Roseiterribacter gracilis gen. nov., sp. nov., a novel filterable alphaproteobacterium isolated from soil using a gel-filled microwell array device.

Our previous studies indicate the abundant and diverse presence of yet-to-be-cultured microorganisms in the micropore-filtered fractions of various environmental samples. Here, we isolated a novel bacterium (designated as strain TMPK1T) from a 0.45-µm-filtered soil suspension by using a gel-filled microwell array device comprising 900 microwells and characterized its phylogenetic and physiological features. This strain showed low 16S rRNA gene sequence identities (<91%) and low average nucleotide identity values (<70%) to the closest validly described species, and belonged to a novel-family-level lineage within the order Rhodospirillales of Alphaproteobacteria. Strain TMPK1T exhibited small cell sizes (0.08-0.23 µm3) and had a high cyclopropane fatty acid content (>13%), and these characteristics were differentiated from other Rhodospirillales bacteria. A comprehensive habitability search using amplicon datasets suggested that TMPK1T and its close relatives are mainly distributed in soil and plant-associated environments. Based on these results, we propose that strain TMPK1T represents a novel genus and species named Roseiterribacter gracilis gen. nov., sp. nov. (JCM 34627T = KCTC 82790T). We also propose Roseiterribacteraceae fam. nov. to accommodate the genus Roseiterribacter.

Two-Dimensional SERS Sensor Array for Identifying and Visualizing the Gas Spatial Distributions of Two Distinct Odor Sources.

The spatial distribution of gas emitted from an odor source provides valuable information regarding the composition, size, and localization of the odor source. Surface-enhanced Raman scattering (SERS) gas sensors exhibit ultra-high sensitivity, molecular specificity, rapid response, and large-area detection. In this paper, a SERS gas sensor array was developed for visualizing the spatial distribution of gas evaporated from benzaldehyde and 4-ethylbenzaldehyde odor sources. The SERS spectra of the gas were collected by scanning the sensor array using an automatic detection system. The non-negative matrix factorization algorithm was employed to extract feature and concentration information at each spot on the sensor array. A heatmap image was generated for visualizing the gas spatial distribution using concentration information. Gaussian fitting was applied to process the image for localizing the odor source. The size of the odor source was estimated using the processed image. Moreover, the spectra of benzaldehyde, 4-ethylbenzaldehyde, and their gas mixture were simultaneously detected using one SERS sensor array. The feature information was recognized using a convolutional neural network with an accuracy of 98.21%. As a result, the benzaldehyde and 4-ethylbenzaldehyde odor sources were identified and visualized. Our research findings have various potential applications, including odor source localization, environmental monitoring, and healthcare.

An Odor Trace Visualization System Using a Two-Dimensional Backside Scattering Localized Surface Plasmon Resonance Gas Sensor.

Odor information fills every corner of our lives yet obtaining its spatiotemporal distribution is a difficult challenge. Localized surface plasmon resonance has shown good sensitivity and a high response/recovery speed in odor sensing and converts chemical information such as odor information into optical information, which can be captured by charge-coupled device cameras. This suggests that the utilization of localized surface plasmon resonance has great potential in two-dimensional odor trace visualization. In this study, we developed a two-dimensional imaging system based on backside scattering from a localized surface plasmon resonance substrate to visualize odor traces, providing an intuitive representation of the spatiotemporal distribution of odor, and evaluated the performance of the system. In comparative experiments, we observed distinct differences between odor traces and disturbances caused by environmental factors in differential images. In addition, we noted changes in intensity at positions corresponding to the odor traces. Furthermore, for indoor experiments, we developed a method of finding the optimal capture time by comparing changes in differential images relative to the shape of the original odor trace. This method is expected to assist in the collection of spatial information of unknown odor traces in future research.

Low-cost gel-filled microwell array device for screening marine microbial consortium.

