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Herein, I review our recent work toward developing methods for generating three-dimensional (3D) droplet arrays driven by capillarity. Microdroplet array-based systems are useful for bioassays and bioengineering because they require only small amounts of samples and reagents and provide the high throughput. Various methods have been developed for preparing droplet arrays, among which methods based on capillarity have attracted considerable attention owing to their simplicity. I and collaborators have developed such methods based on capillary flow, including a method for preparing droplet arrays via oil-water replacement. We recently proposed our own concept of "fluid-fluid interfacial energy driven 3D structure emergence in a micropillar scaffold (FLUID3EAMS)" and its application. FLUID3EAMS allows a 3D droplet (or hydrogel bead) array to be generated in a micropillar scaffold by passing a fluid-fluid interface through the scaffold. This approach is useful for applications requiring ordered or arrayed microdroplets in biosensors, biophysics, biology, and tissue engineering. This review is an extended version of the article "FLUID3EAMS: Fluid-Fluid Interfacial Energy Driven 3D Structure Emergence in a Micropillar Scaffold and Development in Bioengineering" published in Seibutsu Butsuri (vol. 62, p. 110-113, 2022)., Competing Interests: The author declares no conflicts of interest., (2023 THE BIOPHYSICAL SOCIETY OF JAPAN.)

A self-folding method that can fold a thick (~10 μm) metal layer with a large curvature (>1 mm−1) and is resistant to repetitive folding deformation is proposed. Given the successful usage of hinged origami/kirigami structures forms in deployable structures, they show strong potential for application in stretchable electronic devices. There are, however, two key difficulties in applying origami/kirigami methods to stretchable electronic devices. The first is that a thick metal layer used as the conductive layer of electronic devices is too hard for self-folding as it is. Secondly, a thick metal layer breaks on repetitive folding deformation at a large curvature. To overcome these difficulties, this paper proposes a self-folding method using hinges on a thick metal layer by applying a meander structure. Such a structure can be folded at a large curvature even by weak driving forces (such as those produced by self-folding) and has mechanical resistance to repetitive folding deformation due to the local torsional deformation of the meander structure. To verify the method, the large curvature self-folding of thick metal layers and their mechanical resistance to repetitive folding deformation is experimentally demonstrated. In addition, an origami/kirigami hybrid stretchable electronic device with light-emitting diodes (LEDs) is fabricated using a double-tiling structure called the perforated extruded Miura-ori.

We proposed a method to display an intermediate visual texture by spatial mixing. In addition to color information, the visual texture is an important element that characterizes the nature of an object's surface. While the system to display various color information has well matured in engineering, there is no method to reproduce visual textures in ambient light. In our method, the matte and glossy surfaces are used as "primary visual textures", and an intermediate visual texture is displayed by spatially mixing the primary visual textures. In this paper, we first quantified the visual texture of an object's surface based on measured intensities of scattered and reflected lights. Next, based on the quantification, we evaluated spatially mixed surfaces consisting of two primary visual textures, an acrylic plate and a holed sheet of drawing paper, by changing the area proportion of the two primary visual textures. Finally, a sensory evaluation showed significant differences between each intermediate visual texture, and the results corresponded to a trend in the optical evaluation. This study illustrates that visual textures could be quantified based on the intensity of scattered and reflected light and reveals the applicability of our method to the display for intermediate visual texture.

Non-invasive diagnosis on biological liquid samples, such as urine, sweat, saliva, and tears, may allow patients to evaluate their health by themselves. To obtain accurate diagnostic results, target liquid must be precisely sampled. Conventionally, urine sampling using filter paper can be given as an example sampling, but differences in the paper structure can cause variations in sampling volume. This paper describes precise liquid sampling using synthetic microfluidic papers, which are composed of obliquely combined micropillars. Sampling volume accuracy was investigated using different designs and collection methods to determine the optimal design and sample collecting method. The optimized protocol was followed to accurately measure potassium concentration using synthetic microfluidic paper and a commercially available densitometer, which verified the usefulness of the synthetic microfluidic papers for precision sampling., Competing Interests: The authors have declared that no competing interests exist.

