Fumihiro Sassa

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.

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.

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.

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.

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.

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.

Micro titration device and its application to rice freshness measurement

A plug-based on-chip titration device, constructed with a glass substrate and a poly (dimethylsiloxane) (PDMS) substrate, was used for acid-base titration. The device features a volume-regulation unit to produce plugs of a uniform volume used for titration. Although the volume of plugs formed by only injecting a solution and air simultaneously from a T-junction distributed in a wide range, the plug volume became markedly uniform using the volume-regulation unit. Acid-base titration could be conducted using this device. Furthermore, freshness of rice was evaluated by titrating fatty acid in a rice grain with a KOH solution using phenolphthalein as a pH-indicator. Rice samples harvested in different years and from different areas as well as of different species were analyzed using this device. Difference was observed clearly in the titration curves. New rice showed a color change at an early stage, whereas the color change delayed with aged rice.

Microfluidic Device for On-Chip Manipulation of Liquid Plugs for Biosensing Applications

A microfluidic device that forms a row of liquid plugs in a micro flow channel was fabricated, and its applicability to an on-chip enzyme-linked immunosorbent assay (ELISA) was demonstrated. The nanoliter liquid plugs were formed by six independent pumps and were mobilized in the micro-flow channel using a main pump. The operation of the pumps was based on the volume change caused as a result of the electrochemical production of hydrogen bubbles. When the bubbles were produced in the pumps, a polydimethylsiloxane (PDMS) diaphragm at the bottom of the compartment inflated and closed an inlet hole at the top of the reservoir. The solution in the reservoir was forced to be injected into the flow channel forming a liquid plug. After six plugs were formed, they were mobilized to a reaction chamber one by one to allow an antigen-antibody binding, cleansing, and detection. This simple and reliable liquid handling system could be applied to the detection of a tumor maker, oc-fetoprotein (AFP).

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.

Photo-tunable molecular recognizing smart material for gas sensing

We developed a dynamic photo-tunable gas adsorbent based on smart (photo sensitive) material and molecularly imprinted polymer (MIP) technology [1]. Gas adsorption property of the material, mixture of photochromic materials and polymethyl methacrylate (PMMA), can be controlled reversible and continuously by irradiation of with light different wavelength. As a result, gas sensor which is composed of a quartz crystal microbalance (QCM) coated with the developed adsorbent was fabricated. This sensor system can detect a certain gas molecule corresponding to the adsorbent property from gas mixture with only one sensor node.