex potential allows a calculation of the Frank elastic energy, which can be minimized with respect to the defect positions to predict their equilibrium state in the geometry. The proposed calculation method is used to demonstrate a numerical prediction of multiple defects in circular and non-circular geometries, which are consistent with previous experimental results.

Surface-limited reactions for spatial control of kinesin-microtubule motility assays using indirect irradiation of an electron beam.

Gliding of microtubules (MTs) on kinesins has been applied to lab-on-a-chip devices, which enable autonomous transportation and detection of biomolecules in the field of bioengineering. For rapid fabrication and evaluation of the kinesin-MT based devices, optical control techniques have been developed for control of kinesin activity and density; however, use of caged molecules lacks spatial controllability for long-term experiments, and direct irradiations of UV light onto kinesin-coated surfaces are inherently damaging to MTs due to their depth limit since the heights of the kinesin-MT systems are at the tens of a nanometer scale. Considering surface electric fields in electrolytic solutions are shielded at the nanometer scale due to Debye shielding, in this study, we show that fine spatial control of kinesin density and activity is enabled using surface-limited electrochemical reactions induced by indirect irradiations of an electron beam (EB). An EB is indirectly irradiated onto the kinesins through a 100-nm-thick silicon nitride membrane, and the electrons scattered in the membrane can cause localized electrochemical effects to the kinesins. We show that these localized electrochemical effects cause both ablation of kinesins and motility control of kinesin activity by changing the EB acceleration voltage. In particular, the latter is achieved without complete ablation of MTs, though the MTs are indirectly irradiated by the EB. As a demonstration of on-demand control of gliding MTs, we show the accumulation of the MTs on a target area by scanning the EB. The proposed accumulation technique will lead to rapid prototyping of microdevices based on MT-kinesin motility assay systems.

Tough, permeable and biocompatible microfluidic devices formed through the buckling delamination of soft hydrogel films.

Microchannels in soft materials play an important role in developing movable, deformable, and biocompatible fluidic systems for applications in various fields. Intensively investigated approaches to create microscale channel architectures use mechanical instability in soft materials, which can provide intricate yet ordered architectures with low cost and high throughput. Here, for microchannel fabrication, we demonstrate the use of swelling-driven buckle delamination of hydrogels, which is a mechanical instability pattern found in compressed film/substrate layer composites. By spatially controlling interfacial bonding between a thin polyacrylamide (PAAm) gel film and glass substrate, swelling-driven compressive stress induces buckle delamination at programmed positions, resulting in the formation of continuous hollow paths as microchannels. Connecting flow tubes with a 3D-printed connecter provides a deformable microfluidic device, enabling pressure-driven flows without leakage from the connecter and rupture of the channels. Furthermore, by stacking less-swellable bulk gels on the device, we obtained a tough, permeable, and biocompatible microfluidic device. Finally, we performed a cell culture on the device and chemical stimulation to cells through the diffusion of molecules from the microchannels. The results of this work shed light on designing pressure sensitive/resistant microfluidic systems based on diverse hydrogels with intricate 3D morphologies and will be useful for applications in the fields of bioanalysis, biomimetics, tissue engineering, and cell biology.

Analysing diffusion and flow-driven instability using semidefinite programming.

Diffusion and flow-driven instability, or transport-driven instability, is one of the central mechanisms to generate inhomogeneous gradient of concentrations in spatially distributed chemical systems. However, verifying the transport-driven instability of reaction-diffusion-advection systems requires checking the Jacobian eigenvalues of infinitely many Fourier modes, which is computationally intractable. To overcome this limitation, this paper proposes mathematical optimization algorithms that determine the stability/instability of reaction-diffusion-advection systems by finite steps of algebraic calculations. Specifically, the stability/instability analysis of Fourier modes is formulated as a sum-of-squares optimization program, which is a class of convex optimization whose solvers are widely available as software packages. The optimization program is further extended for facile computation of the destabilizing spatial modes. This extension allows for predicting and designing the shape of the concentration gradient without simulating the governing equations. The streamlined analysis process of self-organized pattern formation is demonstrated with a simple illustrative reaction model with diffusion and advection.

Dynamic Creation of 3D Hydrogel Architectures via Selective Swelling Programmed by Interfacial Bonding.

The topological features of material surfaces are crucial to the emergence of functions based on characteristic architectures. Among them, the combination of surface architectures and soft materials, which are highly deformable and flexible, has great potential as regards developing functional materials toward providing/enhancing advanced functions such as switchability and variability. Therefore, a simple yet versatile method for creating three-dimensional (3D) architectures based on soft materials is strongly required. In this study, hydrogels are selected as the soft materials and hydrogel film/rigid substrate layer composites are fabricated to obtain a 3D hydrogel architecture based on swelling instability. When a hydrogel film weakly attached to a rigid substrate is exposed to water, swelling-driven compressive stress induces buckle-delamination of the film from the substrate. Utilizing the chemical modification of a rigid substrate and a conventional photolithography technique, the delamination location is successfully controlled, resulting in a high-aspect-ratio folding architecture at an arbitrary position. In addition, we systematically designed the delamination geometry and chemically tuned the swelling ratio of the hydrogel, leading to the discovery of several new morphology transitions and relationships between the morphologies and the controllable parameters. This work provides a new approach to fabricating highly programmable 3D architectures of soft materials.

Pinpoint Delivery of Molecules by Using Electron Beam Addressing Virtual Cathode Display.

Electroporation, a physical transfection method to introduce genomic molecules in selective living cells, could be implemented by microelectrode devices. A local electric field generated by a finer electrode can induces cytomembrane poration in the electrode vicinity. To employ fine, high-speed scanning electrodes, we developed a fine virtual cathode pattern, which was generated on a cell adhesive surface of 100-nm-thick SiN membrane by inverted-electron beam lithography. The SiN membrane works as both a vacuum barrier and the display screen of the virtual cathode. The kinetic energy of the incident primary electrons to the SiN membrane was completely blocked, whereas negative charges and leaking electric current appeared on the surface of the dielectric SiN membrane within a region of 100 nm. Locally controlled transmembrane molecular delivery was demonstrated on adhered C2C12 myoblast cells in a culturing medium with fluorescent dye propidium iodide (PI). Increasing fluorescence of pre-diluted PI indicated local poration and transmembrane inflow at the virtual cathode position, as well as intracellular diffusion. The transmembrane inflows depended on beam duration time and acceleration voltage. At the post-molecular delivery, a slight decrease in intracellular PI fluorescence intensity indicates membrane recovery from the poration. Cell viability was confirmed by time-lapse cell imaging of post-exposure cell migration.

Dynamic electromechanical control of biomolecules using a nano virtual cathode display.

The dynamic electromechanical control of spatial structures of biomolecules in aqueous solutions was demonstrated using a nano virtual cathode display. By generating a focused electric field around the biomolecules using an electron beam (EB), the molecules' spatiotemporal responses to the electrical stimuli, such as globule transition of DNA random coils and deformation of planar lipid bilayers and vesicles, were successfully observed. The proposed system may be applied to high resolution and high degree-of-freedom manipulations to measure the mechanical and structural properties of bio-nanomaterials.