In order to exploit the microbes present in the environment for their beneficial resources, effective selection and isolation of microbes from environmental samples is essential. In this study, we fabricated a gel-filled microwell array device using resin for microbial culture. The device has an integrated sealing mechanism that enables high-density isolation based on the culture of microorganisms; the device is easily manageable, facilitating observation using bright-field microscopy. This low-cost device made from polymethyl methacrylate (PMMA)/polyethylene terephthalate (PET) has 900 microwells (600 µm × 600 µm × 700 µm) filled with a microbial culture gel medium in glass slide-sized plates. It also has grooves for maintaining the moisture content in the micro-gel. The partition wall between the wells has a highly hydrophobic coating to inhibit microbial migration to neighboring wells and to prevent exchange of liquid substances. After being hermetically sealed, the device can maintain moisture in the agarose gels for 7 days. In the bacterial culture experiment using this device, environmental bacteria were isolated and cultured in individual wells after 3 days. Moreover, the isolated bacteria were then picked up from wells and re-cultured. This device is effective for the first screening of microorganisms from marine environmental samples.

Monolithic processing of a layered flexible robotic actuator film for kinetic electronics.

Low-invasive soft robotic techniques can potentially be used for developing next-generation body-machine interfaces. Most soft robots require complicated fabrication processes involving 3D printing and bonding/assembling. In this letter, we describe a monolithic soft microrobot fabrication process for the mass production of soft film robots with a complex structure by simple 2D processing of a robotic actuator film. The 45 µg/mm2 lightweight film robot can be driven at a voltage of CMOS compatible 5 V with 0.15 mm-1 large curvature changes; it can generate a force 5.7 times greater than its self-weight. In a durability test, actuation could be carried out over 8000 times without degradation. To further demonstrate this technique, three types of film robots with multiple degrees of freedom and a moving illuminator robot were fabricated. This technique can easily integrate various electrical circuits developed in the past to robotic systems and can be used for developing advanced wearable sensing devices; it can be called "Kinetic electronics".

SERS Gas Sensors Based on Multiple Polymer Films with High Design Flexibility for Gas Recognition.

The Surface-Enhanced Raman Scattering (SERS) technique is utilized to fabricate sensors for gas detection due to its rapid detection speed and high sensitivity. However, gases with similar molecular structures are difficult to directly discriminate using SERS gas sensors because there are characteristic peak overlaps in the Raman spectra. Here, we proposed a multiple SERS gas sensor matrix via a spin-coating functional polymer to enhance the gas recognition capability. Poly (acrylic acid) (PAA), Poly (methyl methacrylate) (PMMA) and Polydimethylsiloxane (PDMS) were employed to fabricate the polymer film. The high design flexibility of the two-layer film was realized by the layer-by-layer method with 2 one-layer films. The SERS gas sensor coated by different polymer films showed a distinct affinity to target gases. The principle component analysis (PCA) algorithm was used for the further clustering of gas molecules. Three target gases, phenethyl alcohol, acetophenone and anethole, were perfectly discriminated, as the characteristic variables in the response matrix constructed by the combination of gas responses obtained 3 one-layer and 3 two-layer film-coated sensors. This research provides a new SERS sensing approach for recognizing gases with similar molecular structures.

Draft Genome Sequence of Novel Filterable Rhodospirillales Bacterium Strain TMPK1, Isolated from Soil.

We report the draft genome sequence of novel Rhodospirillales bacterium strain TMPK1, isolated from a micropore-filtered soil suspension. This strain has a genome of 4,249,070 bp, comprising 4,151 protein-coding sequences. The genome sequence data further suggest that strain TMPK1 is an alphaproteobacterium capable of carotenoid production.

Microfabricated electrochemical sensing devices.

Electrochemistry provides possibilities to realize smart microdevices of the next generation with high functionalities. Electrodes, which constitute major components of electrochemical devices, can be formed by various microfabrication techniques, and integration of the same (or different) components for that purpose is not difficult. Merging this technique with microfluidics can further expand the areas of application of the resultant devices. To augment the development of next generation devices, it will be beneficial to review recent technological trends in this field and clarify the directions required for moving forward. Even when limiting the discussion to electrochemical microdevices, a variety of useful techniques should be considered. Therefore, in this review, we attempted to provide an overview of all relevant techniques in this context in the hope that it can provide useful comprehensive information.

