Ejection of Large Particulate Materials from Giant Unilamellar Vesicles Induced by Electropulsation.

Electroporation or electropermealization is a technique to open pores in the lipid bilayer membrane of cells and vesicles transiently to increase its permeability to otherwise impermeable molecules. However, the upper size limit of the materials permeable through this operation has not been studied in the past. Here, we investigate the size of the material that can be released (ejected) from giant unilamellar vesicles (GUVs) upon electrical pulsation. We confirm that the volume of GUV shrinks in a stepwise manner upon periodical pulsation, in accordance with previous studies. When the same operation is applied to GUVs that encapsulate microbeads, we find that beads as large as 20 µm can be ejected across the membrane without rupturing the whole GUV structure. We also demonstrate that functional bioactive particulate materials, such as gel balls, vesicles, and cells can be encapsulated in and ejected from GUVs. We foresee that this phenomenon can be applied to precisely regulate the time and location of release of these particulate materials in the microenvironment.

A fluidics-based impact sensor.

Microelectromechanical systems (MEMS)-based high-performance accelerometers are ubiquitously used in various electronic devices. However, there is an existing need to detect physical impacts using low-cost devices with no electronic circuits or a battery. We designed and fabricated an impact sensor prototype using a commercial stereolithography apparatus that only consists of a plastic housing and working fluids. The sensor device responds to the instantaneous acceleration (impact) by deformation and pinch off of a water droplet that is suspended in oil in a sensor cavity. We tested the various geometrical and physical parameters of the impact sensor to identify their relations to threshold acceleration values. We show that the state diagram that is plotted against the dimensionless Archimedes and Bond numbers adequately describes the response of the proposed sensor.

Deformation Modes of Giant Unilamellar Vesicles Encapsulating Biopolymers.

The shapes of giant unilamellar vesicles (GUVs) enclosing polymer molecules at relatively high concentration, used as a model cytoplasm, significantly differ from those containing only small molecules. Here, we investigated the effects of the molecular weights and concentrations of polymers such as polyethylene glycol (PEG), bovine serum albumin (BSA), and DNA on the morphology of GUVs deflated by osmotic pressure. Although small PEG (MW < 1000) does not alter the mode of shape transformation even at >10% (w/w), PEG with MW > 6000 induces budding and pearling transformation at above 1% (w/w). Larger PEG frequently induced small buddings and tubulation from the membrane of mother GUVs. A similar trend was observed with BSA, indicating that the effect is irrelevant to the chemical nature of polymers. More surprisingly, long strands of DNA (>105 bp) enclosed in GUVs induced budding transformation at concentrations as low as 0.01-0.1% (w/w). We expect that this molecular size dependency arises mainly from the depletion volume effect. Our results showed that curving, budding, and tubulation of lipid membranes, which are ubiquitous in living cells, can result from simple cell-mimics consisting of the membrane and cytosolic macromolecules, but without specific shape-determining proteins.

One-step micromolding of complex 3D microchambers for single-cell analysis.

Herein we examined the extent of replicability of the PDMS microchamber device transferred from the master mold with complex 3D structures fabricated via micro stereolithography. Due to the elastomeric properties of PDMS, the reversely tapered micromold, with the diameter ratio of ∼5 from the largest to the narrowest part, was precisely transferred without breaking. We obtained the mathematical model to estimate the stress exerted on the mold during the demolding process. Finally, we tested the applicability of this unusual microchamber for single-cell trapping and an enzyme assay.

Cell-free protein synthesis in a microchamber revealed the presence of an optimum compartment volume for high-order reactions.

The application of microelectromechanical systems (MEMS) to chemistry and biochemistry allows various reactions to be performed in microscale compartments. Here, we aimed to use the glass microchamber to study the compartment size dependency of the protein synthesis, one of the most important reactions in the cell. By encapsulating the cell-free protein synthesis system with different reaction orders in femtoliter microchambers, chamber size dependency of the reaction initiated with a constant copy number of DNA was investigated. We were able to observe the properties specific to the high order reactions in microcompartments with high precision and found the presence of an optimum compartment volume for a high-order reaction using real biological molecules.