The global increase in autoimmunity, together with the emerging autoimmune-related side effects of cancer immunotherapy, have furthered a need for understanding of immune tolerance and activation. Systemic lupus erythematosus (SLE) is the archetypical autoimmune disease, affecting multiple organs, and tissues. Studying SLE creates knowledge relevant not just for autoimmunity, but the immune system in general. Murine models and patient studies have provided increasing evidence for the innate immune toll like receptor-7 (TLR7) in disease initiation and progression. Here, we demonstrated that the kinase activity of the TLR7-downstream signaling molecule, interleukin-1 receptor associated kinase 4 (IRAK4), is essential for mild and severe autoimmune traits of the Sle1 and Sle1-TLR7 transgenic ( Sle1 Tg7) murine models, respectively. Elimination of IRAK4 signaling prevented all pathological traits associated with murine lupus, including splenomegaly with leukocyte expansion, detectable circulating antinuclear antibodies and glomerulonephritis, in both Sle1 and Sle1 Tg7 mice. The expansion of germinal center B cells and increased effector memory T cell phenotypes that are typical of lupus-prone strains, were also prevented with IRAK4 kinase elimination. Analysis of renal leukocyte infiltrates confirmed our earlier findings of an expanded conventional dendritic cell (cDC) within the kidneys of nephritic mice, and this was prevented with IRAK4 kinase elimination. Analysis of TLR7 at the protein level revealed that the expression in immune cells is dependent on the TLR7-transgene itself and/or autoimmune disease factors in a cell-specific manner. Increased TLR7 protein expression in renal macrophages and cDCs correlated with disease parameters such as blood urea nitrogen (BUN) levels and the frequency of leukocytes infiltrating the kidney. These findings suggest that controlling the level of TLR7 or downstream signaling within myeloid populations may prevent chronic inflammation and severe nephritis.

Objective: Toll-like receptors (TLRs) 7 and 9 are important innate signaling molecules with opposing roles in the development and progression of systemic lupus erythematosus (SLE). While multiple studies support the notion of a dependency on TLR-7 for disease development, genetic ablation of TLR-9 results in severe disease with glomerulonephritis (GN) by a largely unknown mechanism. This study was undertaken to examine the suppressive role of TLR-9 in the development of severe lupus in a mouse model., Methods: We crossed Sle1 lupus-prone mice with TLR-9-deficient mice to generate Sle1TLR-9 -/- mice. Mice ages 4.5-6.5 months were evaluated for severe autoimmunity by assessing splenomegaly, GN, immune cell populations, autoantibody and total Ig profiles, kidney dendritic cell (DC) function, and TLR-7 protein expression. Mice ages 8-10 weeks were used for functional B cell studies, Ig profiling, and determination of TLR-7 expression., Results: Sle1TLR-9 -/- mice developed severe disease similar to TLR-9-deficient MRL and Nba2 models. Sle1TLR-9 -/- mouse B cells produced more class-switched antibodies, and the autoantibody repertoire was skewed toward RNA-containing antigens. GN in these mice was associated with DC infiltration, and purified Sle1TLR-9 -/- mouse renal DCs were more efficient at TLR-7-dependent antigen presentation and expressed higher levels of TLR-7 protein. Importantly, this increase in TLR-7 expression occurred prior to disease development, indicating a role in the initiation stages of tissue destruction., Conclusion: The increase in TLR-7-reactive immune complexes, and the concomitant enhanced expression of their receptor, promotes inflammation and disease in Sle1TLR9 -/- mice., (© 2018 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of American College of Rheumatology.)

This paper presents a precise sampling microstructure formed as a paper strip. We have been developing dialysis system that can be implanted into a human body as an artificial kidney. For the safe use of our artificial kidney, the patients' urine needs to be constantly monitored to detect the abnormal value of ion concentration essential for human life. We are conceiving the monitoring system based on sampling by a paper strip. In this study, we fabricated the strip consisting of slanted and interlocked micropillars for the sampling, known as synthetic microfluidic paper. The paper-like substrate can be fabricated with a well-controlled geometry and subsequently enables precise sampling. Through the conducted experiments, it was shown that synthetic microfluidic paper had better mechanical properties, showed more precision in sampling than paper filter as well as corresponding liquid holding capability to the paper filter. Our proposed paper-based sampling system is expected to lead to the development of minimally invasive ion monitoring system with quantitative sampling strip.