A Robot Equipped with a High-Speed LSPR Gas Sensor Module for Collecting Spatial Odor Information from On-Ground Invisible Odor Sources.

Improving the efficiency of detecting the spatial distribution of gas information with a mobile robot is a great challenge that requires rapid sample collection, which is basically determined by the speed of operation of gas sensors. The present work developed a robot equipped with a high-speed gas sensor module based on localized surface plasmon resonance. The sensor module is designed to sample gases from an on-ground odor source, such as a footprint material or artificial odor marker, via a fine sampling tubing. The tip of the sampling tubing was placed close to the ground to reduce the sampling time and the effect of natural gas diffusion. On-ground ethanol odor sources were detected by the robot at high resolution (i.e., 2.5 cm when the robot moved at 10 cm/s), and the reading of gas information was demonstrated experimentally. This work may help in the development of environmental sensing robots, such as the development of odor source mapping and multirobot systems with pheromone tracing.

Molecularly Imprinted Filtering Adsorbents for Odor Sensing.

Versatile odor sensors that can discriminate among huge numbers of environmental odorants are desired in many fields, including robotics, environmental monitoring, and food production. However, odor sensors comparable to an animal's nose have not yet been developed. An animal's olfactory system recognizes odor clusters with specific molecular properties and uses this combinatorial information in odor discrimination. This suggests that measurement and clustering of odor molecular properties (e.g., polarity, size) using an artificial sensor is a promising approach to odor sensing. Here, adsorbents composed of composite materials with molecular recognition properties were developed for odor sensing. The selectivity of the sensor depends on the adsorbent materials, so specific polymeric materials with particular solubility parameters were chosen to adsorb odorants with various properties. The adsorption properties of the adsorbents could be modified by mixing adsorbent materials. Moreover, a novel molecularly imprinted filtering adsorbent (MIFA), composed of an adsorbent substrate covered with a molecularly imprinted polymer (MIP) layer, was developed to improve the odor molecular recognition ability. The combination of the adsorbent and MIP layer provided a higher specificity toward target molecules. The MIFA thus provides a useful technique for the design and control of adsorbents with adsorption properties specific to particular odor molecules.

Microfluidic Separation of Redox Reactions for Coulometry Based on Metallization at the Mixed Potential.

Coulometric detection of an analyte in a solution at nanoliter scale was conducted by having redox reactions proceed simultaneously on a platinum electrode. The analyte was oxidized on a part of the electrode in one flow channel and silver was deposited on an array of circular microelectrodes formed in another flow channel at a mixed potential. Coulometric determination of the deposited silver showed a steep change in the generated charge as a result of the complete oxidation of silver. The short measurement time after the start of the coulometry suppressed the increase in background charge, resulting in significant lowering of the detection limit. The lower detection limit for H2O2 was 30 nM (3σ). To improve selectivity and minimize the influence of coexisting interferents, the shifting of the mixed potential, application of a permselective membrane, and electrochemical elimination of the interferents were effective modifications.

Coulometric detection of components in liquid plugs by microfabricated flow channel and electrode structures.

Coulometry has been demonstrated to be effective for determining the analyte in a liquid plug on the nanoliter-scale confined in a flow channel. A plug prepared in a rhombus structure of an auxiliary flow channel was placed on a thin-film three-electrode system, and hydrogen peroxide was detected as a model analyte. Under a fixed potential, the current decayed rapidly, particularly in shallow flow channels, thus making reproducible amperometric detection difficult. On the other hand, the increasing charge during coulometry facilitated the measurements. A constricted flow channel structure with an array of platinum strips for the working electrode was effective at efficiently consuming the analyte to improve the sensitivity and lower the detection limit. Compared to the case of a single short working electrode with the same area, a 4-fold increase in sensitivity was observed for the best combination of flow channel height and interstrip distance. With an increase in the generated current while maintaining the background at a low level, the detection limit was lowered from 1.3 µM to 410 nM using working electrodes with the same area. Furthermore, the processing of solutions containing L-glutamate or L-glutamate oxidase and the detection of L-glutamate were demonstrated.

Engineering of capillary-like structures in tissue constructs by electrochemical detachment of cells.