Cell-free protein synthesis from a single copy of DNA in a glass microchamber.

To achieve a cell-mimetic reaction environment, we fabricated and tested quartz microchambers for conducting protein synthesis using an in vitro transcription and translation system, the PURE system. By introducing a glass microchamber and blocking the surface of the chamber with amino acids, the concentration of the synthesized marker protein (green fluorescent protein, GFP) was significantly improved compared to that in the poly(dimethylsiloxane) (PDMS) microchamber. The concentration was below the detection limit in the PDMS microchambers, whereas the glass microchambers yielded 700 nM GFP, representing 41% of the bulk reaction. There was no detectable difference when the GFP synthesis was performed in microchambers with sizes ranging from 40 fL to 7 pL, indicating that the present microchamber system can serve as a cell-sized test tube with a variable reaction volume. Finally, we demonstrated that two different proteins, GFP and ß-galactosidase, can be expressed from single genes in our experimental setup. Quantized and distinctive signals from proteins synthesized from 0, 1, or 2 copies of genes were obtained. The microchamber presented here can be utilized not only to study the effects of compartment volume on protein synthesis but also for the comprehensive analysis of complex biochemical reactions in cell-mimetic environments.

Control of noise-induced coherent behaviors in an array of excitable elements by time-delayed feedback.

We investigate feedback-controlled coherent dynamics in a two-dimensional array of excitable elements. We demonstrate that one can freely enhance or reduce the spatiotemporal coherence of noise-induced oscillation, such as coherence resonance and phase synchronization, by controlling both the delay time and the feedback gain. Furthermore, we find that noise-induced oscillations are entrained by the feedback force in a certain range of the delay time. Experimental observations are approximately reproduced in a numerical simulation with a forced Oregonator model.

Feedback-controlled dynamics in a two-dimensional array of active elements.

We investigate collective behaviors in a two-dimensional array of active elements controlled by time-delayed feedback, where elements are prepared by localizing the Belousov-Zhabotinsky reaction in a gel matrix. We demonstrate that both the spatial and temporal coherence can be effectively controlled by varying feedback parameters, such as the time delay and the gain. For a sufficiently high feedback gain, the fully synchronized state with low temporal coherence appears, which might be the state induced only by the delay feedback. Experimental results are approximately reproduced in a numerical simulation with a forced Oregonator reaction-diffusion model.

Array-enhanced coherence resonance and phase synchronization in a two-dimensional array of excitable chemical oscillators.

We investigate the spatiotemporal dynamics in a two-dimensional array of excitable elements subjected to independent external noise, where elements are prepared by localizing the Belousov-Zhabotinsky reaction in a gel matrix. We experimentally demonstrate that the coherence of noise-induced firings is improved with increasing the array size, i.e., the occurrence of array-enhanced coherence resonance. Furthermore, it is found that synchronization among oscillators which are barely coupled can be achieved via coherence resonance. Experimental observations are approximately reproduced in a numerical simulation with a forced Oregonator reaction-diffusion model.

Successive splitting of autowaves in a nonlinear chemical reaction medium.

The phenomenon of wave splitting is investigated in a two-dimensional excitable light-sensitive Belousov-Zhabotinsky reaction medium after extremely changing the intensity of illuminated light for a short time. It is found that successive wave splitting and nonannihilation collision between two waves of different amplitudes occur spontaneously under narrow experimental conditions. Experimental observations are approximately reproduced in the specific parameter range by a numerical simulation with a Bär-Eiswirth model.

Noise-induced spatiotemporal dynamics in a linear array of excitable chemical oscillators.

The effects of additive noise on spatiotemporal dynamics are investigated in a one-dimensional array of excitable elements in which the Belousov-Zhabotinsky reaction is localized. At the appropriate separation between adjacent elements, we find that the resonance effect becomes larger for the element being more apart from the first element when only the first element is subjected to the external noise. This phenomenon is a sort of array-enhanced resonance. Furthermore, we find that phase locking between the first element and the other elements is induced via coherence resonance of the first element. Experimental observations are approximately reproduced in a numerical simulation with a forced Oregonator reaction-diffusion model.