Two-dimensional (2D) microdroplet arrays with indexed sample concentration gradients have been receiving considerable attention for high-throughput biological and medical analyses. However, the preparation of such an array by conventional methods mandates precise pipetting and/or pumping. In this paper, we introduce a method to spontaneously generate 2D-arrayed aqueous droplets using a well array, for which coarse pipetting is sufficient. The wells are connected in rows and columns via narrow channels. Aqueous solutions impregnated in the well array are split into droplets in every single well as a subsequently introduced immiscible solvent self-propagates and divides the solution at the channels. A concentration gradient of the samples can be formed across the connected solution in the well array; once droplets are generated, each droplet possesses a different sample concentration depending on its position in the array. We experimentally determined the optimal well dimensions and solvent species to obtain a high yield of droplet generation. We next demonstrated a 2D droplet array with a two-sample concentration gradient. Finally, the applicability of the system was demonstrated through a cell viability assay using a sample that induced apoptosis. We believe the proposed method contributes to simplification and miniaturization of the system to generate droplet arrays and thus is applicable to biological and medical analysis.

DNA logic circuits utilizing DNA hybridization and/or enzymatic reactions have drawn increasing attention for their potential applications in the diagnosis and treatment of cellular diseases. The compartmentalization of such a system into a microdroplet considerably helps to precisely regulate local interactions and reactions between molecules. In this study, we introduced a relay approach for enabling the transfer of DNA from one droplet to another to implement multi-step sequential logic operations. We proposed electrical fusion and mechanical splitting of droplets to facilitate the DNA flow at the inputs, logic operation, output, and serial connection between two logic gates. We developed Negative-OR operations integrated by a serial connection of the OR gate and NOT gate incorporated in a series of droplets. The four types of input defined by the presence/absence of DNA in the input droplet pair were correctly reflected in the readout at the Negative-OR gate. The proposed approach potentially allows for serial and parallel logic operations that could be used for complex diagnostic applications.

Logical operations using biological molecules, such as DNA computing or programmable diagnosis using DNA, have recently received attention. Challenges remain with respect to the development of such systems, including label-free output detection and the rapidity of operation. Here, we propose integration of biological nanopores with DNA molecules for development of a logical operating system. We configured outputs "1" and "0" as single-stranded DNA (ssDNA) that is or is not translocated through a nanopore; unlabeled DNA was detected electrically. A negative-AND (NAND) operation was successfully conducted within approximately 10 min, which is rapid compared with previous studies using unlabeled DNA. In addition, this operation was executed in a four-droplet network. DNA molecules and associated information were transferred among droplets via biological nanopores. This system would facilitate linking of molecules and electronic interfaces. Thus, it could be applied to molecular robotics, genetic engineering, and even medical diagnosis and treatment.

We introduce Synthetic Microfluidic Paper, a novel porous material for microfluidic applications that consists of an OSTE polymer that is photostructured in a well-controlled geometry of slanted and interlocked micropillars. We demonstrate the distinct benefits of Synthetic Microfluidic Paper over other porous microfluidic materials, such as nitrocellulose, traditional paper and straight micropillar arrays: in contrast to straight micropillar arrays, the geometry of Synthetic Microfluidic Paper was miniaturized without suffering capillary collapse during manufacturing and fluidic operation, resulting in a six-fold increased internal surface area and a three-fold increased porous fraction. Compared to commercial nitrocellulose materials for capillary assays, Synthetic Microfluidic Paper shows a wider range of capillary pumping speed and four times lower device-to-device variation. Compared to the surfaces of the other porous microfluidic materials that are modified by adsorption, Synthetic Microfluidic Paper contains free thiol groups and has been shown to be suitable for covalent surface chemistry, demonstrated here for increasing the material hydrophilicity. These results illustrate the potential of Synthetic Microfluidic Paper as a porous microfluidic material with improved performance characteristics, especially for bioassay applications such as diagnostic tests.

Glomerulonephritis is a common and debilitating feature of systemic lupus erythematosus (SLE). The precise immune mechanisms that drive the progression from benign autoimmunity to glomerulonephritis are largely unknown. Previous investigations have shown that a moderate increase of the innate Toll-like receptor 7 (TLR7) is sufficient for the development of nephritis. In these systems normalization of B-cell TLR7 expression or temporal depletion of plasmacytoid dendritic cells (pDCs) slow progression; however, the critical cell that is responsible for driving full immunopathology remains unidentified. In this investigation we have shown that conventional DC expression of TLR7 is essential for severe autoimmunity in the Sle1Tg7 model of SLE. We show that a novel expanding CD11b(+) conventional DC subpopulation dominates the infiltrating renal inflammatory milieu, localizing to the glomeruli. Moreover, exposure of human myeloid DCs to IFN-α or Flu increases TLR7 expression, suggesting they may have a role in self-RNA recognition pathways in clinical disease. To our knowledge, this study is the first to highlight the importance of conventional DC-TLR7 expression for kidney pathogenesis in a murine model of SLE.