A major challenge in the development of functional thick tissues is the formation of vascular networks for oxygen and nutrient supply throughout the engineered tissue constructs. This study describes an electrochemical approach for fabrication of capillary-like structures, precisely aligned within micrometer distances, whose internal surfaces are covered with vascular endothelial cells. In this approach, an oligopeptide containing a cell adhesion domain (RGD) in the center and cysteine residues at both ends was designed. Cysteine has a thiol group that adsorbs onto a gold surface via a gold-thiolate bond. The cells attached to the gold surface via the oligopeptide were readily and noninvasively detached by applying a negative electrical potential and cleaving the gold-thiolate bond. This approach was applicable not only for a flat surface but also for various configurations, including cylindrical structures. By applying this approach to thin gold rods aligned in a spatially controlled manner in a perfusion culture device, human umbilical vein endothelial cells (HUVECs) were transferred onto the internal surface of capillary structures in collagen gel. In the subsequent perfusion culture, the HUVECs grew into the collagen gel and formed luminal structures, thereby forming vascular networks in vitro.

Preparation of coculture system with three extracellular matrices using capillary force lithography and layer-by-layer deposition.

Micropatterned cocultures were fabricated with 3 extracellular matrices, hyaluronic acid (HA), fibronectin, and collagen. The feature of the fabrication processes is to avoid the use of potentially cytotoxic materials and utilize capillary force of the solution and interactions between the extracellular matrix components. The coculture system can be used to investigate the effects of heterocellular interactions on cellular fate. Direct heterocellular connections between hepatocytes and fibroblasts were visualized by the transcellular diffusion of fluorescein in this coculture system. The interactions between hepatocytes and fibroblasts were crucial for the maintenance of albumin synthesis by hepatocytes. The coculture system was also beneficial for investigating the effects of cell-cell interactions on the induction of embryonic stem (ES) cell differentiation. In cocultures grown in a sea-island pattern, ES cells formed isolated colonies surrounded by PA6 cells and differentiated into neurons with branched neurites that extended from the colonies. This versatile and biocompatible coculture system could potentially be a powerful tool for investigating cell-cell interaction and for tissue engineering applications.

On-chip culturing of hepatocytes and monitoring their ammonia metabolism.

Ammonia metabolism of hepatocytes was monitored at nanolitre-scale volumes of a medium in a microsystem with microfluidic and sensing functions.

Microprocessing of liquid plugs for bio/chemical analyses.

A microfluidic device and operation to handle liquid plugs for biochemical analyses were developed for efficient handling of plugs of many solutions. A major part of the device was a T-junction consisting of a main flow channel and a handling flow channel. Unit operations including attachment of plugs, division of a plug, sorting of plugs, and formation of plugs of various lengths enabled controlled sequential reactions in a microflow channel. Rapid mixing could easily be achieved by moving a plug formed by merging two plugs back and forth. The device could be used for efficient characterization of performance in bio/chemical sensing. In experiments using L-glutamate oxidase, plugs containing an enzyme or a substrate were formed, mixed sequentially, and the intensity of fluorescence from plugs of different concentrations of L-glutamate or pHs could be measured simultaneously. Cross-contamination of plugs by neighboring plugs poses a problem in using the same flow channel repeatedly. However, the influence could be minimized by using a cleansing plug placed between them in a sufficiently hydrophobic flow channel and by processing the plugs at a low velocity. The device can be a critical component for microprocessing in various bio/chemical analyses.

Electrochemical techniques for microfluidic applications.

Electrochemical principles provide key techniques to promote the construction of bio/chemical microsystems of the next generation. There is a wealth of technology for the microfabrication of bio/chemical sensors. In addition, microfluidic transport in a network of flow channels, pH regulation, and automatic switching can be realized by electrochemical principles. Since the basic components of the devices are electrode patterns, the integration of different components is easily achieved. With these techniques, bio/chemical assays that require the exchange of solutions can be conducted on a chip. Furthermore, autonomous microanalysis systems that can carry out necessary procedures are beginning to be realized. In this article, techniques developed in our group will be comprehensively introduced.